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St. 2..“ .: lllllllllllllllllllllllllllllllllU!Illllllllllllllllllllllll (\ ,«5 .> 31 293 014201531 This is to certify that the dissertation entitled ISOLATION AND CHARACTERIZATION OF BOVINE B-MANNOSIDASE CDNA presented by Hong Chen has been accepted towards fulfillment of the requirements for Ph.D degree in Mica— amid/(510M Major professor hurl/wt 27. no»; MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 «v. w‘ ..—-. <—' ‘~ .—-..._—.~——‘-—-.-’4- v—v-‘V v —'— av—nu. ——-— ‘— —‘ 4—. f ‘24— —-~~—‘ .- LIBRARY Michigan State University PLACE ll RETURN BOX to roman this Mouth!“ your record. TO AVOID FINES Mun on or More data duo. DATE DUE DATE DUE DATE DUE MSU It An Afflmnttvo Adlai/Emu! Opportunity Intuition ISOLATION AND CHARACTERIZATION OF BOVINE B-MANNOBIDABB cDNA BY Kong Chen A DISBBRTATION Submitted to Michigan State university in partial fulfillment of the requirements for the degree of DOCTOR O? PHILOSOPHY Genetics Interdepartmental Doctoral Program 1994 Karen Friderici ABSTRACT ISOLATION AND CHARACTERIZATION OF BOVINB B-NANNOSIDASE cDNA BY Hong Chen Lysosomal B-mannosidase is an acidic glycosidase involved in the degradation of N-linked glycoproteins. The deficiency of B-mannosidase activity results in an autosomal recessive inherited disorder, B-mannosidosis. This lysosomal storage disease has been found to cause a severe and fatal neurovisceral storage disorder in goats and cattle. The human counterpart is milder and exhibits extreme clinical heterogeneity. The B-mannosidase cDNA had not been previously cloned from any species. Cloning and characterization of the normal B-mannosidase gene is essential in order to better understand the genetic defects underlying the B- mannosidoses, the regulation of expression of normal 6- mannosidase, and the nature of the enzyme. In this dissertation, a variety of methods have been attempted to isolate a mammalian B-mannosidase cDNA. The availability of B-mannosidase peptide sequencing finally led to the isolation and characterization of a full—length bovine B- mannosidase cDNA. This cDNA contains a 3852-bp insert, comprising a 74-bp 5' non-coding region, a 2637-bp coding region encoding 879 amino acids, a 1141-bp 3' non-coding region, and a 13-bp poly (A) tail. A 17-residue signal peptide sequence and possible polyadenylation signal sequences were identified. The deduced amino acid sequence was colinear with all peptide sequences determined by protein microsequencing. Northern analysis demonstrated a 4.2 kb single transcript in both normal and affected animals and in various tissues. The mRNA level was decreased in tissues from goats and cattle affected with B-mannosidosis, especially in the thyroid gland which contains the highest expression of B-mannosidase. The gene encoding B- mannosidosis was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. Copyright by Hong Chen, M.D. 1994 This dissertation is dedicated to my parents, who have given me so much love and support in my whole life. Also to my dear husband, Wumin, and our lovely son Allan. ACKNOWLEDGMENTS I sincerely thank Dr. Rachel Fisher for accepting me as her graduate student and for her advice and support. Her generous financial support through the five-year period of my Ph.D studies made it possible for me to explore new research areas and work with a group of friendly people that I will never forget. I thank Dr. Margaret Jones for being a member of my guidance committee. Her assistance, valuable suggestions, dedication to this project, and time spent reviewing my dissertation are deeply appreciated. I am also grateful for her financial support in the last year of my Ph.D studies and her friendship towards my family. I thank Dr. Emanuel Hackel for his guidance and willingness to be the chairperson of my guidance committee. I thank, as well, Dr. Steven Triezenberg for his valuable discussions and suggestions. I am greatly indebted to Dr. Karen Friderici. She was my advisor, my mentor, and my friend. She led me to master the molecular biology technology which will help my future career. I thank her for excellent academic advice, technical assistance, understanding, and encouragement vi throughout the development of this dissertation. I appreciate her sharing the frustrations and joys of this project with me. I thank Jeff Leipprandt (my co-worker) for his important contribution to this dissertation during the last crucial year. My appreciation extends to Dr. Kevin Cavanagh and Christine Traviss for their contributions, particularly, related to the work of amino acid sequencing analysis. My thanks also go to Nancy Truscott, Jack Truscott, Dr. Susan Lootens, Dr. Kathy Lovell, Dr. Keiji Marushige, Dr. Bryce Sopher, Dr. Yasuko Marushige, Dr. P. Storto, Dr. Xiaotan Qiao, Ralph Common, Barbara Hoefler, Helen Wells, members of The AFP laboratory and the Department of Pathology. I was fortunate to have had these friendships and associations. Studying in the Genetics Program and working in the Department of Pediatrics and Human Development, and particularly in the Department of Pathology at Michigan State University, will always remain memorable. vii TABLE OF CONTENTS Page LIST OF TABLES............................. ......... ... xi LIST OF FIGURES........................................ xii LIST OF ABBREVIATIONS.................................. xiv INTRODUCTION........................................... 1 CHAPTER ONE LITERATURE REVIEW 1.1 MOLECULAR ANALYSIS OF NORMAL LYSOSOMAL GENES.. 5 1.1.1 The size and sequence features......... 5 1.1.2 Sequence homologies.................... 11 1.1.3 Alternative transcripts................ 12 1.1.4 Promoters.............................. 16 1.1.5 Pseudogenes............................ 19 1.2 STRATEGIES FOR THE MOLECULAR CLONING OF LYSOSOMAL ENZYME GENES........................ 23 1.2.1 Identification of cDNA clone by hybrid-selected translation............ 24 .2.2 Nucleic acid hybridization............. 25 .2.3 Identification of genes by antibody probes........................ 25 1.2.4 Identification of cDNA clones by oligonucleotides....................... 28 1.2.5 Isolation of genes by polymerase chain reaction (PCR)........ 31 1.3 fl-MANNOSIDOSIS................................ 34 1.4 B-MANNOSIDASE................................. 40 CHAPTER TWO ISOLATION OF A HUMAN CDNA CLONE ENCODING AN UNKNOWN PROTEIN 2.1 INTRODUCTION.................................. 48 2.2 MATERIALS AND METHODS......................... 50 2.2.1 Materials.............................. 50 2.2.2 Library screening...................... 50 2.2.3 Subcloning......... ..... . ....... . ...... 51 viii 2.2.4 Nucleotide sequencing.................. 52 2.2.5 In vitro expression.................... 53 2.3 RESULTS AND DISCUSSION........................ 54 2.3.1 Cloning the human homologues of goat clones......................... 54 2.3.2 Sequence of human p5m11 clones......... 56 2.3.3 Expression of human p5m11.............. 62 2.4 SUMMARY....................................... 63 CHAPTER THREE ISOLATION AND CHARACTERIZATION OF BOVINE B-MANNOSIDASE CDNA CLONES 3.1 SCREENING WITH POLYCLONAL ANTIBODIES.......... 65 3.1.1 Introduction........................... 65 3.1.2 Materials and methods.................. 67 3.1.2.1 Titer of polyclonal antisera.... 67 3.1.2.2 Purification of IgG fractions and affinity purification of polyclonal antibodies........... 68 3.1.2.3 Removal of anti-E.coli antibody. 69 3.1.2.4 Screening cDNA libraries........ 70 3.1.2.5 Identification of expressed fusion proteins................. 70 3.1.3 Results................................ 71 3.1.4 Discussion............................. 75 3.1.4.1 Screening with antiserum 228.... 75 3.1.4.2 Screening with antisera 259 and ZG9..................... 77 3.1.5 Summary................................ 79 3.2 ISOLATION AND CHARACTERIZATION OF BOVINE B-MANNOSIDASE CDNA............................ 80 3.2.1 Introduction........................... 80 3.2.2 Experimental procedures................ 82 3.2.2.1 Partial amino acid sequencing... 82 3.2.2.2 Construction of synthetic oligonucleotide probes.......... 83 .2.2.3 Labeling probes................. 87 .2.2.4 cDNA library screening.......... 87 .2.2.5 Polymerase chain reaction (PCR). 89 .2.2.6 RNA isolation and Northern blot hybridization................... 91 3.2.2.7 DNA sequencing and computer analysis........................ 92 3.2.2.8 Southern hybridization of chromosome blot and zoo blot.... 93 3.2.3 Results................................ 94 3.2.3.1 Peptide sequencing.............. 94 3.2.3.2 Isolation and characterization of cDNA clones.................. 95 3.2.3.3 Northern analysis............... 114 UUUU ix 3.2.3.4 Southern genomic blot analysis.. 3 O 2 O 4 DiSCUSSion. O O O O O O O O I O O O O O O O O O O O O O O O O O O O 3 O 2 O 5 summary. 0 O O I O O O O O O O O O O O O O O O O O O O O O O O O O O O 3.4 BEYOND THE ISOLATION AND CWflERIZATION.00.0.0000...0.00.00.00.00...O BIBLIWRAPHYOOCO0.0.00.0......OOOOOOOOOOOOOOOOOO0...... 114 115 128 130 136 Table Table Table Table Table Table Table Table LIST OF TABLES cDNA clones of normal lysosomal enzyme genes.O.............OOOOOOO......OOOOOOO (cont’d).........OOOOOOOOOOOOOOOOO...... Putative promoter regions of genes encoding lysosomal enzymes.............. (cont’d) ................................ Western analysis of putative clones..... CNBr/tryptic peptide sequences of B-mannosidase........................ Oligonucleotides used for screening of cDNA libraries....................... Selected oligonucleotide primers used in PCR analYSis.......OOOOOOOOOOOOOOOOOO xi 17 18 73 84 85 86 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES The restriction map and sequencing strategy of human cDNA clones.............. Partial nucleotide sequence of human #lpsmll CloneOOOOOOOOOIOOOOOOO00.00.0000... Western analysis of fusion proteins with antiserum 228............... ....... ........ Panel A: Coomassie staining of purified B-mannosidase peptides. Panel B: Western analysis of purified B-mannosidase peptides with antisera 259 and z30......... The reverse phase HPLC profile of CNBr/tryptic cleaved peptides of B-mannosidase.................... ..... ..... The restriction map and sequencing strategy for B-mannosidase cDNA clones ..... Nucleotide and deduced amino acid sequences of B-mannosidase full length CDNAOOOOCOOOOOO0.000.000.0000.0.0.0.0....O The hydropathy plot of the B-mannosidase polypeptides predicted from the full length CDNAOOOO......OOOOOOOOOOOOOOOOO DNA sequence comparison between the human expressed sequences and the B-mannosidase CDNAOOOOOOOOOOOOOOOOOO ..... 0.0.0.0.... ..... The strategy of the 5' RACE for the isolation of the 5' region of fl-mannosidase cDNA................ ......... Analysis of the 5’ RACE products........... Northern hybridization analysis of normal tissues and affected animals ........ xii 55 57 72 96 97 99 100 104 108 112 113 116 Figure Figure Figure Figure Southern hybridization of genomic DNA fromvarious speCieSOOOOOOOOOOOO......OOOOO Chromosome localization of B-mannosidase CDNAOOOOO0......0.00.0.0.........OOOOOOOOOO A nonsense mutation in B-mannosidosis calves...................... Summary of PCR analysis of cDNAs from normal and affected B-mannosidosis goats... xiii 117 119 132 133 AP hp cDNA CNBr Con A cpm DMEM dNTP DTT EDTA eonII Gd! GAPDH HPLC 19 IPTG MAbs M-MLV MOPAC PAGE PCR RACE SDS TBS TMAC TNT UAP LIST OF ABBREVIATIONS alkaline phosphate base pair complementary DNA cyanogen bromide concanavalin A counts per minute Dulbecco's modified Eagle media deoxyribonucleoside triphosphate dithiothreitol disodium ethylenediaminetetra-acetate exonuclease III GalNAcBl~4(NeuAca2~3)GalBl~4Glc31~ceramide glyceraldehyde-3-phosphate-dehydrogenase high performance liquid chromatography immunoglobulin isopropyl-thiogalactopyranoside monoclonal antibodies Moloney murine leukemia virus mixed oligonucleotide primed amplification of cDNA polyacrylamide gel electrophoresis polymerase chain reaction rapid amplification of cDNA ends sodium dodecyl sulfate tris-buffered saline tetramethylammonium chloride Tris-NaCl-Tween 20 universal amplification primer xiv INTRODUCTION INTRODUCTION Lysosomalstorage diseases are a group of inherited or induced disorders caused by deficient activity of one or several lysosomal enzymes (e.g. I-cell disease and multiple sulfatase deficiency) (Neufeld, 1991; von Figura et al., 1984). The concept of lysosomal storage disease was first introduced in the context of a-glucosidase deficiency, known as the Pompe disease (Hers, 1965; 1973), on the basis of classical studies on the biochemistry of lysosomes. So far, three dozen lysosomal storage diseases have been discovered (Neufeld, 1991). The classification of lysosomal storage diseases is typically based on the main chemical type of accumulated material or the function of the defective proteins and enzyme involved (Watts and Gibbs, 1986; Neufeld, 1991). Currently, lysosomal storage diseases are divided into six major categories (Neufeld, 1991) comprising: (1) disorders of sphingolipid degradation; (2) disorders of glycoprotein degradation; (3) disorders of glycosaminoglycan degradation; (4) disorders of one single enzyme deficiency; (5) disorders of lysosomal enzyme biosynthesis; and (6) disorders of lysosomal membrane transport. Other classification methods, e.g. according to 1 2 the principles that cause the deficiency of the specific lysosomal enzyme activities, were also introduced (von Figura et a1., 1984). Within each of the human lysosomal storage diseases, considerable clinical, biochemical, and molecular heterogeneity has been observed. The pathogenetic mechanisms for lysosomal storage diseases are still unclear. The storage of undegraded macromolecules in lysosomes enlarges and probably perturbs the physiology of the cell, which presumably contributes to the various clinical manifestations. The impairment of a specific lysosomal enzymatic function is attributed to gene mutations that may (1) decrease either the amount or the rate of enzyme synthesis; (2) produce a catalytically inactive enzyme which either fails to bind its substrate or fails to catalyze reactions after binding to the substrate; (3) impair the transport of a newly synthesized enzyme into lysosomes; (4) increase the rate of enzyme degradation; or (5) decrease the concentration of a protecting or an activating factor (Watts ' and Gibbs, 1986). In addition, in some lysosomal storage diseases, i.e. mucopolysaccharidoses, the accumulated substrates interact with an enzyme outside the main pathways affected by the genetic deficiencies and cause a "secondary lysosomal disease" (von Figura et a1., 1984) B-Mannosidosis, a recently defined inherited autosomal 3 recessive disorder (Jones and Laine, 1981; Jones and Dawson, 1981), is one of the lysosomal storage diseases. It is caused by decreased activity of B-mannosidase which is involved in glycoprotein catabolism. fl-Mannosidosis was first described in Nubian goats and has more recently also been found in humans and Salers cattle (fiartley et a1., 1973; Healy et a1., 1981; Jones and Laine, 1981; Jones and Dawson, 1981; Bryan et a1., 1990; Jolly et a1., 1990; Abbitt et a1., 1991; Wenger et a1., 1986; Cooper et a1., 1986; 1991; Dorland et 31., 1988; Kleijer et a1., 1990; Wijburg et a1., 1991; Poenaru et a1., 1992; Levade et a1., 1991; 1994). The disease in goats and cattle has been extensively characterized in this laboratory (Jones and Laine, 1981; Jones and Dawson, 1981; Matsuura, Laine and Jones, 1981; Jones et a1., 1982; 1983; 1984; 1986; 1992; Masturra and Jones, 1985; Jones and Abbitt, 1993; Lovell and Jones, 1983; 1985; Lovell and Boyer, 1987; Lovell, 1990; Lovell et a1., 1991; 1994; Dahl et a1., 1986; Kumar et a1., 1986; Fisher et a1., 1987). B-Mannosidase from goats and cattle has also been purified and characterized (Cavanagh et a1., 1982; 1985; 1992; Dunstan et a1., 1983; Frei et a1., 1988; Sopher et a1., 1992; 1993). However, nothing is known about the molecular defect of this disease. The gene encoding B- mannosidase has not been cloned from any species, although an attempt was made to clone guinea pig B-mannosidase (McCabe and Dawson, 1990; Sopher, 1992a). The aim of this 4 project was to isolate B-mannosidase cDNA. The cloning of the B-mannosidase gene will assist with the characterization of the structure, function, and expression of the gene product. Identification of mutations may then be possible, and sequentially, gene therapy. CHAPTER ONE LITERATURE REVIEW CHAPTER ONE LITERATURE REVIEW 1.1 MOLECULAR ANALYSIS OF NORMAL LYSOSONAL GENES Cloning and characterization of normal lysosomal genes is a first step toward understanding the structure and function of lysosomal enzymes and toward the analysis of mutations underlying lysosomal storage diseases. Studies of these normal lysosomal genes and transcripts revealed some common characteristics. 1.1.1 The size and sequence features Nearly 20 complementary DNAs encoding lysosomal enzymes have been cloned and characterized (Table 1.1) during the past ten years. The sizes of full-length cDNAs vary from 1.4 kb to 3.6 kb. The consensus sequence (GCCA/GCCATGG) (Kozak, 1986) for the translation initiation site was present in most of the lysosomal genes. It has been well demonstrated that within the consensus sequence, the purine at -3 position was most highly conserved (Kozak, 1986). This purine is also conserved in all the lysosomal enzymes except arylsulfatase A (Stein et 31., 1989). 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Like the secretory proteins, lysosomal enzymes contain a short signal peptide at the N-terminus, which typically contains 15-30 amino acids and has the following characteristic compositions (von Heijne, 1983; 1986; 1990): (a) a positively charged amino acid within the first five residues; (b) a hydrophobic core (7-15 residues); and (c) a more polar C-terminal region (3-7 residues). The signal peptide directs the nascent protein across the membrane and into the lumen of endoplasmic reticulum by interacting with a signal recognition particle (Kornfeld, 1987; Lewin, 1990). It is usually cleaved immediately after its transfer into the endoplasmic reticulum and before the synthesis of the peptide is completed. The typical signal sequence has been observed in all the lysosomal enzyme genes cloned. All lysosomal enzymes studied so far are synthesized as polypeptides that undergoes limited proteolytic processing which may involve removal of a signal sequence, an N- terminal sequence, a C-terminal sequence, and/or internal cleavage (Kornfeld, 1987; Holtzman, 1989). Apart from the signal sequence cleavage, cleavage at the C-terminus seems to be the most common proteolytic processing event associated with the maturation of lysosomal precursors (Yamamoto et a1., 1990; Erickson and Blobel, 1983; Gottschalk et a1., 1988; Quinn et a1., 1987). To date, arylsulfatase A enzyme is the only lysosomal enzyme for 9 which post-translational proteolytic processing appears to be restricted to cleavage of the signal peptide only (Stein et a1., 1989). This was demonstrated by three lines of evidence: (1) the predicted molecular mass after removal of the signal peptide sequence was close to the size of deglycosylated arylsufatase A on SDS-PAGE; (2) an arylsulfatase A peptide corresponded to the predicted C- terminal sequences and lacked only the three residues located just before the stop codon (The three residues were thought to be cleaved by the staphylococcal V8 proteinase during the step of proteinase digestion in amino acid sequencing (Stein et a1., 1989)); and (3) the N-terminal sequence was found to follow immediately after the signal peptide sequence. Proteolytic processing is not essential for activation as it was found that the precursor forms of many lysosomal enzymes except proteases were active (Holtzman, 1989). It has been speculated that the precursor forms may be important for folding, stabilizing, or sorting the proteins in their early stages. Most cDNAs encoding lysosomal enzymes have a 5' untranslated region containing less than 600 bp compared to a 3' untranslated region which is usually more than 500 bp long. cDNA containing 3' untranslated regions as long as 2 kb in length has been documented for the human N- acetylgalactosidase gene (Wang et a1., 1990). The 3' untranslated region is believed to be important to mRNA 10 turnover in eukaryotic cells (Ross et a1., 1988). However, this may not be true in all cases. The termination codon of human a-galactosidase transcript is followed immediately by a poly (A) tail without a 3’ untranslated region according to the analyses of both cDNA and genomic clones, which indicates that the 3' untranslated region is not absolutely critical for its stability (Bishop et a1., 1986; 1988). Like most eukaryotic mRNAs, all full length cDNAs of lysosomal enzyme genes possess a poly A tail at their 3' termini. The function of the poly A tail is still not fully understood. It may affect mRNA stability and translation (Bernstein et a1., 1989; Proudfoot, 1991; Jackson et a1., 1990). The addition of a poly A tail occurs in the nucleus, and involves cleavage of the primary transcript with subsequent addition of a poly A tail to the newly formed 3' end (Wickens, 1990). Polyadenylation requires the sequence AAUAAA. This hexanucleotide is usually located at 15-35 bp upstream of the poly A addition site of mRNAs and is highly conserved. Mutation analysis indicated that any base change in AAUAAA affects the accuracy and efficiency of cleavage and polyadenylation (Sheets et a1., 1990). However, polyadenylation signal sequences other than the consensus AAUAAA such as CUUAAA, AAUGAA, AAUAAC, or AUUAAA have been found in a few lysosomal enzyme genes (Stoltzfus et a1., 1992; Pohlmann et a1., 1988; Stein et a1., 1989; Proia et a1., 1986). 11 1.1.2. Sequence homologies There is little, if any, homology among the cloned lysosomal genes at either the DNA or amino acid sequence levels. However, it has been observed that there are striking sequence similarities between lysosomal enzymes and non-lysosomal enzymes with similar catalytic functions. Sequence homology has been observed between lysosomal acid phosphatase and prostatic acid phosphatase (Pohlmann et a1., 1988); between a-glucosidase and intestinal sucrase- isomaltose complex (Hoefsloot et a1., 1988); and between lysosomal arylsulfatases A and B, arylsulfatase in the sea urchin, and steroid sulfatase (Stein et a1., 1989; Schuchman et a1., 1990; Sasaki et a1; 1988). There is strong sequence conservation in lysosomal enzymes from different species including lower eukaryotes and even prokaryotes (Peters et a1., 1990; Schuchman et a1., 1990; Stein et a1., 1989; Wilson et a1., 1990; Wang et a1., 1990; Stoltzfus et a1., 1992). As summarized by Neufeld (1991), there are families of B-hexosaminidases, B-glucuronidases, a-galactosidases, a- glucosidases, phosphatases, sulfatases, a-L-iduronidases and other enzymes. Genes in each family presumably originated from the same ancestral gene by duplication and their divergence may reflect their different specificities regarding locations and substrates. 12 1.1.3. Alternative transcripts In most eukaryotic genes, coding regions are interrupted by intervening sequences, known as introns. Introns have to be spliced out from the primary transcript to produce a mature mRNA. Splicing out of introns occurs by the formation of a lariat intermediate in which the left end of the intron is joined to a site near the right end of the intron (Lewin, 1990). Sequences at the left and right splice junctions appear to be conserved. The location of the branch point (i.e. the site near the right end of the intron) seems to play a role in the constitutive splicing (Smith et a1., 1989). Alternative splicing by various mechanisms is an efficient way to generate protein isoforms and may play important roles in gene regulation (Smith et a1., 1989). Transcripts that may be generated by alternative splicing have been reported for several lysosomal enzyme genes (Morreau et a1., 1989; Oshima et a1., 1987; Schuchman et a1., 1991; Wang et a1., 1990; Ikonen et a1., 1991; Scott et a1., 1991). Two cDNAs with different lengths for fl-galactosidase were isolated by Morreau et a1. (1989). The short one lacked two stretches of coding sequences (212 bp and 181 bp) in the 5' region. The reading frame was shifted by missing the 212 bp, but it was restored after exclusion of another 181 bp. The study of a genomic DNA sequence spanning the two missing areas demonstrated that the 212 bp sequence was 13 encoded by two separate exons of 151 and 61 bp. The 181 bp sequence was encoded in another exon. Furthermore, an exon specifying a 95 bp sequence between the two missing stretches was found. These results confirmed that the shorter B-galactosidase cDNA resulted from alternative splicing of its precursor mRNA by skipping two adjacent exons that encode a total of 212 bp and another exon specifying the 181 bp region. The short cDNA was only represented in anti-B-galactosidase antibody selected mRNA. No B-galactosidase activity was expressed by the corresponding protein (Morreau et a1., 1989). Two cDNAs of the human a-N-acetylgalactosaminidase gene have been reported (Yamauchi et a1., 1990). The transcript encoding a cDNA which contained a 70-bp insertion in the coding region was present only in brain and in a small amount. The possibility of alternative splicing in this gene was studied by another group (Wang et a1., 1990). The region flanking the 70 bp insertion was amplified by PCR. cDNA transcribed from mRNAs in various tissues, cloned a-N- acetylgalactosaminidase cDNAs, and genomic clones were used as DNA templates in the PCR. No alternative transcripts in various tissues were observed. A different PCR product in one of cDNA clones was found. However, the PCR product contained an in-frame 45 bp deletion instead of the 70 bp insertion reported by Yamauchi et a1. (1990). Strikingly, both the 70 bp insertion and the 45 bp deletion occurred at 14 the same intron. The investigators proposed that a unique sequence and/or secondary structure in this intron or surrounding region may impair the fidelity of the constitutive splicing. Studies of the B-glucuronidase gene demonstrated that there were two types of mRNA corresponding to the two types of B-glucuronidase cDNA clones isolated from human placenta (Oshima et a1., 1987). Only the protein specified by the longer mRNA showed B-glucuronidase activity. Cloning and characterization of the genomic B-glucuronidase gene illustrated that there was an exon corresponding exactly to the 153-bp deletion in the shorter cDNA. This observation supported the previous hypothesis that the short transcript was generated from alternative splicing by exclusion of that exon (Miller et a1., 1990). More recently, multiple transcripts generated from alternative splicing have been demonstrated for the human acid sphingomyelinase gene (Quintern et a1., 1989; Schuchman et a1., 1991). Three types of cDNA were isolated. Type 1 represented the majority of the acid sphingomyelinase clones identified and expressed lysosomal activity of sphingomyelinase in COS cells. It contained an insert of 1879 bp and possessed a unique 172 bp sequence encoding 57 amino acids. Type 2 contained an insert of 1382 bp and an unrelated 40 bp in-frame sequence that substituted for the 172 bp in-frame sequence presented in type 1. Type 3 15 contained a truncated open reading frame and lacked both the 172 bp and 40 bp sequences. The PCR results of a genomic sequence proved that the 172 bp was encoded by an exon, whereas the 40 bp sequence was of intronic origin. Furthermore, a weak donor splice site adjacent to the 172 bp exon was observed. The authors thought that the occurrence of type 2 cDNA may be due to the involvement of this cryptic 5’ donor splice site, which excluded the entire 172-bp exon and left 40 bp of intron sequence. The type 3 resulted from exon skipping, which excluded the 172-bp exon as well as the whole intronic sequence flanking the exon. The use of a cryptic acceptor splice site within an exon may also be responsible for an alternative canine a-L- iduronidase transcript (Stoltzfus et a1., 1992). In addition to alternative splicing, multiple transcripts can arise from differential polyadenylation. This has been observed in iduronate-z-sulfatase (Wilson et a1., 1990), a- N-acetylgalactosaminidase (Wang et a1., 1990), and B- hexosaminidase (Proia et a1., 1987). The physiological functions of these alternative transcripts remain unclear. The proteins resulting from alternatively spliced transcripts do not have the same catalytic activity as the corresponding hydrolases (Oshima et a1., 1987; Morreau et a1., 1989). 16 1.1.4. Promoters A promoter is a region of DNA involved in binding RNA polymerase to initiate transcription. "Housekeeping" genes produce proteins that have a wide tissue distribution and provide the essential functions needed for many types of cells (Dynan, 1986). The promoters for housekeeping genes typically have the following characteristic features: (1) no TATA box; (2) a high G/C content; and (3) the presence of CpG islands. To date, about a dozen lysosomal enzyme genes have been isolated and characterized (Table 1.2). All except the B- glucocerebrosidase gene have promoters that are characteristic of housekeeping genes. The promoters of the human acid phosphatase gene (Geier et a1., 1989), the human arylsulfatase A gene (Kreysing et a1., 1990), the B-subunit of fi-hexosaminidase (Neote et a1., 1988), the a-glucosidase gene (Martiniuk et a1., 1990; Hoefsloot et a1., 1990), the B-galactosidase (Suzuki et a1., 1991; Morreau et a1., 1991), and the a-L-iduronidase gene (Moskowitz et a1., 1992) all lack a TATA box and have a high G/C content with possible sp1 binding sites, i.e. the GGCGGG motif. Multiple initiation sites of transcription were observed in promoters of the acid phosphatase gene, the arylsulfatase A gene, and the mouse B-galactosidase gene. This is typical of promoters lacking a TATA box. Some lysosomal enzyme genes whose promoters are G/C rich do contain a TATA box. These 17 OOH: omma ..a- a. man: roan vocauuooa can: «ma mm: «own 0:0: ca can: ouaoauouaaonu non: aha: mnau maau avau moan mama ..ac a. anal aaoo moan «can nmu seaso naau ama not as: man ma can a ouauauououaaouo vaau anau man amma ..au «u can wuoux man anon anon cc Gamma-oushlnlu omma ..aq «0 mcanhoux can 0:0: ococ mam: ou man: a oasuauasuamaa man: man: man: saw: nmma ..an a. aavu asz aaan saan «man aaaaosaua moan ama oman «or: naas ma as unucaaomaomcasmu oaoa omen mama ..a1 a. man: can: uoaoo can omau econ mm an: on an: unauusmoosm vaua ammu amma ..aa a. cam: oavu mean unavacasa-ouocaum man: can: aa van: aha: econ am pen: namuouanzuu no «0 ...ouan unau- aOOGOHOuom auosuo aha 81:0 £919 ommusoouem soauaauoasdua mecca c a.ahwca adaoaoamq unavoosn noose no acoaoom Heuoaoum o>au¢usm a.a Candy 18 .mmma ..Nu «0 cuenosnuav sand» o.cnfinos:om Gown namesake ma Candy case a Oman smut «am: can: maal Nmma ..HI 00 aha: Nfld§OOv~ Om H l OCOC 0606 s. 5H l ODGUACOHSUH anal maal aama ..H1 u. moan anal ouooz mm- ama mm- aamu «no: a: ouuaacasuuoxoaua amma .Hs «0 ¢al aman heamasa coal «at avmn coo: ococ mm on: casuacousosaona aama ..a- no Hucaom can: amal mama ..au as NUHSHOE OCOC Gm hl Ole h m 0|! 00104 IORQOHOOOOSHU aama aaal aaal amma .Hu «0 «ma: snouuox aaau «ma can mom: 0:0: am am: on an: o-uaauououauoua ...auan unau- eeocouOuoa auonuo ama 9440 £949 emaucoouom coaumauoucuua cocoa 4.6.»:00. a.a canny 19 include the a-subunit of the B-hexosaminidase (Proia et a1., 1987), a-galactosidase (Bishop et a1., 1988), acid sphingomyelinase (Schuchman et a1., 1992), and murine B- glucuronidase (D'Amore et a1., 1988) genes. The human glucocerebrosidase gene is not a typical housekeeping gene. Its promoter contains a TATA box and CAAT-like box, and lacks the spl binding site (Reiner et a1., 1988a; Grabowski et a1., 1990). TATA and CAAT-like boxes are common in regulated genes. Reiner et al. (1988b) reported that the glucocerebrosidase gene had different levels of expression in a variety of cell types and the level of mRNA was well correlated to the level of chloramphenicol acetyltransferase (CAT) activity directed by the promoter in different cells. 1.1.5 Pseudogenes Pseudogenes are defined by stable genomic sequences that are related to those of functional genes, but they can not be translated into functional proteins. There are two types of pseudogenes. The first type includes those that retain the introns found in their functional counterparts. They are typically derived by duplications and mutations of ancestral active genes. The second type are so called ”processed" pseudogenes. They contain no intervening sequences and resemble the RNA transcripts of their active counterparts. Processed pseudogenes presumably resulted from integration of their mRNAs or cDNA copies into the 20 genome. They may be located anywhere in the genome. Reiner et a1. (1988) reported two different genomic clones for glucocerebrosidase gene that had different lengths. These two clones were later found to have 96% identity at the nucleotide level and were located on the same chromosome (Horowitz et a1., 1989). However, the promoter of the shorter clone showed very low or negligible activity when coupled to a bacterial gene. So far, no protein product has been found to be produced by the shorter gene. This pseudogene lost the open reading frame due to a large deletion in several introns and exons. Presumably, most pseudogenes do not have transcriptional and translational activities. However, some exceptions were reported in which pseudogenes had transcripts or protein products (Sorge et a1., 1990). The glucocerebrosidase pseudogene was found to be transcribed at a similar level to its active gene counterpart (Sorge et a1., 1990). Extra precaution is needed in molecular diagnosis by PCR due to the presence of high levels of transcripts of pseudogenes. Besides the glucocerebrosidase pseudogene, a pseudogene for human a-L-fucosidase gene was discovered by O'Brien’s group (Kretz et a1., 1992). The a-L-fucosidase pseudogene has 80% identity with the active gene but is located on a different chromosome than the functional gene. The a-L- fucosidase pseudogene contains no introns and the sequence diverges at the beginning and the end of the transcript of 21 the functional gene. These phenomena are considered to be part of the general structural characteristics of the processed pseuogenes (Vanin, 1985). However, the a-L-fucosidase pseudogene lacks a poly A tract at the 3’ end and the usual direct repeats flanking a pseudogene sequence. More recently, multiple pseuogenes for human B- glucuronidase gene were identified by PCR when mutation analysis was carried out in a patient with mucopolysaccharidosis (Shipley et a1., 1991; Vervoort et a1., 1993). These pseudogenes shared high sequence homologies with the functional counterparts, but the normal reading frame was disrupted in most cases. Some of the B- glucuronidase pseudogenes containing uninterrupted open reading frames may, in fact, represent gene families with closely related functions. Amplification of genomic DNA from a panel of human/rodent somatic cell hybrid lines demonstrated that the multiple unprocessed pseudogenes for human B-glucuronidase were located on six different chromosomes. Three types of B-glucuronidase pseudogenes were identified with respect to the exon 11 region. Each of these pseudogenes was found to be located in two different chromosomes: type 1 was located in chromosome 5 and 6, type 2 in chromosome 22 and 22, and type 3 in chromosome 7 and Y. A conflict arose regarding the chromosomal location of the gene encoding the Gw,activator protein. In 1985, Burg 22 et a1. (1985) mapped the gene for human Ga activator protein to chromosome 5 by expressing the protein in human [mouse somatic cell hybrids. However, in 1991, Kleyn et a1. (1991) using PCR analysis reported that the gene for the G“, activator protein was not located on chromosome 5. This discrepancy was elegantly explained by the discovery of a processed pseudogene coding for the 6.2 activator protein (xie et a1., 1992). A 7300-bp intron between nucleotides Gm and G,“ of the cDNA was present in the functional gene but missing in the pseudogene. The primers chosen by Kleyn et a1. (1991) happened to flank the intron region. Therefore, amplification of genomic DNAs by these primers could only generate products originating from the pseudogene but not from the functional gene due to the presence of a large sized intron. By using primers specific to the functional gene or the pseudogene, Xie et a1. (1992) were able to place the functional gene on chromosome 5 and the pseudogene on chromosome 3. These studies illustrate that caution is required in mutation analysis of genomic DNA. However, it is still possible to use genomic DNA for mutation analysis even if a pseudogene is present. 23 1.2 STRATEGIES FOR THE MOLECULAR CLONING OE LYSOSOMAL ENZYME In order to better understand the fundamental abnormalities of lysosomal storage diseases at the gene level, it is necessary to isolate and characterize the normal structural gene which codes for the specific lysosomal enzyme or enzyme protector. Not only will the molecular cloning of the normal lysosomal gene facilitate the understanding of the structure, function, and expression of the gene products, but it will also lead to the possibility of molecular diagnosis and gene therapy. Lysosomal enzymes are encoded by mRNAs that are present at low abundance (O'Brien et a1., 1984). Therefore, cloning of the genes for these enzymes has proven to be difficult. Nevertheless, thanks to the rapid progress in molecular cloning techniques, 16 lysosomal enzyme genes whose deficiencies lead to known lysosomal storage diseases have been isolated (Table 1.1). The isolation of cloned cDNA involves several major steps: enrichment of mRNA, synthesis of cDNA, insertion into a cloning vector, and identification of a clone. This review is to discuss the various techniques involved in the final step of molecular cloning. 24 1.2.1 Identification of a cDNA clone by hybrid-selected translation B-Glucuronidase, one of the first lysosomal enzymes to be cloned, was isolated by the technique of hybrid-select translation (Hieber, 1982). First, a cDNA library was screened using a population of mRNAs as probes. The probes were either total mRNA fractions or B-glucuronidase-enriched mRNA. DNA was isolated from positive clones, bound to nitrocellulose filters and hybridized with the population of mRNAs. After washing, the bound mRNAs were eluted from the filters. The selected mRNAs were used to direct cell-free protein synthesis in a rabbit reticulocyte lysate system. Positive clones were identified by looking for bands of total in vitro translated protein which had the same mobility as the immunoprecipitated and translated proteins on SDS-polyacrylamide gel (PAGE). The same technique has been used by other researchers for verifing clones coding for a-glucosidase (Konings, 1984) and the a-chain of B- hexosaminidase (Myerowitz et a1., 1984). Hybrid-selected translation is a valuable technique both as a screening tool and as an independent means of confirming the authenticity of a clone. However, this method is time- and labor-consuming. In addition, it may be inefficient for large mRNA because of the possible degradation of mRNA and inefficient transcription (Horwich et a1., 1984). Today, this technique is mainly used to 25 verify positive clones isolated by other methods. 1.2.2 NUcleic acid hybridization When a cDNA probe is available, it is often used to screen cDNA libraries from the same species to isolate the full-length cDNA, or it is used to screen cDNA libraries from other species. High or relatively high stringency hybridization conditions can usually be applied when homologous or partially homologous cDNA probes are used. Therefore, screening with cDNA probes seldom has the problem of false positive clones. cDNA probes synthesized from a population of mRNAs can also be used to screen clones. However, for rare genes, it is necessary to enrich the mRNA before the preparation of cDNA probes. fi-Glucuronidase (Nishimura et a1., 1986), a-glucosidase (Konings, 1984), and the a-chain of B-hexosaminidase (Myerowitz et a1., 1984) were successfully isolated by differential hybridization with cDNA probes prepared by reverse transcription of both immunoselected and depleted polysomal mRNA. 1.2.3 Identification of genes by antibody probes The technique of screening cDNA libraries with antibodies was introduced after the development of the phage Agtll as an expression vector (Young et a1., 1983). In this system, double stranded cDNA is inserted into a restriction site in the E. coli lacz (B-galactosidase) gene carried by 26 Agt11, AZAPII, or other expression vectors. If the foreign DNA is inserted in the correct orientation and reading frame, a fusion protein will be produced with B- galactosidase at its amino terminal and the foreign protein at the carboxyl terminus. To isolate cDNA clones by immunoscreening, E. coli cells transformed with recombinant phage from a cDNA expression library are plated and induced to make fusion proteins when phage plaques are just visible. The plates are overlaid with nitrocellulose filters to absorb the fusion proteins. Immunoreactions are carried out by sequential incubation of filters with a primary antibody specific for the enzyme being cloned and a secondary antibody against the primary one. Finally, positive clones are identified by either chromogenic immunodetection or auto-radiography depending on the secondary antibody. By screening a human hepatoma cDNA library constructed in a Agt11 expression vector, O'Brien's group was able to isolate partial cDNAs encoding lysosomal enzymes a-fucosidase, galactosidase, the a-chain of fl-hexosaminidase (O'Brien et a1., 1984; de Wet et a1., 1984). This technique was also used by other groups to identify genes encoding lysosomal enzymes such as acid phosphatase, a-glucosidase, a- galactosidase A, a-fucosidase, and fl-glucocerebrosidase (Pohlmann et a1., 1988; Hoefsloot et a1., 1988; Martiniuk et a1., 1986; Calhoun et a1., 1985., Sorge et a1., 1985., Ginns et a1., 1984; Fisher et a1., 1989; Fukushima et a1., 1985). 27 There are several problems which may affect the success of immunoscreening. First, most human and rabbit antisera usually contain significant amounts of antibodies against the E. coli or phage proteins (Snyder et a1., 1987). These nonspecific antibodies can be removed by pseudoscreening, i.e., by preincubating the diluted antisera with filters coated by lysed E. coli for several times or, more efficiently, by passing the polyclonal antisera through an affinity column to which a large amount of lysed E. coli proteins has been coupled (Sambrook et a1., 1989). Secondly, false positive clones may be detected by contaminating antibodies generated from impure proteins. The use of affinity purified antisera may overcome this problem. However, any protein possessing a common epitope to the protein of interest may still be isolated. Monoclonal antibodies usually generate less background than polyclonal antibodies. However, screening with polyclonal antibodies has a higher possibility of isolating a gene since it recognizes multiple epitopes. All in all, the ideal probes are pools of monoclonal antibodies directed against different epitopes of the protein of interest. Although antibody screening is frequently used, the success of this approach relies not only on the quality of antibody used, but also the abundance of the protein expressed. Generation of monoclonal or polyclonal antisera is labor- intensive and time-consuming. More plaques or colonies may 28 need to be screened in the isolation of a gene encoded in very low abundance compared with other methods, since only one out of six recombinant clones, in theory, will produce fusion proteins and react with antibody. The most difficult and tedious part of isolating genes by antibody screening is to verify the immunoreactive clones. A cDNA selected by antibody probes is by no means a true positive. Failure to isolate genes by antibody screening was documented (Moreman, 1989) and is not uncommon. 1.2.4 Identification of cDNA clones by oligonucleotides Using degenerate oligonucleotides to screen cDNA libraries is currently one of the most commonly employed techniques for cloning genes. To apply this technique, a portion of the amino acid sequence of the protein is required. Based on the known peptide sequence information, either a set of short degenerate oligonucleotides or a unique long guessmer can be synthesized chemically. Short degenerate oligonucleotides are usually 17 to 20 nucleotides long and include all possible codon choices for each amino acid. Only one sequence in the mixture will perfectly match the coding region in the cDNA. The disadvantages of using short degenerate oligonucleotides are: (1) An amino acid peptide with low degeneracy is necessary in order to synthesize an oligonucleotide mixture with low complexity. The more complicated the 29 oligonucleotide mixture is, the more false positives will be isolated. (2) The sequence information of amino acid peptides should be correct. A single mismatch of nucleotides will substantially affect the hybridization of the probe. The long guessmer is typically 30 to 100 nucleotides long with a unique sequence. The best guesses are made for each amino acid based on the typical codon usage. The guessmer overcomes the disadvantages of short degenerate oligonucleotides because the long length in the guessmer compensates the possible effect of nucleotide mismatches. Thus the amino acid sequence need not be absolutely correct and amino acids with high codon degeneracy (e.g. arginine, leucine, and serine) need not be avoided when designing a guessmer. However, at least ten amino acid residues are needed for syntheSizing a guessmer. Screening with oligonucleotides is done by either plaque or colony hybridization. It appears that plaque hybridization with short mixed oligonucleotides was seldom used because there is less DNA per plaque than per bacterial colony. For short oligonucleotides, however, the recombinant plaques may be amplified in situ before hybridization. By amplification of plaques in situ, there are more copies of the phage DNA per plaque fixed on the filters. Therefore, the intensity of hybridization signals is increased substantially. Generally, it is not necessary 30 to prepare duplicate filters after in situ amplification. However, the phage need to be plated at much lower density than in the unamplified method (Wozney, 1990). In some oases, tetramethylammonium chloride (TMAC) has been used as the hybridization solvent. In TMAC, the melting temperature of an oligonucleotide is independent of the oligonucleotide sequence, i.e. base composition, and is a function of probe length (Wood et a1., 1985; Jacobs et a1., 1988). Therefore, by hybridization in TMAC, the preferential hybridization of G/C rich sequences may be abolished. Most of the lysosomal enzyme genes isolated by oligonucleotides were screened by plaque hybridization with unique oligonucleotide guessmers. They include arylsulfatase A (Stein et a1., 1989), arylsulfatase B (Peters et a1., 1990), a-L-iduronidase (Scott et a1., 1991; Stoltzfus et a1., 1992), iduronate-z-sulfatase (Wilson et a1., 1990), cathepsin B (Segundo et a1., 1985), N- acetylgalactosaminidase (Tsuji et a1., 1989), aspartylglucosaminidase (Ikonen et a1., 1991); and a- mannosidase (Schatzle et a1., 1992). Colony hybridization with a short oligonucleotide mixture was used in the cloning of a-N-acetylgalactosaminidase (Wang et a1., 1990), the 6- subunit of B-hexosaminidase (O'Dowd et a1., 1985), the a- subunit of B-hexosaminidase (Korneluk et al.,1986), co-B- glucosidase (Rorman et a1., 1989), and sphingomyelinase 31 (Quintern et a1., 1989).. Glucosamine-G-sulfatase was the only lysosomal enzyme gene isolated by plaque hybridization with short mixed oligonucleotides (Robertson et a1., 1988). 1.2.5 Isolation of genes by polymerase chain reaction (PCR) False positive clones are often encountered when screening with degenerate oligonucleotides, while screening with guessmers require a decrease in the stringency of hybridization due to codon uncertainty. Recently, molecular cloning of genes using PCR techniques has been introduced, which diminishes the disadvantages of these conventional methods described above. A mixed oligonucleotide primed amplification of cDNA (MOPAC) method was first introduced by Lee et a1 (1988). Unlike conventional PCR, the amplification reaction in this system is primed by degenerate oligonucleotides specified by peptide sequences rather than by two unique oligonucleotides. The template is either cDNA synthesized by reverse transcription or DNA prepared from cDNA libraries. By the MOPAC technique, specific PCR products can be generated, which can then be used as unique probes for screening DNA libraries. The advantage of screening cDNA libraries using longer, unique nucleic acid probes instead of mixed oligonucleotides or guessmers is obvious, i.e. hybridization can be carried out in high stringency conditions so that fewer false positive clones are produced. However, a relatively long piece of 32 amino acid sequence, or the information regarding relative orientation of two different peptide sequences is generally required so that a sense and an antisense mixed oligonucleotide based on the amino acid sequences can be designed. Multiple PCR products are expected due to the use of mixed oligonucleotides. Mixed oligonucleotides with low degeneracy are recommended in order to increase the specificity of PCR. However, priming by mixed oligonucleotides with as high as 8200 fold degeneracy is possible (Ottolie et a1., 1991). The authenticity of PCR products can be confirmed by the following methods: (1) Southern hybridization if an internal mixed oligonucleotide that lies between two primers used in the PCR reaction is available; (2) the size selection if the length between the two PCR primers is known; and (3) sequencing (Lee et a1., 1988; 1991; Moremen, 1989; Schuchman et’a1., 1990). Other ways of identifying the complex PCR products are possible (Strub et a1., 1989). In addition to the generation of a unique DNA fragment as a probe, the MOPAC method can also be used for direct production of a portion of the DNA using either two gene specific oligonucleotides (Moreman, 1989) or one gene specific oligonucleotide primer and a vector primer (Gonzalez and Chan, 1993; Ikonen et a1., 1991). The MOPAC technique has been used successfully in the isolation of a number of genes including the gene encoding 33 lysosomal arylsulfatase B enzyme and the gene encoding the endoplasmic reticulum-targeting protein of B-glucuronidase (Schuchman et a1., 1990; Ovnic et a1., 1991; Camirand et a1., 1991; Bischoff et a1., 1990; Moremen, 1989). More recently, a lysosomal gene encoding galactocerebrosidase enzyme was isolated by screening with a PCR product generated by the MOPAC technique (Wenger et a1., 1993). This approach is especially useful for cloning members of gene families. Conventional screening of cDNA libraries is time- and labor-consuming, especially when the gene is expressed at a very low level, necessitating the screening of a large number of clones. This problem can be overcome by using the MOPAC technique for diagnostic screening, i.e. a group of minilysates, each containing a certain number of phage plaques from a library, is first prepared, followed by amplification of DNA with gene specific primers. Library subsets represent by positive minilysates are then screened (Moremen, 1989). Recently, a PCR method, called rapid amplification of cDNA ends (RACE) (also known as anchored- or one-sided PCR) has been developed (Frohman et a1., 1988; 1993; Ochara et a1., 1989). This method has been used for rapid cloning of the 3' or 5' end of cDNA (Frohman et a1., 1988; Casella et a1., 1989). By conventional screening, it may take weeks or months to screen a cDNA library and to isolate and analyze 34 candidate cDNAs in order to clone a full-length cDNA. Using the RACE method, a gene specific primer based on a portion of internal DNA sequence is required. In both 5' and 3' RACE systems, mRNAs are first copied by reverse transcription, and the first strand cDNAs are then amplified. The amplification is accomplished by two primers: a gene specific oligonucleotide and a synthetic homopolymer tail (poly-T for the 3' RACE; poly-T or G for the 5' RACE) tagged by restriction sites. For the 5' RACE system, a terminal deoxynucleotidyl transferase (TdT) tailing reaction is required before amplification. The RACE technique provides a rapid and efficient way not only for cloning full-length cDNAs, but also for obtaining information regarding alternative splicing, alternative polyadenylation, or alternative promoters. Kits for RACE are commercially available. 1.3 fl-MANNOSIDOSIS B-Mannosidosis is an inherited glycoprotein storage disorder. It was first described in Nubian goats (Jones et a1., 1981a; Hartley and Blakeman, 1973; Healey et a1., 1981). Affected goats all have the following phenotypic features: inability to stand, dome-shaped skulls, carpal contractures, pastern joint hyperextension, narrowed 35 palpebral fissures, deafness, and intention tremor (Jones et a1., 1982, 1983; Kumar et a1., 1986). Pathologically, the affected goat displayed widespread cytoplasmic vacuolation in a variety of cell types of the central nervous system and viscera as well as central nervous system axonal spheroids and myelin deficits (Jones et a1., 1983; Lovell et a1., 1983). Goats with B-mannosidosis were found to have a deficiency of the enzyme B-mannosidase in plasma and various tissues such as kidney, brain, liver, and skin fibroblasts (Jones and Dawson, 1981; Jones et a1., 1984). The main storage products associated with the enzyme deficiency are a disaccharide (ManBl-4GlcNAc) and a trisaccharide (ManB1- 4G1cNAcBl-4GlcNAc) (Jones and Laine, 1981; Matsuura et a1., 1981; Jones et a1., 1983; 1984). Minor accumulations of tetrasaccharide and pentasaccharide were also observed (Matsuura and Jones, 1985). Studies of accumulated oligosaccharides suggested that the di- and tri-saccharides were generated independently (Hancock et a1., 1986). Two catabolic pathways involving the activities of an endo-B-N- acetylglucosaminidase and an aspartylglucosaminidase in goat are partially responsible for the heterogeneity in storage and excreted material (Hancock and Dawson, 1987). In 1990, B-mannosidosis was identified in Salers calves in Australia, New Zealand, and the U.S.A. (Jolly et a1., 1990; Abbitt et a1., 1991; 1990; Bryan et a1., 1990; Orr, 1990). The clinical, pathological, and biochemical features 36 of affected calves are very similar to these of affected goats and include: facial dysmorphism, inability to stand, intention tremors, profound central nervous system dysmyelination, extensive tissue cytoplasmic vacuolation, di- and tri-saccharide accumulation in tissues and urine, and marked deficiency of B-mannosidase activity in plasma and various tissues. However, affected cattle had less hearing defect, but more profound enlargement of the kidney and thyroid (Bryan et a1., 1990; Abbitt et a1., 1991). The latter difference is believed to be due to the longer gestation period of the bovine species and thus more accumulation of the undegraded or uncatabolized oligosaccharides (Abbitt et a1., 1991; Jones and Abbitt, 1993). fl-Mannosidosis in the caprine and bovine species has a rapidly fatal course. Affected animals die in the neonatal period if intensive care is not administered. In 1986, Cooper et a1. and Wenger et al. described the first cases of human B-mannosidase deficiency. Cooper et a1. (1986) described 44-year-old and 19-year-old brothers. Wenger et al (1986) reported a four-year-old boy. In both cases, the patients suffered from mental retardation and hearing loss but no other neurological symptoms. Coarse facial features were observed in Wenger's patient. There was no detectable B-mannosidase activity in leukocytes, fibroblasts, and plasma in these patients. The four-year- old boy also had heparin sulfamidase deficiency. Excess 37 disaccharide was observed in these patients' urine. So far, eleven patients from eight families have been diagnosed with B-mannosidosis (Cooper et a1., 1986; 1991; Wenger et a1., 1986; Dorland et a1., 1988; Kleijer et a1., 1990; Wijburg et a1., 1991; Poenaru et a1., 1992; Levade et a1., 1991; 1994). There is great clinical heterogeneity among these patients. The most characteristic symptoms are mental retardation and deafness. The course of B-mannosidosis in human is generally much milder than that in either ruminant form, although three patients died before age 20. More recently, Levade et a1. (1994) reported a 14-year-old patient with complete deficiency of fl-mannosidase activity in plasma and leukocytes, but no mental retardation or hearing loss were revealed. Moreover, there was no detectable disaccharide in the patient's urine. The major storage product in other patients is disaccharide rather than the trisaccharide which is the major accumulating oligosaccharide in ruminants. Recently, a new oligosaccharide complex (sialyl-a(2-6)- mannosyl-B(1-4)-N-acetylglucosamine), resulting from an a2- 6-sialylation of the accumulated disaccharide product, was isolated from the urine of a patient with B-mannosidosis (Hokke et a1., 1990; van Pelt et a1., 1990). The more complex oligosaccharides similar to those accumulated in affected animals have not been found in human cases. The striking biochemical differences between ruminant and human B-mannosidosis (i.e. accumulation of trisaccharide versus 38 disaccharide) are probably due to the different catabolic pathways for glycoproteins (Hancock et a1., 1986; Jones, 1992). In ruminants, removal of the reducing-end GlcNAc from oligosaccharides that have been cleaved by an amidohydrolase is catalyzed by B-hexosaminidase, while in human and rats, lysosomal chitobiase is responsible for this activity (Aronson and Kuranda, 1989; Hancock et a1., 1986). Dysmyelination in the central nervous system is a consistent feature among affected B-mannosidosis animals. The degree of myelin deficits in regions of the central nervous system varies and it correlates with the time of myelination and with more severe myelin paucity in regions that develop myelin at a later stage (Lovell et a1., 1983; 1987; Patterson et a1., 1991). The dysmyelination appears to be associated with a defect in oligodendrocytes (Boyer et a1., 1990; Lovell et a1., 1987). The observation of profound cytoplasmic vacuolation in thyroid and decreased thyroid hormone in affected animals raises the possibility that hypothyroidism may play a role in the central nervous system dysmyelination (Boyer et al., 1990b; Lovell et a1., 1991). However, the factors responsible for the severity of dysmyelination in various regions of the central nervous system are still unclear. B-Mannosidosis is identified by its characteristic clinical manifestations and urinary excretion of di- and tri-saccharides. The final diagnosis relies on the 39 deficiency of B-mannosidase activity in plasma, lymphocytes, or tissues. Intermediate levels of B-mannosidase activity are generally observed in heterozygotes (Jones et a1., 1981; Cavanagh et a1., 1992; Cooper et a1., 1987), however, age matched controls should be used to detect carriers. The determination of plasma B-mannosidase activity within the same herd can provide useful information for B-mannosidase carrier detection (Cavanagh et a1., 1992; Taylor et a1., 1993). Further studies are needed to discover a more efficient method for carrier detection. Preliminary studies suggested that prenatal diagnosis of B-mannosidosis by oligosaccharide detection in allantoic or amniotic fluid was possible (Jones et a1., 1984; Dahl et a1., 1986). The high expression of B-mannosidase activity in amniotic cells and in chorionic villi make it feasible to do prenatal diagnosis by amniocentesis or chorionic villi sampling (Jones et a1., 1984; Poenaru et a1., 1992). B-Mannosidosis is inherited as a simple autosomal recessive genetic defect. The disease has been found in both sexes. Obligate carriers generally show an intermediate level of B-mannosidase activity. To study the possibility of a defect in a common factor in a patient with a combined deficiency of B-mannosidase and heparin sulphate sulfamidase (Wenger et a1., 1988), complementation studies were carried out by Hu et a1. (1991) using cultured skin fibroblasts from the patient with combined deficiency, other 40 human B-mannosidosis cases from five different families, and one caprine B-mannosidosis case. No complementation was observed between the combined deficiency cells and other human B-mannosidase cells, between the combined deficiency case and caprine one, and between these patient cell lines. The results indicated that an allelic mutation in the same gene is responsible for these human as well as caprine B- mannosidosis cases. The molecular basis for the combined deficiency case is still unclear. 1.4 p-xauuosznasn The degradation of N-linked glycoproteins involves three mechanisms: (1) the sequential removal of the sugar monomers from the non-reducing end of carbohydrate side chains by exo-glycosidases; (2) cleaving the chitobiose link between the two N-acetylglucosamine residues located in the "core" portion by endo-B-N-acetylglucosaminidase; and (3) the hydrolysis of the bond between N-acetylglucosamine and asparagine by aspartylglycosaminidase (Watts and Gibbs, 1986). Species differences in this pathway have been reported by Aronson et a1. (1989). B-Mannosidase (EC 3.2.1.25) is one of the exo- glycosidases involved in the catabolism of glycoproteins. It cleaves the B-linked mannosyl residue at the final step of the degradation of N-linked oligosaccharides. B- 41 Mannosidase is widely distributed in fungi, insects, plants, and animals. Its activity has been detected in plasma and in various tissues such as brain, liver, kidney, thyroid, spleen, urine, skin fibroblast, granulocytes, and lymphocytes (Cavanagh et a1., 1982; 1992; Bernard et a1., 1986; Dunstan et a1., 1983; Pearce et a1., 1987; Cooper et a1., 1987; Colin et a1., 1987; Jones et a1., 1984). B- Mannosidase activity is highest in thyroid, with decreasing levels in kidney, liver, muscle, and brain (Pearce et a1., 1987; Lovell et a1., 1994). The enzyme activity can be influenced by age, sex, reproductive status, and other factors (Cooper et a1., 1987; Dunstan et a1., 1983). It was found that the B-mannosidase activity in goats as well as in humans was significantly decreased during the period of sexual maturation and leveled off in adulthood. Lysosomal enzymes are glycoproteins with acidic pH optima. Neutral counterparts of a-mannosidase, B- mannosidase, B-glucosidase, B-galactosidase, and sphingomyelinase have been discovered. Except sphingomyelinase, all neutral forms are present exclusively in liver (Chatterjee et a1., 1989). The discovery of considerable residual activity of B-mannosidase in the liver of affected kids (Jones and Dawson, 1981; Cavanagh et a1., 1982) led to Dawson's investigation of the neutral form of B-mannosidase in goat liver tissue (Dawson, 1982). By concanavalin A (Con A)-sepharose 4B chromatography, the 42 unbound and bound forms of B-mannosidase were separated. The latter represents typical lysosomal enzyme with acidic pH optimum (5.0-5.5), while the former one showed a broad and high pH optimum (6.0-8.0). The study of liver 3- mannosidase activity of affected animals demonstrated that it consisted exclusively of the unbound forms. Further studies of the lysosomal and non-lysosomal forms of B- mannosidase in goats by Cavanagh et a1. (1985) demonstrated that these two forms have different molecular weights, isoelectric points, and substrate specificities and reacted differently towards inhibitors. Pearce et a1. (1987) reported that the activity of the non-lysosomal form increased progressively in liver during the second half of gestation. These reports clearly illustrated that the lysosomal and non-lysosomal forms are genetically, structurally, and functionally distinct from each other. The substantial residual activity found in the affected animal liver was due to the activity of the non-lysosomal form which does not hydrolyse trisaccharide. The biological function of liver specific B-mannosidase is still unknown. The neutral form of fi-mannosidase has not been found in humans (Noeske et a1., 1983; Iwasaki et a1., 1989). Apart from the non-lysosomal form, multiple isoforms of lysosomal B-mannosidase have been demonstrated in goats and humans by different groups (Pearce et a1., 1987; Frei et a1., 1988; Percheron et a1., 1992). The difference among 43 these isoforms largely reflects the degree of sialylation. B-Mannosidase has been purified to various degrees from several mammalian sources including guinea pig liver, human placenta, and bovine and goat kidney (Sukeno et a1., 1972; Noeske et a1., 1983; Kyosaka et a1., 1985; Frei et a1., 1988; Iwasaki et a1., 1989; Sopher et a1., 1992; 1993). The first mammalian B-mannosidase purified to homogeneity was from guinea pig liver. The molecular size of the purified B-mannosidase protein was found to be 97-110 kDa (Kyosaka et a1., 1985; McCabe et a1., 1990). The protein was thought to be monomeric since the molecular weight estimated by gel filtration and SDS-PAGE were very close. However, additional studies suggested that it consisted of at least 3 subunits (McCabe and Dawson, 1991). McCabe and Dawson prepared a specific polyclonal antibody using Kyosaka's purified pig liver B-mannosidase protein. Western analysis of a fresh liver homogenate using this polyclonal antibody revealed a major band at 150 kDa. However, when frozen proteins were used, the immunoreaction pattern changed: the 150 kDa band disappeared, but two bands at 120 kDa and 20 kDa were observed. These were replaced finally by three bands at 97, 37, and 20 kDa after more cycles of freezing and thawing of proteins. Fresh proteins from a crude lipid extraction showed the 97, 37, and 20 kDa immunoreactive bands, too, which led the authors believe that the three subunits of guinea pig B-mannosidase were stabilized by a 44 lipid environment. The 120 and 150 kDa proteins were not observed in guinea pig kidney. The reason for this heterogeneity was not addressed. The functional and structural relationship between the two smaller subunits and the 97 kDa subunit was not clear. A five-step purification of B-mannosidase from human placenta yielded a 10,000-fold purification (Iwasaki et a1., 1989). However, SDS-PAGE still revealed multiple bands with molecular masses from 98 to 57 kDa. The molecular weight calculated by gel filtration was 110 kDa, which suggested that the human B-mannosidase may consist of multiple subunits. More recently, specific monoclonal and polyclonal antibodies against caprine and bovine B-mannosidases were produced in our laboratory. B-Mannosidase can now be highly purified (12,000 to 16,000 fold) from bovine and goat kidney tissues by a five-step purification procedure including an immunoaffinity chromatography step using a monoclonal antibody (Sopher et a1., 1992; 1993). Upon separation by SDS-PAGE, the purified goat protein revealed three bands (80, 90, and 100 kDa) by silver staining and Western analysis. The two major bands (90 and 100 kDa) were clearly associated with B-mannosidase activity. They both reacted with a monoclonal antibody specific against B-mannosidase and exhibited similar peptide patterns under limited proteolysis. Neither the 90 nor the 100 kDa peptide were 4s detectable in caprine fi-mannosidosis kidney as indicated by silver staining and Western analysis of affinity purified protein. The 80 kDa peptide which copurified with the B- mannosidase peptides represents a small amount of the total amount of this protein in the Con A bound fraction. This peptide was clearly not related with the catalytic activity of the B-mannosidase and was also found to be present in B- mannosidosis goats by Western analysis. The purification of bovine B-mannosidase produced a very similar result. Three bands with the size of 84 kDa, 100 kDa, and 110 kDa were observed by silver staining and Western analysis. Glycosylation studies indicated that the size difference of the peptides between the two species was due to the different amounts of carbohydrate side chains. Removal of N-linked carbohydrate decreased the sizes of the major peptides to 86 kDa and 91 kDa in both species. Besides the size difference, another difference observed between cattle and goat was that the affected goats lack any 90 and 100 kDa but contain the 80 kDa protein as documented by silver staining and Western analysis. In contrast, the 84 kDa peptide found in normal bovine tissues and cattle with B- mannosidosis reacted with anti-B-mannosidase antiserum and did not react with anti-80 kDa antiserum. It was proposed that the 84 kDa peptide found in variable amounts in bovine kidney probably resulted from degradation-of B-mannosidase peptides. The reason for this difference and the functional 46 relationship between the 80 kDa peptide and B-mannosidase are still unclear. Like Kyosaka’s guinea pig (Kyosaka et a1; 1985), the bovine and goat B-mannosidases appear to be monomeric. The structural gene encoding B-mannosidase had not been previously cloned from any species. In 1987, Lundin (1987) localized a gene controlling mouse B-mannosidase activity in the distal part of mouse chromosome 3. However, whether it is a B-mannosidase structural or regulatory gene is unclear. In the same year, Fisher et a1. (1987) tentatively localized the gene responsible for human 6- mannosidase to human chromosome 4 whose long arm is syntenically related to the distal part of mouse chromosome 3. A putative cDNA was cloned from guinea pig using a specific anti-B-mannosidase polyclonal antibody (McCabe and Dawson, 1991). However, chromosome mapping and linkage studies suggested that this clone did not represent the B- mannosidase gene (Sopher, 1992a). This investigation was designed to isolate and characterize the cDNA encoding B-mannosidase. It is an important step towards the achievement of the goal of development of an animal model system for gene therapy of a lysosomal storage disease characterized by neurodegeneration and early onset. The isolation of B-mannosidase cDNA was attempted by several approaches. First, a human cDNA library was 47 screened with putative goat cDNA clones in order to clone a human B-mannosidase cDNA and to verify the goat cDNA clones. Secondly, goat and bovine polyclonal antibodies were used to screen goat and bovine cDNA libraries after the generation of polyclonal antibodies and the failure of the isolation human B-mannosidase cDNA with goat clones. Finally, purified bovine B-mannosidase was microsequenced and peptide sequences were obtained. Bovine cDNA libraries were then screened with degenerate oligonucleotides. By this method a cDNA encoding bovine B-mannosidase was finally isolated and characterized. CHAPTERTWO ISOLATION AND CHARACTERIZATION OF HUMAN cDNA HOMOLOGOUS TO GOAT CLONES CHAPTER TWO ISOLATION AND CHARACTERIZATION OF HUMAN cDNA HOMOLOGOUS TO GOAT CLONES 2.1 INTRODUCTION Since the discovery of B-mannosidosis in Nubian goats (Jones and Laine, 1981; Jones and Dawson, 1981), extensive studies regarding B-mannosidosis and B-mannosidase have been carried out in this laboratory (Matsuura et a1., 1981; Matsuura and Jones, 1985; Jones et a1., 1982; 1983; 1984; 1986; 1992; Jones, 1989; Jones and Abbitt, 1993; Lovell and Jones, 1983; 1985; Lovell and Boyer, 1987; Lovell, 1990; Lovell et a1., 1991; 1994; Dahl et a1., 1986; Kumar et a1., 1986; Fisher et a1., 1987; Cavanagh et a1., 1982; 1985; 1992; Dunstan et a1., 1983; Frei et a1., 1988; Sopher et a1., 1992; 1993). The partial purification and characterization of goat B-mannosidase protein in 1990 led to the preparation of monoclonal antibodies (MAbs) (Sopher et a1., 1992). Three MAbs (43F1OS, 44A6, and 44D9) which were capable of reacting with both native and denatured goat fl-mannosidase proteins were produced (Sopher et a1., 1992). The production of MAbs not only resulted in the generation 48 49 of an immunoaffinity column and high purity B-mannosidase protein (Sopher et a1., 1992; 1993), but also initiated the molecular cloning of the B-mannosidase gene. Two goat thyroid cDNA libraries constructed in the Agt11 vector in this lab were screened by a combination of two of the three MAbs (44D9 and 43F1OS) in an attempt to find a caprine B- mannosidase clone. A total of 4 positive clones were identified. Preliminary studies suggested that two of the clones (p5m8 and p5m11) might be good candidates for B- mannosidase (Sopher, 1992a). However, these two clones were not long enough to encode the whole B-mannosidase protein if they were authentic clones for B-mannosidase. Cloning and characterization of human B-mannosidase cDNA were the original goal of this project. A high quality human cDNA library was chosen for isolation of a full-length clone. Two goat cDNA clones were used to probe a human placenta cDNA library in order to isolate human B- mannosidase gene. A full-length clone could be transferred to a expression system for verification of B-mannosidase activity. Thus, it was postulated that screening of a high quality human cDNA library might enable us to isolate human B-mannosidase cDNA and verify the goat clones. 50 2.2 MATERIALS AND METHODS 2.2.1 Materials A human placenta cDNA library cloned in AZAPII vector and XL1-Blue E. coli cells were obtained from Stratagene (La Jolla, CA). pSVL vector and DEAE-dextran were purchased from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Sequenase Version 2.0 T7 DNA polymerase, M13 reverse and -21 primers, exonuclease III, and mung bean nuclease were purchased from United States Biochemical Corp. (Cleveland, OH). T4 ligase, random primed DNA labeling kit, and restriction enzymes were from Boehringer Mannheim Biochemicals (Indianapolis, IN). Nitrocellulose paper BA85 (0.45 pm) was from Schleicher & Schuell (Keene, NH). [a- 32P]dCTP (3000 Ci/mmol; 111 TBq/mmol) was from Amersham Life Science (Arlington Heights, IL). 35Sequetide (1500 Ci/mmol; 55.5 TBq/mmol) was from NEN/Du Pont (Wilmington, DE). DH5a competent cells and Dulbecco's Modified Eagle Media (DMEM) were purchased from Gibco BRL (Gaithersburg, MD). Chloroquine, polyethylene glycol, 4-methylumbelliferyl B-D- mannopyranoside, and 4-methylumbelliferyl a-D- mannopyranoside were purchased from Sigma (St. Louis, MO). 2.2.2 Library screening Approximately 1 x 106 phage plaque-forming units of the human placenta cDNA library were plated at a density of 51 5 x 10‘ phage plaque-forming units per 150 mm plate and screened with two goat cDNA probes. Duplicate nitrocellulose filters were prehybridized (2 hours) and hybridized (16-20 hours) in a solution containing 0.4 M NaCl/0.01 M Pipes (pH 6.5)]50% formamide/0.5% SDS/100 ug/ml denatured herring sperm DNA at 42°C. The cDNA probes were labeled using the random primed DNA labeling method according to the manufacturer's instructions. Approximately 1 x 10‘ counts per minute (cpm) of radioactive DNA probe per milliliter were used during hybridization. The hybridized filters were washed in a step-wise fashion with the final wash in 0.2 x SSC/0.1% SDS at 65°C. Positive recombinant clones were isolated by plaque purification and phage DNA was prepared by the standard procedures (Sambrook et a1., 1989). 2.2.3 Subcloning Positive clones were subcloned into pBluescript plasmid vector by in vivo excision following the manufacturer’s instructions. Plasmid DNA was prepared by a boiling method according to the Stratagene instructions. Clone #1p5m11 was subcloned into pSVL vector essentially as described in Current Protocols in Molecular Biology (Ausubel et a1., 1989). Briefly, 4 ug of plasmid DNA from clone f1p5m11 were digested by Ach enzyme, then treated with Klenow enzyme to generate blunt ends. Phosphorylated XhoI 52 linker was ligated to the plasmid DNA followed by digestion with XhoI enzyme. pSVL vector DNA was digested by XhoI and dephosphorylated by calf intestine alkaline phosphatase. Both the insert of clone #1p5m11 and vector DNA were then gel purified. Finally, the plasmid DNA of clone f1p5m11 was ligated with pSVL vector. One fourth of the ligation was used to transform subcloning efficiency DHSa competent cells following the manufacturer's procedures. Subclones were identified by in situ hybridization with #1p5m11 insert as a probe by the standard procedures (Sambrook et a1., 1989). 2.2.4 Nucleotide Sequencing Sequencing was performed using M13 reverse and -21 primers on the intact plasmid. To obtain internal sequence, eonII/mung bean nuclease deletion was chosen to prepare deletion clones. Large amounts of f1p5m11 DNA were first purified by precipitating with polyethylene glycol essentially according to the method described in Molecular Cloning (Sambrook et a1., 1989). Approximately 20 ug of plasmid DNA was subjected to double restriction enzyme digestions. For preparation of the 5' deletion, restriction enzymes ClaI and KpnI were selected. For the 3' deletion, BamHI and SacI were used. The DNA was first digested by one restriction enzyme, then extracted once with phenol/chloroform (1:1) and once with chloroform. DNA was reprecipitated by adding 1/10 volume of 3 M NaOAC, pH 5.6 53 and two volumes of ethanol. The resuspension was then subjected to the second restriction enzyme digestion. After the completion of double enzyme digestions, eonII/mung bean nuclease deletions were carried out at 37°C following the Stratagene instructions. DNA that had been treated by eonII for different times and thus fragmented into different lengths was ligated and transformed into XLI-Blue or DHSa competent cells according to the manufacturers' instructions. DNA from overlapping clones was prepared and sequenced by the dideoxy chain-termination method using T7 sequenase DNA polymerase, 3‘S-labelled dATP, and M13 universal primers. DNA sequence data and homology searches were analyzed using the GCG program, version 7 by the Genetics Computer Group DNA Sequencing Analysis Software, Madison, Wisconsin. 2.2.5 In_21;szExpression COS-7 cells (gift from Dr. D. Dewitt) were grown in DMEM containing 10% fetal bovine serum (FBS) in 60 mm culture dishes. After the cells were about 75% confluent, 10 ug of plasmid DNA from recombinant clones pSVL-15, pSVL- 32, and pSVL were transfected into the cells by a DEAE- dextran method (Oshima et a1., 1988). After 10 hours of incubation with DEAE-dextran, the cells were washed with DMEM/10% fetal bovine serum and treated for 3 hours with 100 uM chloroquine in 2 ml of DMEM/10% FBS. Cells were 54 harvested after 48 to 72 hours of incubation. Cell lysates and supernatant were assayed for B-mannosidase and a- mannosidase activities with 4-methylumbilliferyl substrates as described previously (Jones et a1., 1984). 2.3 RESULTS AND DISCUSSION 2.3.1 Cloning the human homologues of goat clones By screening 1 x 10° recombinants from the human placenta AZAPII cDNA library, twelve positive clones were identified by a cDNA probe from the goat cDNA clone p5m8, and five clones by a cDNA probe from the goat cDNA clone p5m11. Six clones from the p5m8 group and three from the p5m11 group were excised as pBluescript plasmids. Restriction enzyme digestion analysis suggested that the two groups of clones had different types of restriction maps (Fig. 2.1). This result was consistent with previous Southern hybridization data (Friderici, unpublished data) that indicated there was little homology between the two goat clones p5m8 and p5m11. Except for clone 15p5m8 which showed a weak signal in Southern hybridization analysis (data not shown), identical or overlapping restriction maps within each group were observed (Fig. 2.1). 55 closesvee Ismsoecou acoeeumou numb oaaoa .aoua .aa aasua .a .aaaacaa .a ..usmuco coauoauuuca coauuauaca anneauaaucuuu o>auau=m on» .oaa omsose aammma\ cacao .aamm (zoo uuom a an uoumaoea meccao ”a aessm necOau sons» «4 aesdm aammmas aammmas asmmiaamuocme mammahtuovaafl aaammse aasmmmae aaamma‘ .N.N .Uah Ca .Hunh .m aHUQN .x aaonx .nx aHQnm .m aHonda .£ «Haa>m .m .ecoauuoaao oocosvue acoeeumou esouuc .enoum aama auas soauuuauaunms camcusom a ma adamae 3103 a .aaamm aznu uuom a an aouaaona .eesoao tuna ails: mo hmevuuae meannesvee use mil soauoauueeu CAB a." .mah .souou . f: Iv +¢ All V IV . A a m an omu J. ___ «a a a as a V A _.___ _____.w a a a as a x a it ___ . a a a an a V A _ __ ___ _ v a a a a a a a. _.___ __ _i _ a a m a a a a unaanaanm manna -eaanaanm auasaanm 56 2.3.2 Sequence of human p5m11 clones Clone #1 from the p5m11 group, which contains a 3.6 kb insert was subjected to eonII/mung bean nuclease deletions and sequenced from both directions by the dideoxy chain- termination method. A partial DNA sequence (3070 bp) (Fig. 2.2) revealed a putative initiation codon, a poly (A) tail, and three stretches of open reading frames that were interrupted by three short gaps of unsequenced DNA. The sequence flanking the initiation codon (AGCATGG) was in good agreement with the consensus sequence (A/GCCATGG) for a eukaryotic initiation codon by Kozak (1986). No typical signal peptide sequence was found. Sequencing searches did not reveal any significant sequence similarity with proteins in the database. However, sequence similarity was found between the goat clone p5m11 and the human clone #1p5m11. Interestingly, the deduced amino acid sequences from the goat and human clone had repetitive sequences consisting of 5 residues with a proline residue at the beginning (Fig 2.2 underlines). The molecular mass of human B-mannosidase peptide was 98 kDa (Iwasaki et a1., 1989). Clone f1p5m11 was long enough to encode a 98 kDa protein. Sequencing both ends of clones #7p5m11 and #15p5m11 with M13 reverse and -21 primers confirmed their homologies with clone #1p5m11. Partial sequencing (both ends) of clone #9 from the p5m8 group did not reveal any open reading frames or possible translational initiation codon. 57 Fig. 2.2 Partial nucleotide sequence of human lips-11 clone. Three fragments were displayed in a 5' 4 3' direction. Nucleotides and amino acids are numbered noncontinously between each fragment. Gaps are marked with ///////. The repetitive proline sequences in fragment pb11h1d4- are marked with underlines (the human clone) and double underlines (the goat clone). The putative initiation codon is marked by *** 61 v121 181 241 301 361 14 421 34 481 54 541 74 601 94 661 114 721 134 781 154 58 ggctgcaggaattcctgaacttgtgcaaataactttattaccataaacctatgaatactc atgaatagtttcccaattctggggcactcagatagagagcaaaagcaaatgtttcaattt ttgtttacaaaagtatactttaccaattgctgaagaaaaaaagttcataaatctggagaa taaaacattccaagaatcagcacattttccastaaaaaattatgaaaacnttatcctttt gattatttagtccaataac;ttgagtttttttcttctaattcatctcttgttttatcagg tgtgtgtggtttcagcgcagcatggctgtggtcatccgtttgcaaggtctcccaattgtg tit M A V V I R L Q G L P I V gcggggaccatggacattcggcacttcttctctggattgaccattcctgatgggggcgtg A G T M D I R H F F S G L T I P D G G V catattgtagggggtgaactgggtgaggctttcatcgtttttgccactgatgaagatgca H I V G G E L G E A F I V F A T D E D A aggcttggtatgatgcgcacaggtggtacaattaaagggtcaaaagtaacactattgttg R L G M M R T G G T I R G S K V T L L L agtagtaagacggaaatgcagaatatgattgaactgagtcgtaggcgttttgaaactgcc s S K T E M Q N M I E L S R R R F E T A aacttagatataccaccagcaaatgccagtagatcaggaccaccacctagctcaggaatg N L D I P P A N A S R S G P P P S S G M agtagcagggtaaacttncccacaacagtatccaactttaataatccatcacccagtgta S S R V N x P T T V s N F N N P S P S V gttactgccaccacttctgttcatgaaagcaacaaaaacatacagacattttccacagcc V T A T T s V H E S N K N I Q T F S T A agcgtaggaacagctcctccaaatatgggggcttcctttgggagcccaacgtttagctca s V G T A P P N M G A s F G s P T F S s Fig. 2.2 841 174 901 194 961 214 1021 234 1081 254 1141 274 1201 294 1261 314 1321 334 1381 354 1441 374 1501 394 1561 414 59 actgttccaagcacagcctctccaatgaacacagtcccgccgccaccaattcctccaatt T V P S T A S P M N T V P P P P I P P I ccagcgatgccatctctgccaccaatgccatccattcccccaattccagttcctcctcca P A M P S L P P M P S I P P I P V P P P gtacctacattgcctcctgtncctcctgtncccccgattnccccagttccttctgtgcca V P T L P P V P P V P P I x P V P S V P cccatgaccccactgccacccatgtcgggcatgccgcccttgaatccgccacctgtggca P M T P L P P M S G M P P L N P P P V A cctctacctgctggaatgaatggctctggagcacctatgaatttgaacaataatctgaat P L P A G M N G S G A P M N L N N N L N cctatgtttcttggtccgttgaatcctgttaaccctatccagatgaactctcagagcagt P M F L G P L N P V N P I Q M N S Q S S gtgaagccactccccatcaaccctgatgatctgtatgtcagtgtgcatggaatgcccttt V K P L P I N P D D L I V S V H G M P F tctgcaacggaaaatgatgtcagagatttttttcatgggctccgtgttgatgcagtgcat S A T E N D V R D F F H G L R V D A V H ttgttgaaagatcatgtaggtcgaaataatgggaatggattggttaagtttctctcccct L L R D H V G R N N G N G L V K F L S P caagatacatttgaagctttgaaacgaaacagaatgctgatgattcaacgctatgtggaa Q D T F E A L K R N R M L M I Q R I V E gttagccctgccacagaaagacagtgggtagctgctggaggccatatcacttttaagcaa V S P A T E R Q W V A A G G H I T F R Q aatatgggaccttctggacaaactcatcccctcctcagacacttccaggtcaaatcgcca N M G P S G Q T H P L L R H F Q V K S P gtggcagaaagatcaggtcaaga (pbllhlrl) V A E R S G Q Fig. 2.2 61 21 121 41 181 61 241 81 301 101 361 121 421 141 481 161 541 181 601 201 661 221 60 //l//////////// agcagaaaacaaacatgtcattgatttttttaaaaagctggatattgtggaagatagtat A E N K H V I D F F E K L D I V E D S I ttatatagcttatggacccaatgggaaagcaactggcgaaggctttgtagagttcagaaa I I A I G P N G K A T G E G F V E F R N tgaggctgactataaggctgctctgtgtcgtcataaacagtacatgggcaatcgctttat E A D Y E A A L C R H E Q I M G N R F I tcaagttcatccaattactaagaaaggtatgctagaaaagatagatatgattcgaaaaag Q V H P I T K K G M L E K I D M I R K R actgcagaacttcagctatgaccagagggaaatgatactaaatccagagggggatgtcaa L Q N F s I D Q R E M I L N P E G D V N ctctgccaaagtctgtgcccacataacaaatattccattcagcattacaaagatggatgt S A K V C A H I T N I P F S I T K M D V tcttcagttcctagaaggaatcccagtggatgaaaatgctgtacatgttcttgttgataa L Q F L E G I P V D E N A V H V L V D N caatgggcaaggtctaggacaggcattggttcagtttaaaaatgaagatgatgcacgtaa N G Q G L G Q A L V Q F K N E D D A R K gtctgaacgcttacaccgtaaaaaacttaatgggagagaagcttttgttcatgtagttac S E R L H R K K L N G R E A F V H V V T cctagaagatatgagagagattgagaaaaatccccctgcccaaggaaaaaagggattaaa LEDMREIEKNPPAQGKKGLK gatgcctgtgccaggtaatcctgcagttccaggaatgcccaatgcgggactgcccggtgt M P V P Q N P A V P G M 2 N Lh.§__L 2__§__! WWW gggactgcccagtgcaggacttcccggtgcaggcctgcccagcacaggactgcctggttc _G._LF__$_A_§._LP_G__A_§__LB_§_1_9_.L_L§__$_ MMMLLAJ—HLH. Fig. 2.2 721 241 781 261 61 21 121 41 181 61 61 121 181 241 301 361 421 61 agcaataaccagtgcaggactgcctggtgcgggaatgcccagtgcaggaatacctngtgc A 1 T S A G L 2 Q A E M E.Ji_JL_JL_JL EL_3__A_ .2..§ £..E..A..§..fl, z..3..a..2..3 z._n..3..§..1 .2..§..1 aggaggtgaagagcat Q g E E H (pbllhld4-) .9..H /////////////// gcggggcctttggtgatgctaggcctggtatgccttcagttggaaacagtggtttgcctg G A F G D A R P G M P S V G N S G L P G gtctagnactggatgttccgggttttggaggtggaccaaacaatttaagtgggccatcgg L x L D V P G F G G G P N N L s G P S G gatttggagggggccctcagaattttggaaatggccctggtagcttaggcggtcccccgg F G G G P Q N F G N G P G S L G G P P G ggtttggaagtcccggc 197 (dB-19) F G S P G 65 /////////////// taaagggaacaaaagctggagccatggtggcctttgagtctcgggatgaagccacagctg ctgtcattgacttaaatgacaggcctataggttcaagaaaagtaaaacttgtattagggt agccattcacatcattttttatngggtagatcttcatattgctgtgattaatgcatccag attgttttcctagtatttccaggttagaacctgtggattgtttcaattgcatatagcttg gtttccataacatagagcattggttgactgtttacagaagactcactcaccaggatgggc attgctgtatgttacagtaaagctatctggagagaacac3tgggtgattttggcatacca ttagagaaaccatttgtaaaactcaaatgaccacataaagcttatcaaggagtctagatt ggtttttg 429 (pbllhld3-) Fig. 2.2 62 2.3.3 Ekpression of human p5m11 In order to determine whether clone #1p5m11 was 8- mannosidase, the insert was excised by EcoRI digestion, gel purified, and subcloned into an eukaryotic expression vector pSVL. A recombinant clone (pSVL-15) which contains an insert with the correct size and orientation and a clone (pSVL-32) containing an insert with the right size but reverse orientation were identified by in situ hybridization and restriction enzyme analysis. Transfection of clones pSVL-15 and pSVL-32 into COS-7 cells led to no elevation of the B-mannosidase activity in the cells and supernatant compared to the transfection of pSVL only. Northern analysis indicated the level of RNA extracted from cells that were transfected by pSVL-15 after 72 hours was increased (data not shown). Positive results of in vitro expression would certainly confirm the authenticity of clones which were analyzed, however, negative results could not rule out the authenticity of clones. The successful expression of an enzyme activity in vitro not only relies on a full-length cDNA, but may also be influenced by others factors, such as a cofactor needed for expression of the activity or for stabilizing. While the verification of the human cDNA clones was underway, characterization of the goat clones p5m8 and p5m11 in parallel experiments (Sopher and Friderici, unpublished data) provided more evidence that these goat clones were not the B-mannosidase gene. The 63 evidence for this included: (1) when genomic DNA from several human-rodent cell hybrids was hybridized with clones p5m8 and p5m11, none of these clones were located on human chromosome 4 as was expected by previous work (Fisher et a1. 1987; Lundin, 1987); (2) the fusion proteins produced by the two goat clones could not be recognized by any of the specific B-mannosidase polyclonal antisera that became available later on. Therefore, it was clear that the human clones identified by p5m8 or p5m11 did not correspond to the B-mannosidase gene. 2.4 SUMMARY A human placenta AZAPII cDNA library was screened by two goat cDNA probes, p5m8 and p5m11, separately. Of 106 plaques screened, twelve positive clones were hybridized with p5m8 probe, and five with p5m11 probe. Six clones in the p5m8 group and three in the p5m11 group were excised as pBluescript plasmids and subjected to further analyses by restriction mapping and DNA sequencing. The possibility that these clones might be candidates for the B-mannosidase gene was ruled out by in vitro expression in COS cells and more importantly by the further analyses of the two goat cDNA clones (p5m8 and p5m11). The two goat cDNA clones were originally thought to be B-mannosidase but subsequently it was found that they more likely represented proteins sharing 64 the same or similar epitopes with the B-mannosidase protein rather than the B-mannosidase itself. CHAPTERTHREE ISOLATION AND CHARACTERIZATION OF BOVINE fl-MANNOSIDASE cDNA CLONES CHAPTER THREE ISOLATION AND CHARACTERIZATION OF BOVINE B-MANNOSIDASE GDNA CLONES 3.1 SCREENING WITH POLYCLONAL ANTIBODIES 3.1.1 Introduction Immunoscreening is one of the common strategies used for the isolation of cDNA clones, especially when no amino acid sequences are available. Since screening goat libraries with MAbs did not yield B-mannosidase clones, attention was redirected toward identifying the B- mannosidase using polyclonal antiserum to purified enzyme. Polyclonal antisera specifically against B-mannosidase were generated as the consequence of the purification of B- mannosidase protein (Sopher et a1., 1992; 1993). Antiserum to caprine B-mannosidase was prepared by immunizing rabbits with crushed gel slices containing the 90-kDa B-mannosidase peptide. A polyclonal antiserum, 228, referred to as anti- 80/90/100 in a previous paper (Sopher et a1., 1992) was produced. This antiserum could detect as little as 1 ng of purified B-mannosidase protein by dot blot analysis. It reacted mainly to the 90- and 100-kDa peptides, but also had 65 66 cross-immunoreaction to an 80 kDa peptide which was copurified with B-mannosidase as judged by Western analysis. Due to limited availability of goat kidneys, large scale enzyme purification was redirected to use bovine kidneys as an enzyme source (Sopher et a1., 1993). Studies of purified bovine B-mannosidase revealed three peptides. To generate very specific bovine polyclonal antibodies, the purified bovine protein was deglycosylated and the major band was gel purified and injected into rabbits. As a result, two anti- B-mannosidase antisera were generated from the deglycosylated bovine peptide by Sopher et a1. (1993). The bovine specific antisera, 259 and 269 (referred to as the anti fl-mannosidase in the published paper) were more specific than the goat antiserum 228, with little cross- immunoreaction to the 80 kDa peptide in goat kidney. The bovine antisera could recognize B-mannosidase peptides from goat, and furthermore, goat antisera could react with bovine B-mannosidase peptides. Polyclonal antibodies generally recognize multiple epitopes, therefore more peptides can be identified. The project was directed to bovine and caprine B-mannosidase at that time. In order to isolate the mammalian B-mannosidase gene, goat polyclonal antiserum 228 and bovine antisera 259 and 269 were used to screen cDNA expression libraries in the following studies. 67 3.1.2 Materials and methods W The titer and specificity of each polyclonal antiserum were determined by either dot blot test or Western analysis. Partially purified bovine protein consisting mainly of the 100 kDa with small amounts of the 110 kDa and 84 kDa B- mannosidase peptides was used as an antigen. In dot blot tests, serial dilutions of the antigen (50 ng to 100 pg) were spotted onto strips of nitrocellulose filter (Bio-Rad Laboratory, Melvill, NY). After air drying, the filter strips were blocked in a solution containing 10 mM Tris.Cl, pH 7.5/150 mM NaCl/0.05% Tween 20 (Bio-Rad) (TNT) and 5% dry milk for 1 hour at room temperature followed by incubation with serial dilutions (1:250 to 1:1000) of polyclonal antisera, individually, for 2 hours. The filters were then washed in TNT solution three times, each for 5 min, with the final wash in a solution containing 20 mM Tris-Cl, pH 7.5/150 mM NaCl (TBS) for 5 min. After washing, the filters were incubated with alkaline phosphatase (AP)-conjugated goat anti-rabbit antibody (Bio-Rad) for 1 hour followed by the washing-step described before. Finally, color was developed by reacting with 5-bromo-4-chloro-3-indoly1 phosphate/nitro blue tetrazolium (BCIP/NBT) (Promega, Madison, WI) solution. 68 MW Wis: The 196 fraction was purified by using a protein A column (1 ml) (Pierce, Rockford, IL) according to the procedures described by Harlow et al. (1988). Briefly, 1-2 ml of crude antiserum was adjusted to pH 8.0 by adding 1/10 volume of 1 M Tris.Cl, pH 8.0 and then applied to a protein A column which was preequilibrated with 0.1 M Tris, pH 8.0. After sequential washing with 10 volumes of 100 mM and 10 mM Tris.C1, pH 8.0, the IgG fraction was eluted with 0.1 mM glycine pH 3.0. Approximately 1 ml elution fractions were collected into microcentrifuge tubes containing about 100 pl of 1 M Tris.Cl, pH 8.0. IgG-containing fractions were pooled together and bovine serum albumin (BSA) was added up to 1%. i To purify small amounts of epitope-selected anti-B- mannosidase antibodies, a total of 1 ug protein was fractionated on 7.5% SDS-PAGE (10 ng/well) and transferred onto PVDF membrane (Millipore, Bedford, MA) after electrophoresis. The filter was blocked and incubated with 1:500 diluted antiserum 228 essentially as described above. The strips between 100 and 110 kDa were cut and the bound antibodies were eluted by incubating with 400 pl of 0.2 M glycine pH 2.6 for 10 min at room temperature. The elution was immediately adjusted to pH 8.0 by adding tris base solution. The activity of the elution was assayed by 69 Western analysis with 10 ng of TSK-butyl purified bovine protein. Wind! Before being used in library screening, diluted polyclonal antisera or IgG fractions were preincubated with nitrocellulose filters (Schleicher & Schuell, Keene, NH) coated with XL1-blue E. coli (Stratagene, La Jolla, CA) cell lysate until no substantial background was present. In some cases, the anti-E. coli antibodies were removed from polyclonal antisera by affinity chromatography (Sambrook et a1., 1989). A large amount of lysate of XLl-blue cells was prepared first according to the procedures described by (Sambrook et a1., 1989). The cell lysate was chilled to 0%: and bound to cyanogen bromide (CNBr)-activated sepharose 4B (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) according to the manufacture's instructions. After equilibration with TBS solution containing 0.2% sodium azide, the slurry of CNBr-activated sepharose 4B coupled with E. coli lysate was mixed with 196 fractions or antiserum for more than 12 hours at room temperature. The slurry was then loaded into a column and eluted with TBS solution. Fractions containing antibody were pooled, diluted, and used to screen libraries. 70 WW Approximately 5 x 10‘ phage plaque-forming units from goat kidney AZAPII (Clontech, Palo Alto, CA) or bovine liver AZAPII cDNA library (Stratagene) per 150-mm petri dish were plated. After about 3.5 hours incubation at 42%L nitrocellulose filters (Schleicher & Schuell) presoaked in 10 mM B-D-isopropyl-thiogalactopyranoside (IPTG) (Boehringer Mannheim Biochemicals, Indianapolis, IN) were overlaid on the plates, and plaques were allowed to grow for another 3.5 hours. Duplicate filters were applied at the first round of screening. The immunoreaction procedures were essentially as described above in the dot blot test. Polyclonal antisera were diluted 300-500 fold in TNT solution containing 5% dry milk. .- .t,-. . -39 -7.-. .f., . . - ,; Clones identified by antibodies were plaque purified by several rounds of rescreening at lower density and excised into pBluescript plasmid (Stratagene) according to the manufacturer's instructions. To express fusion proteins, 10 ml of cell cultures inoculated by colonies from positive clones were grown at 37°C overnight. An aliquot of 50 ul of the overnight cell culture was added into 5 ml of LB medium containing ampicillin (100 ug/ul). After two hours of incubation at 37°C, fusion proteins were induced by adding IPTG solution up to a final concentration of 1 mM and 71 incubation of the cultures at 37°C for another two to four hours. An aliquot of 1 ml culture was removed after two and four hours of induction, respectively, and collected by centrifugation at 12,000 g for 1 min. The pellet was then resuspended in 100 pl of 1 x SDS loading buffer (2% SDS/10% glycerol/32.5 mM Tris.Cl (pH 6.8)/O.1% bromophenol blue/5% B-mercaptoethanol), boiled for 3 min, and centrifuged at 12,000 g for 1 min. Finally, 30 ul of suspension was loaded on 10% SDS-PAGE. After electrophoresis, the proteins were transferred to the Hybond-N membrane. The blot was stained with Ponceau S and subjected to Western analysis essentially according to Current Protocols in Molecular Biology (Ausubel et a1., 1987). 3.1.3 Results Approximately 1 x 10‘ plaques from a goat kidney AZAPII cDNA library were first screened with the antiserum 228. Nine positive clones were identified and plaque purified. Restriction enzyme analysis and Southern cross-hybridization suggested that clones Gi26228 and Gi27228 were virtually identical. Clones G#8228 and G#11228 also appeared to be identical and had sequence homologies with clone Gf16228. The remaining clones each appeared to represent a different gene. Of the nine clones, five produced fusion proteins (Fig. 3.1). Clones Gf8228, Gf11228 (not shown on Fig. 3.1), and GI16228 all gave rise to a 50 kDa fusion protein, clone 72 3.1 Western Analysis of fusion proteins with antiserum 228. Seven goat cDNA clones isolated by antiserum 228 were analyzed. Clone #26 and #11 were identical clones of #27 and #8 respectively and were not included in this figure. Fusion proteins were induced by 1 mM IPTG for 2 hours. The protein lysates were fractionated by 10% SDS-PAGE. The gel was run at 25 mA for 2 hours, then 40 mA for 4 hours. The number on top of the figure represents each clone. Low range SDS-PAGE standard from Bio-Rad was used as a molecular weight marker. 73 G#22228 produced a 25 kDa fusion protein, while clone G#21228 generated a 38 kDa and a 39 kDa protein. These fusion proteins were all recognized by antiserum 228 as judged by Western analysis (Fig. 3.1). Fusion proteins produced by clones G#8228, G#11228, G#16228, and G#22228 were later subjected to Western analysis to check cross- immunoreaction with two anti-fi-mannosidase antisera (259 and 269) and antiserum 230. The latter antiserum reacted specifically with the copurified caprine 80 kDa peptide. Cross-immunoreaction to all three antisera in various degrees was observed in these four putative clones (table 3.1). The cross-immunoreaction to antiserum 259 by the Table 3.1 Western analysis of putative clones Clones fusion protein polyclonal antisera 228 259 269 230 G#8228 50 kDa + + + +? G#16228 50 kDa + + + +? G#l7228 - + - G#21228 38, 38 kDa + - G#22228 25 kDa + - +? + G#24228 -? + - - G#27228 - + - Based on the results of plaque immunoreactions and fusion protein analyses. fusion proteins from clones G#8228, G#11228, and G#16228 was also demonstrated by immunoscreening of plaques. However, it appeared these fusion proteins could be recognized 74 neither by monoclonal antibodies 44D9 and 43F10 nor by affinity purified antibody against bovine 100 kDa and 110 kDa peptides. This suggested that these putative clones were not likely to be fl-mannosidase. After specific anti-B-mannosidase antisera 259 and 269 became available, the goat kidney cDNA library was rescreened using 196 fractions purified from polyclonal antibody 269. In addition, 259 antibody was used to screen a bovine liver A2APII cDNA library. Upon screening of approximately 1 x 10‘ phage plaques from each library, only one positive clone (B#9259) was identified by antiserum 259 after plaque purification, and 19 positive clones were selected by 269-IgG. Clone B#9259 did not show any cross- immunoreaction with antiserum 228 at all. Two out of the 19 clones identified by 269-IgG had weak cross-immunoreaction to antiserum 259. When these two clones (G#9269 and G#12269) were probed with antisera 230 and 228, it appeared that both clones had cross-immunoreaction to antiserum 230. However, only clone G#12269 had weak immunoreaction with antiserum 228. No obvious fusion proteins were produced by these clones as judged by a Ponceau S staining. Nevertheless, unexpected smeared bands were observed by Western analysis. Moreover, similar patterns of immunoreactions were produced when different antibody probes (230, 269 IgG, and 269 preimmunoserum) were used in the Western analysis. The smear patterns were not observed in 75 lanes containing proteins from clone G#8228 and plasmid pBluescript only. 3.1.4 Discussion MW Before other specific polyclonal antisera were available, the goat polyclonal antiserum 228 was chosen to immunoscreen goat kidney cDNA AZAPII library. Nine positive clones were identified. Six clones showed no sequence homologies as indicated by Southern hybridizations. Restriction enzyme analysis provided different restriction maps among these six clones. Of the remaining three clones, two were identical (G#8 and G#11) and one (G#16) was related to the identical clones. These results suggested that there were seven different groups in the nine positive clones. False positive clones were common during immunoscreening as any fusion proteins containing the same or similar epitopes may be recognized by polyclonal antibodies. Different preparations of antisera raised against the same protein may contain antibodies against different epitopes, therefore a clone, recognized by two separate preparations of antisera, is more likely to represent the given protein (Helfman and Hughes, 1987). By reacting with other polyclonal antisera acquired subsequently, a total of four clones out of the original nine putative clones (G#8228, G#11228, #16228, and G#22228) appeared to cross-immunoreact with antisera 259 and 76 269. However, they also cross-immunoreacted with polyclonal antiserum 230. Antiserum 230 was raised against only the 80 kDa peptide from goats and did not react with B- mannosidase. The 80 kDa protein in goat was relatively abundant in crude homogenate and Con A fractions. It did not express B-mannosidase enzyme activity and co-purified in small amounts with B-mannosidase protein (Sopher et a1., 1992; 1993). As antiserum 228 contained some anti-80 kDa antibodies, it was possible that clones G#8228 (G#11228), G#16228, and G#22228 were picked up by the contaminated anti-80 kDa antibodies since they did cross-immunoreact to antiserum 230. However, antiserum 230 did not recognize fl- mannosidase peptides, which suggested that the 80 kDa peptide did not contain similar epitopes to the B- mannosidase protein. The cross-immunoreaction to antisera 259 and 269 observed in the above clones implied that these clones were more likely to represent other proteins sharing the same or similar epitopes with B-mannosidase as well as the 80 kDa protein. That these clones were not B- mannosidase candidates was further demonstrated by their negative immunoreaction to affinity purified anti-B- mannosidase antibody and monoclonal antibodies. This conclusion was further supported by the lack of hybridization of these clones with several different 3- mannosidase oligonucleotides obtained later on. 77 WW Polyclonal antisera 259 and 269 were produced by immunizing a rabbit with deglycosylated 100 kDa and 110 kDa bovine peptides (Sopher et a1., 1993). They had little cross-immunoreaction to the 80 kDa bovine peptide. The specificity of an antibody is important for successful immunoscreening. The only positive clone recognized by antiserum 259 upon screening 10‘ plaques from bovine liver AZAPII cDNA library could not be identified by antiserum 269. Preliminary epitope mapping suggested that 269 recognized the same B-mannosidase epitopes as 259 did and recognized more peptides than 259 (data not shown). Therefore, it was unlikely that the clone, recognized by 259 but without cross-immunoreaction with the antiserum 269, would encode B-mannosidase. The whole antiserum 269 and its IgG fraction appeared to contain a quite large amount of preimmune-antibodies. Most of the positive clones identified by 269-IgG through rescreening the goat kidney cDNA library showed strong cross-immunoreaction with preimmunoserum of 269. Two clones containing some cross- immunoreaction with 259 antiserum and one without the cross- immunoreaction were chosen for further analysis. Although no obvious fusion proteins were seen by Ponceau S staining, multiple smear bands were observed by immunostaining with various antisera. The immunostaining pattern of each clone detected with 269-IgG and preimmunoserum of 269 was very 78 similar. The possibility of inefficient blocking of non- specific protein binding sites was implausible since two internal controls of protein lysates (pBluescript plasmid without insert and clone G#8228) prepared at the same time did not show the smear pattern. This result implies that these smear patterns were related to the clones isolated by 269-196. For successful immunoscreening, there are two crucial factors (Helfman and Hughes, 1987). First, the library should be large enough to contain sufficient copies of the gene of interest because not all the recombinant clones will express fusion proteins. In theory, only one out of six recombinant clones may react with antisera. Secondly, the antibody should be specific in recognizing epitopes of the proteins of interest, and of high titer. The failure to isolate the B-mannosidase gene by polyclonal antisera was probably due to the low abundance of expression of the gene. Screening with oligonucleotides in the following section indicated that the frequency of the fi-mannosidase gene in the thyroid library was approximately 0.001%. The activities of the B-mannosidase protein in different tissues have been studied (Lovell et a1., 1994). Thyroid displays the highest enzyme activity, followed by kidney and liver which were about four fold less than thyroid. Therefore, the frequency of the B-mannosidase gene in a kidney or a liver library are expected to be lower than 0.001%. This 79 would limited the success of immunoscreening. The polyclonal antisera used here were able to detect 10 ng purified protein with a suitable signal at about 250-fold dilution. However, antiserum 228 had a significant amount of cross-immunoreaction with the 80 or 84 kDa peptide. This also affected the success of the immunoscreening. 3.1.5 Summary Both goat kidney and bovine liver cDNA libraries were screened by polyclonal antibodies specific for B- mannosidase. Twenty nine putative clones from three independent screenings were identified. Cross- immunoreaction with the other polyclonal antibodies different from that used in the screening was evaluated to determine the authenticity of any of these positive clones for B-mannosidase. The results excluded the candidacy of these clones. Subsequently, Southern hybridization of some of these clones with oligonucleotides obtained later further supported that they did not represent the B-mannosidase gene. The failure to isolate B-mannosidase cDNA by immunoscreening was most likely due to the low level of expression of the fi-mannosidase gene in goat kidney and bovine liver libraries. 80 3.2 ISOLATION AND CHARACTERIZATION OP BOVINE fl-MANNOSIDASE CDNA 3.2.1 Introduction After failing to isolate B-mannosidase cDNA by immunoscreening, efforts were directed to peptide sequencing of the B-mannosidase protein. Previous studies suggested that the N-terminus of B-mannosidase was blocked (Sopher, 1992a). Several approaches had been tried before. Bovine B-mannosidase protein was purified by a four-step purification procedure (Sopher et a1., 1993). After deglycosylation, the B-mannosidase protein was further purified by SDS-PAGE. The major 86 kDa B-mannosidase was subjected to CNBr digestion. Three peptides were sequenced. However, these peptides yielded a limited sequence information. One produced a sequence with six amino acid residues. The second contained two different peptides, and the third yielded only a partial sequence. By in situ digestion with V8 protease on SDS-PAGE followed with transfer to PVDF membrane, multiple amino acid assignments were obtained (Sopher, 1992a). Therefore to obtain large quantities of B-mannosidase peptide, the deglycosylation and gel elution were omitted. B-Mannosidase was purified approximately 15,000 fold using the four-step procedure. This purified protein was digested with CNBr and trypsin and analyzed by the Biotechnology Resource Laboratory at Yale 81 University. Eventually, we obtained more than a dozen peptide sequences. These amino acid sequences, together with the partial sequence obtained earlier (Sopher, 1992a), enabled us to pursue the isolation of cDNA clones by screening cDNA libraries with synthetic oligonucleotides as well as by PCR methods. Cloning a cDNA using synthetic oligonucleotide probes designed from a known amino acid sequence is a popular approach. There are two different types of oligonucleotide probes: one is a mixed short oligonucleotide and the other one is a guessmer. Both probes have been used successfully to isolate genes. Recently, a PCR technique, known as mixed oligonucleotides primed amplification of cDNA (MOPAC), has been developed (Lee et a1., 1988). In this method, degenerate oligonucleotides based on one or two known peptide sequences are used in PCR reactions. Unlike those used for conventional screening, the mixed oligonucleotide probes used in the MOPAC system can be very degenerate. Mixed primers of up to thousands of combinations have been used to clone a cDNA (Lee and Caskey, 1990). Another new PCR technique, known as rapid amplification of cDNA ends (RACE), was introduced first by Frohman et a1. (1988) and has now been commercialized (BRL and Clontech). The RACE system includes 5’ and 3' RACE systems. Even with limited information of amino acid sequences, the RACE system is capable of cloning cDNA ends rapidly. 82 The main advantages of the PCR techniques are their speed, ease, and sensitivity in comparison with the library screening methods. In this paper, the MOPAC method was used in an attempt to generate specific cDNA probes and as an alternative approach for cloning cDNAs. The RACE system was used when conventional screening methods were inadequate for cloning the 5' region of B-mannosidase cDNA. 3.2.2 EXperimental procedures WW B-Mannosidase protein was purified from bovine kidney (Ada Beef Co., Ada, MI) according to the procedures described by Sopher et a1. (1992 and 1993). Briefly, a crude homogenate was prepared from 2.4 kg of sliced bovine kidney tissue and subjected to a four-step chromatography purification procedure including Con A-Sepharose (Sigma, St. Louis, MO), immunoaffinity, TSK-butyl (Pharmacia LKB Biotechnology Inc. Piscataway, NJ), and cation exchange high performance liquid chromatography (HPLC, Mono 8, Pharmacia LKB Biotechnology Inc., Piscataway, NJ). The purity of the protein preparation after the Mono 8 step was confirmed by Coomassie blue staining and Western analysis. The purified protein was dialyzed against 5 mM ammonium bicarbonate solution, lyophilized, and sent to Keck Foundation Biotechnology Resource Laboratory in Yale University for amino acid sequencing. Approximately 740 pmol of purified 83 protein (from a total of 1240 pmol) was subjected to CNBr/trypsin digestion. Peptides were separated by C18 reverse phase HPLC. Peptides which yielded a separable and high peak (Fig. 3. 3) were subjected directly to amino acid sequencing or were repurified by reverse phase HPLC prior to sequencing (Table 3.2). ..3 _ ., . ; , ,- . ...q -. .- . .,-. Mixed and unique oligonucleotides were synthelSsized on 394 and 380 B DNA synthesizers (Applied Biosystems). Regions with minimal codon redundancy were chosen to construct mixed oligonucleotide probes. Guessmers were constructed according to the typical codon usage frequency of human protein (Lathe, 1985). Inosine was placed at residues with three or four fold codon degeneracies in the preparation of the MJ30 oligonucleotide (Table 3.3). Antisense oligonucleotides were constructed for PCR analysis. More than ten oligonucleotides were des'igned based on peptide sequences (Table 3.3 and 3.4). Gene specific oligonucleotides were designed based on the Primer Program (Scientific & Educational Software, PA) to avoid dimer formations and long stretches of G or C. Synthesized oligonucleotides were reprecipitated by two volumes of ethanol and 2 M NH,OAc before being used in PCR. The 2 M lflhOAc solution was substituted by 0.3 M NaOAc (pH 5.6) when the oligonucleotide was going to be labeled at the 5' end. .Uenaaa no: one noososwee onsuxaa enaumem 03» can» once no eeucesvoe aeaunem neuaEaa mcauenmsem nonaummm .eesnamen enesm uses no caeuneocs one mononucenem sa nuaoe osaaa ..eumma .nenmomv mae50a>onm vocasuno aeocesvea enaumem one exaaneuaa 84 o a a s o a m a a a o a a aanmaa is. a a a a a a a a a o a n aanmaa 1a. a a o a a a a > a a a o a a a as. aha is. > o a a a a a a n a canmaa in. a a a a a in. noa a a a a a n a a a a n a a .n meaummm .mc in. an. is. .xv.m\asxav Am. int n a a a a o > a a a a a a Ana .aa «anaau .0. ins Ana is. in. A». in. an. a Amy .2. a an. in. am. is. .u eeaummm is. o o o a a a a a a o n a a a «mnama .a\zv m a s m a a a > a oaa is. a a a n m a n a a aanvoa a a a m a m .a ovauaom ass a z a a a m a n «anmca easesvea euapmea eaenaeossezlc no sensesvea enaumem oaumhna\nazo a.a sandy .exnaneume sua3 ooxnea one uncommon dzno nuas cones nos can uesu eonauooaosz .aesaanoncs manson suaz noxnea ene aneaeaeso .eecaaneocs sua: nexnea ene ancennn enceeause Sonu nesmaueo eononm enauoeaossomaao 85 G I. i i G G oao eon oao oeo oao eon ooo use see one one oso mmmu asnama .C S a. a. CS oaa ooa oao aoo oao oon ooa oao aao one ooa oeo oao oao oao mama aha o o o as sea sac nan aha aoo aoa any one mama «anaea a. a. {I a. {#8 as one oeo oao nan ooo one you oeo oao ooo ooo nan oaa ooa he: vanaaa a o o o o ooz see eye oaa see as mama aanmma a o o e o as sea zoo aao oaa sea was: aanaoa o a o o no: one eao one «ea oo mama aanmaa e o a o as oea nae oao sea zoo on: vanaau ..n:.m. coconooa coauooaoscomaao ononm coaumom eeanennaa ciao no msaceonom now use: eeoauoeaossomaao a.a sanea 86 .Oanz onoaanm .ouae amoow aesnouca oo>eoao e moano>oo ucoemenu e oosoonm on can: one who: ..hmaao mam. men oocosvon nonnonmxo sees: e Bonn nonmaeoo .onoaanm ouenocomon oSOe ca coupe ono: .ooeonoSOav nouae scauoanueom enoaanm Eva: eons ono: moss eno3 man: one aaaz .eocan «zoo onanouoeneso ou noaOamso ono: anoaanm ouenocomon nosno mo scamon .m one occao on nonno ca souome flue“ .m on» ca ooaamme ono3 oaabz one .aoanx .azoo onenanoccealn .msacoonon now ononm e ouenocom on nmeoune ce ca noes one: on: one man: HeQfl aaoaoeeoaoeeoeaoaaoeaoaaumenoooo maamm an amau noeooeoaoenaoeeoeaaoaooa amamn on «can oanaooaoaoaaoeooaoaaaoao madam on amau aoenoeooaoooaoeaeeaooe .maum aaooeooaaooeoeaoaaoeoo .aan: oeaooaooeeooaooaoaao mama an aoomn oooaooaoaaaonoeeoaoo cams on aama: a o o o o 49 yea aao see eye zoo mam imanuvav o o o a aama aae aea eye ooz aoo eao oooemoumoa mmn Aoaa. o a o a aama ooz aae oao one nno oeoemoomoe mam ivmnaamv o o 0 am sea oo nee nee eao zao oeeoouemmeo aanz anoas .ooaumom. .a 1.m noosesuoa noSanm coaueooa ua-zauce mom :a as»: unoaanm coauooauacomaao oouooaoa a.a canes 87 3121211_Labelins_nrehea The 5' end labeling reaction was carried out by T4 polynucleotide kinase (Boehringer Mannheim Biotechnicals, Indianapolis, IN). A twenty microliter reaction contained ten pmol of oligonucleotide, 15 pmol of aP[r-A.TP] (6000 Ci/mmole, 10 mCi/ml, NEN/Du Pont, Wilmington, DE), 1 x buffer (Boehringer Mannheim Biotechnicals), and ten units of T4 polynucleotide kinase. The mixture was incubated at 37%: for one hour. Unincorporated nucleotides were removed using an Nuctrap push column (Stratagene) following the manufacturer's instructions. cDNA fragments were labeled by the random primed method (Boehringer Mannheim Biotechnicals) using 32P [a-dCTP] (3000 Ci/mmole, Amersham, Arlington Heights, IL). WW Normal bovine thyroid tissue was provided to Clontech (Palo Alto, CA) to construct a AZAPII cDNA library. The bovine thyroid cDNA library, consisting of 1.2 x 10‘ independent clones with an average insert size of 2.0 kb, was plated at a density of 1 x 10‘ plaque forming-units per 150 mm petri dish. After 4-5 hours of incubation, a colony/plaque screen filter (137 mm) (NEN research products, Du Pont, Boston, MA) was overlaid on top agarose for 2-5 min. The filter, with the phage plaques facing up, was then transferred to a fresh plate and incubated overnight 88 (approximately 12 hours). Phage DNAs were denatured, neutralized by the standard procedures (Sambrook et a1., 1989) and fixed onto the filters by baking at 80°C for two hours. The filters were washed in a prewarmed (50%» solution of 2 x SSC/0.5% SDS/50 mM EDTA (pH 8.0) prior to hybridization. The hybridization procedure was basically as described in Current Protocols in Molecular Biology (Ausubel et a1., 1989). The filters were prehybridized (at least two hours) and hybridized (2-3 days) at 46-48°C in a solution containing 3 M TMAC (Aldrich Chemical Co. Inc., Milwankee, WI or Sigma, St. Louis, M0) [0.1 M sodium phosphate buffer, pH 6.8/1 mM EDTA, pH 8.0/5 x Denhardt's solution (1% Ficoll/1% polyvinylpyrrolidone/1% bovine serum albumin) [0.6% SDS/100 ug/ml denatured herring sperm DNA (Boehringer Mannheim Biochemicals) in crystallizing dishes. Approximately 1-2 x 10‘ cpm/m1 of end-labelled mixed oligonucleotide probes with specific activity at 2-10 x 106 cpm/pmol were used during hybridization. The hybridized filters were washed once with 3 M TMAC/50 mM Tris.Cl (pH S.0)/0.2% SDS, and then washed at room temperature for 15 min, followed by washing exactly 1 hour with the same solution at 46-50°C. Finally, filters were washed in 2 x SSC/0.1% SDS at room temperature for 2-3 times, each for 10 min. Filters were then wrapped with Saran wrap and exposed to Kodak XOMAT-AR film (Eastman KODAK Co., Rochester, NY) at -80°C for 1-3 days. The hybridized filters were 89 sequentially probed with different oligonucleotides after removal of the previous hybridized probe. Probes were removed by incubating hybridized filters in 0.4 M NaOH for 30 min at 45°C and 0.1 x SSC solution containing 0.1% SDS and 0.2 M Tris.Cl (pH 7.5) for 30 min at 45°C. Putative positive plaques were purified by several rounds of rescreening at lower densities and excised as pBluescript plasmids according to the manufacture's instructions. In order to isolate a full-length cDNA, the 1.6 kb insert of clone #47MJ4 was isolated and labeled by the random primed method to a specific activity of 1.4 x 109 cpm/pg. Up to 1 x 10‘ cpm/ml of the denatured probe was added in a 5 x SSPE hybridization solution containing 50% formamide (Boehringer Mannheim Biotechnicals)/0.5% SDS/5 x Denhardt's solution/10 ug/ml denatured herring sperm DNA to reprobe the filters at 42°C. After approximately 20 hours of incubation, the filters were washed in 2 x SSC/0.1% SDS for two times, each 10 min at room temperature, then washed in 1 x SSC/0.1% SDS at 65°C for 30 min. Finally, the filters were washed in 0.1 x SSC/0.1% SDS at 65°C for 30 min. WWW Ten micrograms of total RNA or 1 ug of poly A+ mRNA were reverse transcribed into single strand cDNAs. RNA in diethylpyrocarbonate (DEPC) treated water was first heated at 65°C for 5 min, then incubated with 10 pl of 5 x reverse 9o transcription buffer (Gibco BRL, Gaithersburg, MD), 5 ul of 0.1 M dithiothreitol (DTT), 5 ul of 10 mM dNTP mixture, 1.5 ul of 40 unit/pl ribonuclease inhibitor (rRNasin) (Promega, Madison, WI), 5 to 75 pmol of antisense oligonucleotides and 200-400 units of M-MLV reverse transcriptase (Gibco BRL) in a 50 ul of reaction mixture at 37°C for one hour. The first strand cDNAs were precipitated by adding an equal volume of 4 M ammonium acetate acid and two volumes of ethanol and resuspended in 50 ul of distilled water. An aliquot (1-2 ul) of cDNAs were amplified by AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT) in the GeneAmp PCR system 9600 (Perkin-Elmer Co.). Generally, 30-35 PCR cycles were performed and each PCR cycle consisted of 45 seconds denaturation at 94°C, 1 min annealing at 46°C-55°C, and a 1 min extension at 72°C of cDNA. A 5 min predenaturation at 95°C and an additional 10 min extension at 72°C were applied before and after the cycle reactions, respectively. One fourth of the amplified product was analyzed by electrophoresis on Nusieve 3:1 agarose gels (FMC BioProducts, Rockland, ME). To perform reamplification and nested PCR, the agarose containing interesting bands was removed using a capillary tube. The agarose was either directly used (5 ul) in PCR reactions or diluted in distilled water, then aliquots of agarose suspension were used in PCR reactions. To analyze putative clones, PCR was performed using 91 either plasmid DNA or crude phage lysates as templates. Crude phage lysates were prepared by adding an equal volume of 0.1 M NaOH to an aliquot of phage stock, incubating for 10 min at 95°C, and then neutralizing by adding 1/20 volume of 2 M Tris.Cl solution (pH 7.5). To determine the orientation of an insert, PCR was accomplished by using a gene specific primer (either unique or degenerate) and a M13 primer (reverse or -21 primer). In order to isolate the 5' region of B-mannosidase cDNA, 0.5 ug poly A+ mRNA of bovine thyroid was copied into single strand cDNAs and amplified using the 5' RACE system kit (Gibco BRL, Gaithersburg, MD) according to the manufacture's instructions. Two gene specific antisense oligonucleotides (MJ100 and MJ101) designed from clone #17MJ48 and a degenerate oligonucleotide MJ48 from peptide 142r12 were used in the 5' RACE system. ;H :. . ., 1,. (. ,- , . . , . . , ., Total RNA was extracted from various bovine and caprine tissues and from both normal and affected animals according to the procedure as described (Ausubel et a1., 1989). Poly A+ RNA was isolated using poly A+ quick mRNA kit (Stratagene). RNA samples (gift from Dr. Karen Friderici) were analyzed by electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde as described (Ausubel et a1., 1989) and 92 blotted on the Hybond-N membrane (Amersham). The hybridization condition is either based on the method described in Current Molecular Protocol (Ausubel et a1., 1989), or is the same as the Southern hybridization described in section 2.2.8. Approximately 1-2 x 10‘ cpm/m1 of cDNA probe(s) with a specific activity of approximately 1 x 10’ cpm/pg was used during the hybridization. Filters were washed in a step-wise fashion according to the background signal. The filters were exposed at -80°C for 3- 10 days. After removal of the B-mannosidase probe, the blot was rehybridized to a cDNA probe of rat glyceraldehyde-3- phosphate-dehydrogenase (GAPDH) as an internal control. WWW Miniplasmid DNAs were prepared using the Magic (Wizard) minipreps DNA purification system (Promega, Madison, WI). Sequencing was carried out by the Tag cycling method using dye terminators and dye primers (M13 -21 primer and M13 reverse primer) on a 373A DNA sequencing system (Applied Biosystems). The entire inserts in clones #46MJ4, #47MJ4, #17MJ48, #11MJ101/UAP, and #2MJ48/UAP were sequenced in both orientations using internal oligonucleotide primers. To sequence PCR products, PCR products were separated on 1% Nusieve GTG low melting agarose gel (FMC BioProducts, Rockland, ME), bands of interest were excised under long wave-length UV light, and purified using Magic (Wizard) PCR 93 DNA purification system according to Promega's instructions. The purified PCR products were sequenced directly by the dye terminator sequencing method. In some cases, the PCR products were subcloned into PCR‘ vector using a T/A cloning system (Invitrogene, San Diego, CA). Plasmid DNAs were then prepared by the Magic (Wizard) minipreps system and sequenced by dye dideoxyribonucleotide terminator. DNA sequence analysis and homology search against GenBank were performed using GCG program version 7, April, 1991 (Genetics Computer Group DNA Sequence Analysis Software, Madison, Wisconsin). '- 91! Q P '. 5.. ’I '- 4"}."9'1; P ' 3.4.. 9' A DNA panel of 24 human/rodent somatic cell hybrids was obtained from Coriell Cell Repositories, Coriell Institute for Medical Research (Camden, NJ). Except for hybrids NA07299 and NA10478, all hybrids retain one human chromosome under either mouse or Chinese hamster background. Fifteen micrograms of DNA isolated from each of the hybrids were digested in 100 pl reactions by restriction enzyme PstI (120 units) at 37°C. After overnight digestion, DNA was reprecipitated by two volumes of ethanol and 0.3 M NaOAc (pH 5.6) and separated in a 1% agarose gel at 25 V for 24 hours. DNA was transferred overnight to Hybond-N membrane (Amersham Co., Arlington Heights, IL). A PCR product of clone #46 MJ4 94 was labeled by the random primed method to a specific activity of 4 x 10' cpm/pg. Approximately 3 x 10‘ cpm/ml of the probe was denatured and added to 15 m1 of hybridization solution. Hybridization was carried out in 50% formamide/6 x SSC/5 x Denhardt’s/0.5% SDS/100 ug/ml denatured herring sperm DNA for 20 hours at 42°C. The final wash was in 0.2 x SSC/0.1% SDS for 15 min at 65°C (high stringency wash) or 1 x SSC/0.1% SDS for one hour at 65°C (low stringency wash). The blot was exposed to Kodak XOMAT-AR film at -80°C for three to ten days. Genomic DNAs from different species and affected animals were prepared previously by Sopher (1992a). The restriction enzyme digestion, gel electrophoresis, DNA transfer, and hybridization were performed as described above. 3.2.3 Results WW Bovine protein was purified to approximately 15,000 fold by a four-step-chromatography procedure. The final protein preparation revealed three peptides 84, 100, and 110 kDa as judged by Coomassie blue (Fig. 3.2). There was little, if any, 80 kDa peptide as judged by Western analysis (Fig. 3.2) using the antiserum 230 which reacts specifically with caprine 80 kDa protein or bovine 80 kDa protein. Approximately 740 pmol of this purified protein was subjected to CNBr/tryptic digestion. Fourteen peptides 95 including those repurified by reverse phase HPLC were sequenced (Fig. 3.3). This resulted in a total of ten non- overlapping peptides with complete sequences plus additional peptides with incomplete or uncertain sequences. The sequence of the ten peptides is shown on Table 3.2. MW After the failure of immunoscreening, preliminary oligonucleotide screening with several oligonucleotide probes by plaque hybridization, and PCR with various combinations of degenerate oligonucleotide primers were performed without success (data not shown). In an effort to increase detection levels, in situ amplification of plaques was used to intensify the signal. Furthermore, a library was obtained from bovine thyroid tissue showing the highest expression of B-mannosidase activity. In addition, oligonucleotides (e.g. MJ4) that gave a good signal in preliminary studies or that were derived from peptide sequences (e.g. 142r12) produced by two different sequencing sources were used for screening. By screening approximately 5 x 10’ phage from the bovine thyroid cDNA library sequentially with four different degenerate oligonucleotides, a total of 19 positive clones were detected. Among these positive clones, seventeen clones were identified by oligonucleotide probe MJ4 and two by oligonucleotide probe MJ48. No positive clones were found 96 kDa Fig. 3.2 Panel A: Coomassie staining of purified #- mannosidase peptides. Fifty microliters (approximately 1/100) of purified B-mannosidase protein was fractionated on 7.5% SDS-PAGE (lane 2). Lane 1, High range SDS-PAGE standard (Bio-Rad). Panel B: Western analysis of purified B-mannosidase peptides with antisera 259 and 230. Lane 1 and 2, Containing 1 ug of purified protein were reacted with antisera 259 and 230 respectively. 97 ”SF 3 3 “‘ '3 N F. n N 0 F4 4" '4 n H 4 ° ‘ a ' a I l S 3 ‘3 I .4 C i p ‘ H ‘ I 3 n ! s. J J“. .*0.\.. O—ee-n-o ~ as... .e .J-‘Jo'OOO—v'l 0000000 .J-ofi'.~. .' . .. ‘..~e.. '-“ " J L, 1 1 BB . 00 Fig. 3.3 The reverse phase HPLC profile of CNBr/tryptic cleaved peptides of B-mannosidase. Approximately 750 pmol B-mannosidase protein purified by a four-step column purification procedure were subjected to CNBr and trypsin digestions and separated by C18 reverse phase HPLC. Peptides 103, 171, 180, and 253 were sequenced directly. Peptides 104, 142, 151, 169, 218, 251, 265, and 267 were subjected to repurification before sequencing. No sequence was yielded by sequencing peptides 252, 265, and 267. Mixed sequences were obtained from peptide 253. 98 when reprobing the filters with two other oligonucleotide probes: MJ63 and MJ64. Of the 19 positive clones, clones #43MJ4, #46MJ4, and #47MJ4 were also found to hybridize with guessmers corresponding to three different non-overlapping peptides (i.e. MJ7, MJ23, and MJ65). The three clones were plaque purified, subcloned into pBluescript plasmid, and subjected to further analysis by restriction enzyme digestion and sequencing. The results indicated that clones #43MJ4 and #47MJ4 were identical containing a 1.6 kb insert with identical restriction maps, while clone #46MJ4 contained an 1875 bp insert (Fig. 3.4). Clones #43MJ4, #47MJ4, and #46MJ4 all started at the same 5' region at a cleaved EcoRI site and contained 735 bp of open reading frame. There were two nucleotide differences between the two clones: C at position 2124 was replaced by G in clone #47MJ4, while C at position 2417 was substituted by T in clone #47MJ4 (Fig. 3.5). The C/T substitution was neutral. The C/G substitution changes an amino acid residue from histidine (H) to the aspartic acid (D), found in that position in the direct peptide sequence of peptide 151r72. The homology between clone #46MJ4 and #47MJ4 diverged at 1182 bp from their 5' ends. The authenticity of the three clones #46MJ4, #47MJ4, and #43MJ4 was established by colinearity of predicted amino acid sequence of these clones with five microsequenced peptide sequences (103, 218r24, 151r72, 180, and 171) (Table 3.2). 99 aaeu aav mace ..a .aassm .m aaoum .e aaaoeaa .a aaaem .a manna .x aamooa .a aaeao .o nausea .a manna .M «462644 nuaa moamoaoaon 0: .ozaa some one ocaa manooo «soamon msaooo .nen oaaom «soaueanOMsa ousosvom mmnaomau seas: on» mean: oouenozom uoaoonn mom a .vs\~anzaom nauseous aoaa .m no ocoaonom a .avaao .uoqoao £200 eneuaeosseann non hmouenue msaonosvom one mes noauoanuoon one v.a .mah uluv an ace All A A A A V Al eanmn uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu +a IV All Him“ a.a“ «noun v . Iv .llv Iv IV V V V V All Anll. A ll «62544 7 4 V V V V V V A .AMWI 7n L (A A 46:64“ 7 7 All .||.v es\~anzaom A V V All AHIILI aeumsae ‘ 7 V AV T v .llv aeomo 100 Fig. 3.5 Nucleotide and deduced amino acid sequences of 3- mannosidase cDNA. Nucleotides upstream of the predicted initiation codon ATG are given negative numbers. Potential N-glycosylation sites are indicated by *. Colinear CNBr/Tryptic peptides are underlined. Residues which do not match with peptide sequences determined by microsequencing are marked with l ]. Two possible polyadenylation sites are marked with . Signal peptide sequence is underlined. The arrow ind cates the predicted signal peptide cleavage site. are -74 -14 46 17 106 37 166 57 226 77 286 97 346 117 406 137 466 157 526 177 586 197 646 217 706 237 766 257 826 277 886 297 101 ctcaggccgagcgtggcttccgctgcccaccgcatccctcgggttcttgccctgtgcggg . +1 . taccgggcaacaccatgctcctccgcctgctcctgctgcttgcaccgtgcggtgcgggct M L L R L L L L L P F tcgctaccaaggtggtcagcatcagtttgcggggaaactggaagatccacagcgggaacg A T R V V S I L R N W I H N —: gttcgctgcaactccccgcgacggttcccggttgcgtgcacagcgccttgttcaacaaga S L Q L P T P A L P N K R e ggatcatcaaggatccttactacagatttaataaccttgactatagatggatagccttgg I K D P Y Y R [F] N N L D Y R W L 104r85 ataactggacctatatcaagaaatttaaactccactctgatatgagcacatggagtaaag W T Y I R K P R L H S D M S T * taaatttggtttttgagggtatcgatacagttgcagtagtcctgctcaacagtgttccca N L F D T V A V V L L N S V P I ttggcgaaacagacaacatgttcagaagatacagctttgatattacacatacggtcaaag G E T D N M R R Y S P D I T H T V R A cagtgaacatcattgaggtgcgtttccagtcaccagtggtatatgcgaaccagaggagcg V N I I V R F Q S P V V Y Q aacgtcacactgcctactgggtgccccccaactgccctccacctgtgcaggatggcgaat R H T A Y W V P P N C P P P Q C gtcatgtcaactttattcgcaagatgcagtgttcctttggatgggactggggaccttctt H V N F I R K M Q C S F W P S F ttcctacccagggcatctggaaagatgttagaattgaagcctataatgtttgtcatctga P T Q G I W K D V R I E A Y N V C H L N actacttcatgtttacccccatctacgataactatatgaagacatggaatcttaaaatag Y F M F T P I Y D N Y M K T W N L K I E -—————- 142r12 agtcgtcttttgatgttgtcagttcaaagctggtttctggtgaagcaattgtagccatcc S S F D V V S s K S G E A I V A I P ctgaactaaacatacagcagacaaacaacattgaacttcaacatggggaaaggactgttg E L N I Q Q T N N I E L Q N G E R T V E agctctttgtgaaaatcgacaaggctattattgtagaaacttggtggcctcatggacatg L F V K I D K A I I T W H H G gaaaccagactgggtacaacatgagcgttatttttgagctggatggaggcttacgttttg N Q T G Y N M s V I F L L R F E a t Fig. 3.5 ‘946 317 1006 337 1066 357 1126 377 1186 397 1246 417 1306 437 1366 457 1426 477 1486 497 1546 517 1606 537 1666 557 1726 577 1786 597 1846 617 1906 637 1966 657 102 aaaaatcagctaaggtttattttaggacagtggaacttgtagaagagcccatacaaaatt K S A K V Y F R T V L V E E P I Q N S ctcctggtctgagtttctacttcaaaattaatggacttcccatatttctgaaaggctcga P L S F Y F K I N G L P I F L K G S N attggatccctgcagattcattccaggatagagtaacctctgccatgttgcggctcctct W I P A D S P Q D R V T S A M R L L L tgcagtctgttgtggatgctaacatgaatgctcttcgggtctggggaggaggagtttatg Q D A N M N A L Y E agcaggatgaattctacgaactctgtgatgaactaggcataatgatatggcaggatttca Q E P Y E L C D E L G I M W Q D F M tgtttgcctgtgcgctttacccaaccgataaggatttcatggattctgtgagagaagaag F C A L Y P T D K D F M S V R E E 169r65/169r6l tcactcaccaggtccggagactgaaatctcatccctccatcatcacatggagtgggaata T H Q V R R L K S H P S I I T W S N N atgaaaatgaagcagcactaatgatgggttggtatgatacaaagcctggctacttgcaaa N E A A L M T K P G Y L Q T cctacatcaaagactatgtgacactgtatgtgaaaaacatccgaacgatcgtcttagaag Y I R D Y V T L Y V K N I R T I V L E G gagaccagactcgtccttttatcacatccagtcctacaaatggggccaaaaccattgcag D Q T R P F I T S S P T N G A K T I A E aaggttggctctctccaaacccctatgacctgaattatggggacgtacatttttatgatt L s P N P Y D L N Y G D V H F Y D Y atgtgagtgactgctggaattggagaactttccccaaagctcgatttgtatctgagtatg V S D C N W R T F P K A R F V S E Y G gatatcagtcctggccttccttcagtacattagaaaaggtttcctctgaagaggactggt Y Q s W P s F S T L E K V s s E E D W S cttacagaagcagctttgcacttcatcggcaacatttgattaacggtaacaatgaaatgc Y R S S F A L H R Q H L I N G N N E M L ttcaccagattgaacttcacttcaagctcccaaacagtacagatcaactacgcaggttca H Q I E L H F K L P N S T D Q L R R F K * aagacactctttatcttactcaggtgatgcaggcccagtgtgtcaaaacagaaactgaat D T L Y L T Q V M Q A Q C V K T E T E F tctaccgtcgcagtcgcagcgagatagtgaatggaaaagggcacaccatgggggcgcttt R R S R S N K G T M L Y attggcagctcaatgacatctggcaagctccttcctggtcttctctagagtatggaggaa L N D I W Q A P S W S S L Y K Fig 3.5 2026 677 2086 697 2146 717 2206 737 2266 757 2326 777 2386 797 2446 817 2506 837 2566 857 2626 877 2686 2746 2806 2866 2926 2986 3046 3106 3166 3226 3286 3346 3406 3466 3526 3586 3646 3706 3766 103 ngtggaaaatgcttcnttactttgctcggcatttcttcgcccccctgttaccggtgggtt KMLHYFARlHlFFAPLLP F —103——— 218r24 ttgaggataaagatatgcttttcatctatggtgcgtcacaccttcnctcagaccagcag; EDKDMLFIYGASIHILHSDQQM 151r72 tgatgctcactgtgagagtccacacttggagttccctggagctcgtatgctctgagtcaa M L T V R V H T W S S L L c S E S T ctaaccctttcgtgntaaaagctggggagtctgttctcctctatactaagccagtgcctg N P P V I K A G E S V L L Y T K P V agttgctaaaaggatgtcccggatgtacacgacaaagctgtgtggtttccttttacctgt L L K G C P G C T R Q 8 C V V 8 E Y L 8 canctgacggggaactcttgagcccaatcaactatcacttcctgtcctcactgaagaatg T D G E L L S P I N Y H F L S S L K N A ccaaggggctccacaaggcaaatatcactgccaccatctcgcagcaaggggacacatttg K G L H K A N I T A T I S Q Q G D T F V tttttgatctgaaaacctcagctgtcgctccctttgtttggttggatgtaggaagcatcc F D L K T S A V A P F W L S I P cagggagattcagtgacaatggtttcctcatgactgagaagacacggactgtattctttt R F D N G F L M T E K T R T V F F Y acccttggaaacccaccagcaagagtgaattggagcaatcttttcatgtgacttcactgg PWKPTISIKSELEQSFHVTSL 180 171 ctgatacttgctgagggaatcaggttgtaétttcgagagétgaaggcaaétagaaacnag D T Y * ttgaagaagccaggaaatgcatctgcttgctgtcaggtgtctggttagccacttggttct cccagggaaggctgtgtatattcaggtgatgttctcaacaaagcggtgcctgggtgctgt tccgtctgcaccagggctgtgtctttagctcttccttttgcaccttttgcaccacgtgaa tcagttctaacccaactgtctctcctacccccaaaggaggtcctgtccacacgcagtcct ttaagggaatcacaggaacatgaccaagtagccctttaagagaattacaggcacactccc nggtagcccttaagggaatcacagtaatgaccattgtggtatctgtggaatcaaatgtgg aagattgtgagggcatgtaggcccctcaggatagctttgagaaataccaaacgattgaan tgaaactgctttgtcattatttccagaggaaatagagattcagatgttgcaacagaaaga gatgtctgggtggtagccatattggttgttgatgctggaaagtttggtgggattgattat tgccattcgattactttttgagtaggagtcttttttcatttgtgatttttttttttaata auntatttgttttaacaataataatttattttttcaaaggcaattagtgattcttctttg ggaaaaaaaaaactcacattggaatggacatcaccttgatcatgttggaaacttttgggt gtcctgacgtaagtggtcacctgtattaagtatgggcttcagatttggttaagtccagtg aactttccagttcaagactatggtttgatttgcatgtgatgagcctggcagcaaagtggt attgcctttaacttgagattgaaccattttaaaaaacactgattaattataattgctatg anatcattttgttctcatcatcctgtttataaaattacattgatagtgaagcaaggggca anatgttaataagtagtcaatttgagtaaaggtgtataggaatatttttgttctgcttga gcaacttttctgtaagtttgaaatatataaaaatttaagattatataaattgcattgaca aaaaaaaaaaaa 3777 _ — Fig. 3.5 104 .cmuowcmum mum: macaummn mcflccnmm unmanawa 02 .p as: mafia song“: was .A~mma ..mauufiaooo can mu>xv conuma m.mauuwaooo can wuax co cmmmn mm3 Dean hnummouvan was .1200 ouuuwuonndann ona>on any Scum vouowuoum uouwumomhaon oauowaonauann can «0 Dean unannouchn one a.a .Oum UH H Ea: uflaocaz 105 In a parallel experiment, PCR was used to generate a gene specific probe for screening the cDNA library. One of earlier peptides generated by sequencing of CNBr cleaved peptides (peptide 3, Table 3.2) of the deglycosylated 86 kDa protein (Sopher, 1992a) was found to overlap two of the peptides (218r24 and 103, Table 3.2) in this study. We suspected that the two peptides 218r24 and 103 were continuous. Therefore, a sense primer M366 and an antisense primer MJ6 (Table 3.4) were used to amplify a bovine thyroid cDNA reverse transcribed by oligonucleotide primer MJG. As was predicted from the amino acid sequence, multiple PCR products were produced, including one with expected size of 81 bp. The 81 bp product was gel purified and reamplified. The single 81 bp product was then repurified and subcloned into PCR‘ vector using the T/A cloning system. Several subclones containing the 81 bp product were sequenced. The deduced amino acid sequence showed colinearity with the peptide sequence. This approach was not pursued since these data became available at the same time the large clones described above were obtained. In order to isolate a full-length cDNA, the 1.6 kb insert of clone #47MJ4 was gel purified and used as a probe to rescreen the original filters. Three additional clones (rz-la, r8-1, and r20-2) were isolated and excised into pBluescript plasmid. EcoRI digestion of the plasmid DNAs indicated clone #r20-2 lacked an EcoRI site in one of the 106 cloning sites and contained an insert size of approximately 1.4 kb. The insert size of clone ire-1 was close to 1.8 kb (Fig. 3.4). Clone frz-la appeared to have a large insert of approximately 4.3 kb, which was confirmed by Southern hybridization of EcoRI digested plasmid and phage DNA. To analyze whether any of these clones contained sequences upstream of the 5' ends of clones #46M34 and I47M34, a specific oligonucleotide (M374) (Table 3.4) was designed according to the sequence located 105 bp downstream from the 5' end of clone #46M34. This gene specific oligonucleotide was used with vector primers close to the cloning sites to prime PCR reactions of either crude phage lysates or plasmid DNAs of clones #rZ-la, Ira-1, and frzo-z. The PCR results indicated that clone #r2-1a contained approximately 1.2 kb more sequence than the 5' end of clones #46M34 and #47M34. The other two clones appeared to start at the same internal EcoRI site as clones #46M34 and #47M34 (Fig. 3.4). Further studies by PCR using either gene specific (M373, M374) or mixed oligonucleotide primers (M366, M35) located downstream of the EcoRI site of clonef46M34 suggested that clones frz- 1a, #r8-1, and #rZO-Z had sequence homologies with clones #46M34 and #47M34. Partial sequencing of these clones confirmed that clones {rs-1 and #20-2 did indeed start at the same 5' position as clones #46M34 and #47M34, corresponding to a cleaved EcoRI site. Their 3’ end sequences were nearly identical to that of clone #46M34. 107 Clone #r8-1 contains additional 20 base pairs including a poly (A) tail (Fig. 3.5). Partial sequencing of clone irz- 1a with M13 reverse and forward primers and some internal primers derived from the DNA sequence of clone #46M34 demonstrated that it encompassed most of the sequence of clone #46M34. However, the sequence homology diverged at 86 bp upstream of the 3' end of clone #46M34. A long stretch of poly (A) tail was present in the 5’ end of clone r2-1a, and no open reading frame was revealed in the region upstream the EcoRI site of clone #46M34. These results clearly indicated that the EcoRI sites of these clones had not been protected by EboRI methylase during the construction of the bovine thyroid cDNA library. Since an internal EcoRI site of B-mannosidase cDNA was cleaved, these clones were not suitable as probes to rescreen the bovine thyroid cDNA library. Surprisingly, a sequence homology search disclosed that there was high homology between sequence downstream of the EboRI site of a human expressed sequence tag to an unknown gene (EST01397) (Adams et a1., 1992) from GenBank and the 5' ends of clones #46M34 and #47M34 (Fig. 3.7). The insertion of one hp at positions 284 and 305 of the human clone shifted the reading frame twice in a human expressed sequence tag cDNA (Fig. 3.7). We speculated that the human expressed sequence tag obtained from a hippocampal cDNA library was derived from human B-mannosidase. Using this information, two 108 D 1794 act cttacagaa cagcttt cacttcatcggcaacatttgattaa {SI III I! III III HHIHH HIH H 1 gacgggtctttcaatagcaagttttcacttcatcgacaacatcacgaagg 1844 cggtaacaatgaaatgcttcaccagattgaacttcacttcaagctcccaa HHIIH HHHH I H: H IHHI HIH HHI 51 tggtaacaaacaaatgctttatcaggctggacttcatttcaaactccccc ------) ........ “382 --------- -> 1894 acagtacagatcaactacgcaggttcaaagacactctttatcttactcag I H IHHH I HH: H H: B H : H HHIHH 101 aaagcacagatccattacgcacatttaaagataccatctaccttactcag 1944 gtgatgcaggcccagtgtgtcaaaacagaaactgaattctaccgtcgcag 151 gtgatgcaggcccagtgtgtcaaaacagaaactgaattctaccgccgtag #1714348 4 ——>#46M34 1994 tc cagcgagatagtgaatggaaaagggcacaccatgg ggcgctttatt H HHHIHHH H HIHHHI HH IH HHHI 201 tcgcagcgagatagtggatcagcaagggcacacgatgggggcactttatt (stuns-“J54 :ssssxssfiJ73aussssss 2044 ggcagctcaatgacatctggcaagctccttcct.ggtcttctctagagta HH IIHHHHHHH HHHH H HHHI IHH 251 ggcagttgaatgacatctggcaagctccttcctggggcttctcttgagta 2093 tgga.ggaaagtggaaaatgcttcattactttgctcggcatttcttcgcc H: HHHHHHHIHH HHHHH: : HIHH H 301 cggagggaaagtggaaaatgcttcattactttgctcagaatttctttgct 2142 cccctgttaccggt.gggttttgagg.ataaagatatgcttttcatctat H HHI H H H HIH H H H l l : H HHH 351 ccactgttgccagtaggcttttgaggaatgaaaacacggtctatatctat 2190 .ggtgcgtcacaccttcactcagaccagcagatgatgctcactgtgag HH HH 3 HHHH H : IHH : H HHH 401 gggtgtgtcagatcttcactcggattattcgatgacactcagtgtgag I U! El U I: II I II Fig. 3.7 DNA sequence comparison between human expressed sequence tag (EST01397) and bovine fl-nannosidase cDNA. 8 represents the bovine sequence and H represents the human sequence. There is 81.49% identity between the two sequences. The internal EcoRI site is marked with . Sequence upstream of the BcoRI site is from the 3' end of clone #17M348, and downstream is from the 5' end of clone #46M34. The two sense human primers (M381 and M382) and the two antisense bovine primers (M373 and M374) are marked with single dash lines and double dash lines, respectively. 109 oligonucleotide primers M381 and M382 were designed based on the 5' region of the human sequence in order to generate a suitable probe to rescreen the library. Bovine thyroid total RNA was reverse transcribed into first strand cDNA using an antisense primer (M373) located downstream of the EcoRI site in clone #46M34. Amplification was done by priming the first strand cDNA with antisense primer M373 and sense primer M381. A specific product of approximately 215 bp was generated by reamplification of the PCR products with two nested primers (M382 and M374). The above 215 bp of PCR product was gel purified and directly sequenced from both directions. The sequence flanking the EcoRI site had high homology to the human taq sequence (EST01397) at both DNA and amino acid levels (Fig. 3.7). In the meantime, the discovery of the failure of EcoRI methylation led us to reevaluate the other two clones, which had previously been identified by a mixed oligonucleotide probe (M348). The peptide sequence corresponding to probe M348 was not found in the clones #46M34 or r8-1, so it was possible that the clones identified by M348 corresponded to sequences upstream of the cleaved EcoRI site. To quickly determine the authenticity of these clones, PCR reactions were carried out on crude phage lysates from clones #9M348 and #17M348 using vector primers (M13 forward or reverse primer) and various oligonucleotide primers (e.g. M39, M348, M363, M382, and M364) with various combinations (Table 3.2 110 and 3.3). Specific PCR products were produced from clone #17M348. This clone was subjected to sequencing analysis. The sequence data showed that clone #17M348 contained an insert of 1119 bp and encoded 373 amino acids. Four peptide sequences (169r64, 169r65, 169r61, and 142r12) were found to exactly match with predicted amino acid sequences. The 3' region showed high sequence homology with the human EST01397 sequence (Fig 3.7). The 215 PCR product (amplified by the human and bovine primers described above) spanned the 3' region of clone 17M348 and the 5' region of clone 46M34. Besides the expected 3' cloning EcoRI site, an internal EcoRI site was found in this clone. The EcoRI site at the 5' cloning site was defective by missing a C nucleotide. The composite cDNA of clone 17M348 and r8-1 was approximately 3 kb long, however, about 1.2 kb of the 5' region was still missed as indicated by the Northern analysis (see the following section). In order to isolate the missing 5’ end of the B-mannosidase gene, 5' RACE was adopted (Fig. 3.8). Two synthetic oligonucleotide primers (M3100 and M3101) based on the 5' region of clone #17M348 were prepared and used in the 5' RACE system. The DNA template was first strand cDNA transcribed from bovine thyroid poly(A)+ RNA by M3100 oligonucleotide and tailed with homopoly-C. Under a smear background, a discrete band at approximately 950 bp was observed in a PCR reaction that was carried out by a gene specific oligonucleotide primer 111 (M3101) and an anchor primer (Fig. 3.9). The 950 bp product was gel purified and reamplified by a universal amplification primer (UAP) and a nested oligonucleotide primer (M3101) and by UAP and a nested degenerate primer (M348). A PCR product of approximately 950 bp was produced using the first pair of oligonucleotide primers. Two PCR products with a major product being 770 bp, were observed using the second pair of oligonucleotide primers (Fig. 3.9). The reamplified 950 bp product and 770 bp product (Fig. 3.9) were gel purified and subcloned into PCR“ vectors. To identify positive subclones, putative subclones were subjected to PCR using a nested gene-specific oligonucleotide (M3110) and M13 reverse or forward primer. The authenticity of the 5' RACE product was confirmed by both direct sequencing of the PCR products and sequencing positive subclones. A peptide sequence (104r86) was found to be colinear with the deduced amino acid sequence of the 5' RACE products. In addition, sequencing of the 5' RACE products also revealed a possible translation initiation codon at nucleotide 75 followed by an open reading frame. The nucleotides flanking the ATG (ACCATGC) were in good agreement with the consensus sequence for the eukaryotic initiator codon: A/GCCATGG (Kozak, 1986). Furthermore, the 17 amino acid residues following the initiator codon exhibited features characteristic of a signal sequence, i.e. a basic N-terminal region (M—L-L-R), a central hydrophobic 112 ..n oHHoooHHoooHHoooosaosaosuoaoouoaoooocsoaooaaosso .m «Amt. uGEaum uosose usmnoumou Sound used cansou 0:» 05am eoHoudo sumo one ..n osaosaoaooaoomocooooaoocooasosoo .m "Assoc nossus oucoavou Huuum>acs usueoumou soaouao sumo one .mvhzhH$ osoHo mo szno musemoumou oswa mansou use .omeudeossnaln no scenes .m ecu eusunoumou exeuuoume one .1200 eeeudeossellc «0 scum.» .n on» no soda-Hone on» now sues .n on» «0 uneven». one a.a .uqs oaahz Ilv HOHHX 'IIoeoo use ondn: oaanz uoueos Ilv spas mswososvom men: Ittoooo 3 damn: Ilv HOHHX I'Ieeoo Aoeeo uss mumfiaum men: uoumos suqz luv sequooauaamadom dean: ItIoooo Aeooe Hoanz\m¢ .. use no uosuoum mom unsung H00 Head: liloooo 2 Hands uouuos use dean: Hosaum uososd Ilv osu mswms mom comm: saouvl.m 0.00000 n— Meagan wososd Hue uso maou nus; azou Hana cosh: :Aouvu.m .— szso usuuue 00am: new Onwnosusam r ¥¥C¥¥¥ii§0¥¥¥¥IICC 113 A Fig. 3.9 Analysis of the 5' RACE products. Panel A: cDNA reverse transcribed from bovine thyroid 0.5 ug poly (A)+ RNA was amplified by a gene specific primer M3101 and an anchor primer. After 35 cycles (9ft 1 min, 57%:30 seconds, and 7Tb 2 min for each cycle) of amplification, twenty microliters of 50 ul reactions were loaded into 1% agarose gel. Lane 1: negative control without cDNA. Lane 2: PCR of non-tailed cDNA. Lane 3: PCR of poly C tailed cDNA. Lane 4: 1 pl of marker III (lambda DNA/EcoRI/HindIII). Panel B: the major product of 940 bp in lane 3 at panel A was excised and suspended in 30 pl water. Aliquots of the suspension were reamplified with nested primers in a total of 200 pl reaction. DNA was reprecipitated, fractionated on 1% Nusieve GTG agarose gel in order to purify the products for subcloning. Lane 1: PCR with M348 and UAP. Lane 2: PCR with M3101 and UAP. Lane 3 DNA marker III. 114 region with (L-L-L-L-L-A), and a more polar C-terminal region. When A single transcript of approximately 4.2 kb was observed in both normal and affected tissues as well as in both caprine and bovine tissues (Fig. 3.10). The amount of transcript in affected tissues was significantly decreased compared to their normal counterparts. The tissue distribution of B-mannosidase transcript seemed to be consistent with the distribution of enzyme activity of B- mannosidase (Lovell et a1., 1994), i.e, Thyroid > kidney, liver > spleen, brain. W A PCR product of 544 bp generated from clone #46M34 using M366 and M35 (Table 3.4) was labeled and hybridized with genomic DNAs from different species including human, cattle, goat, mouse, rat, and Chinese hamster (Fig. 3.11). Under high stringency wash (Fig. 3.11 Panel A), i.e. in 0.2 x SSC/0.1% SDS at 65°C, EcoRI digested DNA revealed a single band of 7 kb in goat, a band of 3.2 kb in human, and two bands of 5 kb and 7.5 kb in bovine DNA. With PstI digestion, there were two bands of 3.7 and 5.5 kb in goat, one band of 1.7 kb in human and five bands of 1.1, 1.8. 2.7, 3.1, and 8 kb in bovine samples. With XbaI digestion, two 115 bands of 2.8 and 3.4 kb in goat, one band of 1.9 kb in human and three bands of 3, 3.4, and over 10 kb in bovine samples were observed. No bands were observed in DNA from rat, mouse, and Chinese hamster. However, when the filter was washed at 45°C in a solution containing 1 x SSC/0.1% SDS, a smear background in human DNA was found and a new band was appeared in caprine and bovine DNA (Fig. 3.11 Panel 8). Under the low stringency wash, some faint bands could be seen in lanes containing DNAs of rat, mouse, and hamster. Southern hybridization of PstI cleaved DNAs from a panel of 25 human/rodent hybrids showed a 1.7 kb-band in the hybrid NA10115 (Fig. 3.12, lane 4). Its human origin was demonstrated by the observation of a band with the same size as in the control human DNA. Ninety-seven percent of cells from the hybrid NA10115 contain chromosome 4. Two bands of larger size were also found in several other hybrids (Fig. 3.12). The human control showed a smear background too. Similar results were produced with different bovine 3- mannosidase cDNA probes (data not shown). 3.2.4 Discussion In previous studies (Sopher, 1992a), the bovine B- mannosidase peptide of 100 kDa was deglycosylated and gel purified by SDS-PAGE. The peptide was subjected to CNBr digestion and sequencing. Three peptide sequences (peptide 1, 2, and 3) were obtained (Sopher, 1992a). Only peptide 1 116 Fig. 3.10 Northern hybridization analysis of normal tissues and affected animals. Panel A: Poly A+ RNA samples isolated from various bovine and caprine tissues were hybridized to a cDNA probe generated by PCR of clone #46M34 using primers M366 and M35 and a EcoRI fragment of clone #17M348. The blot was hybridized for two days and washed finally in 0.5 x SSC/O.1% SDS at 4Tb for 30 min. The film was exposed for 10 days at -80%:. Lane 1 to 9: affected bovine thyroid ; normal bovine thyroid; affected bovine kidney; normal bovine kidney; normal goat brain; normal goat spleen; normal goat liver; normal goat kidney; affected goat kidney. Panel B: The blot was rehybridized to a rat GAPDH cDNA probe after removal of B-mannosidase probe. The film was exposed for 1 day at -80°C 117 Fig. 3.11 Southern hybridization of genomic DNA from ‘various species. Panel A: Genomic DNA samples from hamster (lane 1, 7, and 13), mouse (lane 2, 8, and 14), rat (lane 3, 9, and 15), goat (lane 4, 10, and 16), cattle (lane 5, 11, and 17), and human (lane 6, 12, and 18) were digested with .EcoRI (Lane 1-6), PstI (lane 7-12), and XbaI (lane 13-18). The blot was hybridized with a cDNA probe generated by PCR of #46M34 using M366 and M35 and washed at high stringency condition (0.2 x SSC/0.l% SDS at 65°C for 1 hour) and exposed for 11 days at -80TL Panel B: The same blot was stripped, rehybridized, and washed at low stringency condition (1 x SSC/0.1% SDS at 42°C for 1 hour) and exposed for 10 days at -80°C. 118 78 9 101112 1314151761718 A . 119 LligiiiiifiiLLi I4 151 1 l8 :9 2 122 x-v m'g e —————————__ -. o —— g u _ ‘IT‘ 9 v i I? - g 2 "' e X h 0 ‘ . r . -. .. .i’ Fig. 3.12 Chromosome localization of fl-mannosidase cDNA. Approximately 15 pg of PstI digested genomic DNA from 24 human/rodent somatic cell hybrids were hybridized with a cDNA probe generated by PCR of plasmid DNA of clone #46M34 using primers M366 and M35. The hybridized blot was washed in 1 x SSC/O.1% SDS for 1 hour at 4Tb and exposed at -8¢T for 10 days. The number on top represents a human chromosome retained in that somatic cell line. H: human control DNA. M: mouse control DNA. C: Chinese hamster control DNA. S: DNA molecular marker III. 120 yielded a complete sequence with six amino acid residues and the other two produced either mixed assignments or incomplete sequence. The sequence of peptide 1 was too degenerate to design a mixed oligonucleotide probe suitable for screening. More peptide sequences were therefore required. The protein used for peptide sequencing was highly purified and contained three bands (84, 100, and 110 kDa). All three peptides were B-mannosidase protein (Sopher et a1., 1993) with the 100 kDa as a major peptide. The 84 kDa was thought to be a proteolytic product from the larger peptides and little or no 80 kDa protein was present in this preparation as judged by Coomassie blue staining and Western analysis. Previous studies by Sopher et a1. (Sopher, 1992a) indicated that the N-terminal of B-mannosidase protein was blocked. In addition, trypsin cleaved peptides appeared to be relatively insoluble. Therefore, internal peptides created by combined CNBr and trypsin digestion were sequenced. The sequence of peptide 142r12 was found to match the sequence of peptide 1 but with two additional residues at its C-terminus. Peptide 151r72 appeared to match the mixed sequence of peptide 2 with differences in the last two residues. The incomplete sequence from peptide 3 appeared to overlap peptides 103 and 218r24. Searches for all the B-mannosidase peptide sequences obtained revealed no significant homologies with existing proteins in the GenBank database. 121 These results indicated that these peptide sequences were derived from the bovine B-mannosidase protein. Knowing the B-mannosidase peptide sequences made it possible to clone the B-mannosidase cDNA. Six independent but incomplete cDNA clones were identified and confirmed by screening a total of 5 x 10‘ plaque-forming units from the bovine thyroid cDNA library. Their relationship is shown in Fig. 3.4. The 3' non-coding region of clone r8-1 may represent the true sequence for B-mannosidase cDNA since its sequence was found in clones 46M34 (only missing the final 20 bp) and r2-1a (except for the final 106 bp). The final parts of 3’ non-coding regions of clone #47M34 (approximately 450 bp) and r2-1a (approximately 1 kb) were not found in other clones and were most likely cloning artifacts. Analysis of genomic DNA, or Northern analysis, or genomic cloning will solve this problem. These clones comprised approximately 70% sequence of the full-length cDNA (4.2 kb). With the addition of the 5’ RACE system, cDNA sequence information close to a full-length of fl-mannosidase was obtained. Three pieces of evidence support the authenticity of the cDNA for B-mannosidase. First, the deduced amino acid sequence from the nucleotide sequence of this cDNA is colinear with that of all ten B-mannosidase peptides (a total of 105 residues) determined by direct amino acid sequencing. Second, this cDNA is located on human 122 chromosome 4, which is in agreement with that of previous report (Fisher et a1., 1987). Third, the RNA transcript of this cDNA in affected B-mannosidosis animals was much lower than in normal animals. The cDNA contains 3852 base pairs. There is a 74 bp 5' non-coding region, followed by a 2637-bp coding region encoding 879 amino acids, then a 1141-hp of 3' non-coding region and finally a l3-bp poly (A) tail. The first in- frame initiation codon is followed by 17 amino acids containing the characteristic features of a signal peptide sequence, i.e. a positively charged amino acid (lysine) within the first 5 amino acids, a hydrophobic core (L-L-L-L- L-A), and a more polar C-terminal region. Since the B-mannosidase protein was N-terminal blocked as suggested by previous studies (Sopher, 1992a), the precise cleavage site of the peptide sequence is unknown. Based on the (-3, -1) rule (von Heijne, 1986; 1990) we predict that the signal peptide is cleaved between residues 17 (A) and 18 (T). Besides the signal peptide sequence, several hydrophobic regions (e.g. residues 96 to 114 and 406 to 422) are predicted according to Kyte and Doolitte (1982) (Fig. 3.6), however, none of them is likely to be a membrane spanning peptide. The hydropathy profile of the B- mannosidase polypeptide shows dispersed hydrophobic or hydrophilic regions except around region 490 to 640 which is 123 mainly hydrophilic. Two possible poly (A) signal sequences (AATATA and ATTATA) were found at 15 bp and 32 bp before the poly (A) tail. None of them is a consensus sequence. Various non- consensus poly (A) signal sequences have been reported in several lysosomal enzymes (Stoltzfus et a1., 1992; Pohlman et a1., 1988; Stein et a1., 1989; Proia et a1., 1986). The deduced peptide sequence from the cDNA matches with all peptide sequences including those containing incomplete or mixed sequences. Although there are four discrepancies between the microsequenced amino acid sequences from CNBr/tryptic peptides and those predicted from the cDNA, two are at residues with uncertainty and thus are possibly due to peptide sequence artifacts. Other two may reflect natural polymorphisms. Previous studies (Sopher et a1., 1993) demonstrated that the size of bovine fl-mannosidase was decreased to 86 and 91 kDa from 100 and 110 kDa, respectively, after deglycosylation with N-glycosidase F. This suggested that seven to nine complex type oligosaccharides may be present. There are six potential glycosylation sites in the cDNA (Fig. 3.5). We predict therefore that all six glycosylation sites may be occupied. The overestimation of glycosylation sites by the deglycosylation study might be due to inaccurate estimation by SDS-PAGE since the presence of carbohydrate chains was found to shift the protein migration (Mahuran et a1., 1988). 124 The 2586 bp coding region (after removal of 17 amino acid residues of the signal peptide) encodes 862 amino acids. This would give a predicted molecular mass of approximately 103 kDa. Therefore, an additional 12 to 17 kDa peptide is presumably cleaved from the B-mannosidase precursor protein as well as the signal peptide sequences. The majority of lysosomal enzymes have been demonstrated or predicted to be involved in proteolytic processing including trimming the N-terminal or C-terminal sequences, or cleaving internal peptides (Gottschalk et a1., 1989; Mahuran et a1., 1988; Erickson and Blobel, 1983; Yamamoto et a1., 1990; Quinn et a1., 1987; Hoefsloot et a1., 1988). So far, arylsulfatase A is the only lysosomal enzyme whose maturation is restricted to the cleavage of its signal peptide sequence (Stein et a1., 1989). A peptide (171) sequence was found to be located next to the stop codon in bovine B-mannosidase cDNA. Therefore, it is likely that the proteolytic processing of bovine fi-mannosidase does not involve the C-terminal cleavage. The expression of the B- mannosidase cDNA should determine the biosynthesis and processing of B-mannosidase and the relationship between the three B-mannosidase peptides. A single transcript species of approximately 4.2 kb was revealed in both normal and B-mannosidosis animals and in both bovine and caprine tissues. There is a slight size difference between the constructed cDNA (3845 bp + 200 bp 125 poly (A) - 4045) of B-mannosidase and the RNA transcript (4.2 kb), which probably reflects some missing 5' non-coding region. There was no size difference between the normal mRNA and B-mannosidosis mRNA. However, the amount of messenger RNA was much lower in affected animals compared to normal after standardizing the RNA loading with GAPDH. The observation of normal size generated from B-mannosidosis animals implies that the B-mannosidosis in ruminants is most likely to be caused by point mutations or small deletions or insertions (1-10 bp). The low yield of transcript observed in affected ruminants indicated that there might be a point mutation producing a premature stop codon (Mahuran, 1991; Zhang, 1994, and Cheng and Maquat, 1993) or mutations in the promoter region affecting the transcription initiation. Southern analysis with several restriction enzyme digestions revealed no gross gene rearrangements in affected and carrier B-mannosidosis animals (data not shown). The same sized RNA transcript was demonstrated in both caprine and bovine tissues. These results were consistent with a previous study which demonstrated that cattle and goats had B-mannosidase peptides of the same size after deglycosylation (Sopher et a1., 1992; 1993). This laboratory has already documented that goats and cattle affected with B-mannosidosis have very similar phenotypes including clinical manifestations, pathological defects, physiological dysfunction, and biochemical perturbation 126 (Jones and Abbitt, 1993; Jones et a1., 1992; Lovell et a1., 1991; Patterson et a1., 1991). Hybridization of restriction enzyme digested genomic DNA with a cDNA fragment of bovine B-mannosidase revealed 2- 6 bands of approximately 1 to 10 kb in the bovine species, two bands of approximately 2.8 to 7 kb in the caprine species, and a single band in humans. Under the conditions of a low stringency wash, a new band in caprine DNA and bovine DNA and a smear background in human appeared. A similar pattern was observed upon hybridization of genomic DNA from a panel containing 24 human/rodent cell hybrids with the same bovine cDNA probe and with a different 6- mannosidase cDNA probe. Similar complex results of genomic Southern analysis were observed in studies of the B-subunit of human fi-hexosaminidase (O'Dowd et a1., 1985). These results may reflect the presence of closely related gene sequences, e.g. gene families or pseudogenes. However, this speculation remains to be proven by genomic cloning studies. The reason for this smear is unclear now. A 1.75 kb fragment was located only on human chromosome 4. The 1.75 kb was the only band presented under the condition of a high stringency wash. Our result supported the previous chromosome mapping (Fisher et a1., 1987; Lundin, 1987). The two larger bands of approximately 4 and 20 kb that appeared under low stringency washing were seen in several cell hybrids. The incomplete restriction enzyme digestion might 127 account for this result. No significant sequence homologies between the fi- mannosidase and other lysosomal enzymes were found by searching against GenBank. However, a striking homology between a human expressed sequence tag (EST01397) to unknown gene (Adams et a1., 1992) was observed. There was 80.6% identity in a 454 bp overlap. High homology at amino acid level was observed mostly in the central region of the human cDNA. However, the open reading frame in the human sequence is shifted by adding a nucleotide at 284 bp and 305 bp. We believe that the human tag sequence actually represents a partial cDNA sequence of human B-mannosidase. The reading frame shift in the human expressed sequence tag is most likely due to sequence errors since it occurs in regions containing G stretches. B-Mannosidase had not previously been cloned from any species. The availability of the cDNA encoding bovine B- mannosidase enables us to isolate B-mannosidase cDNA from other species including human and goat. The expression of the cDNA allows studies of fl-mannosidase processing and transport, as well as possible association with other proteins in the lysosome. The availability of the B- mannosidase cDNA should also permit us to characterize the gene structure and gene regulation. In addition, the cloning of B-mannosidase cDNA will facilitate the identification of molecular lesions underlying B- 128 mannosidosis in humans, goats, and cattle. Finally, it opens the door to gene therapy of several early onset neurodegenerative disorders. 3.2.5 Summary Approximately three dozen positive clones were identified by screening a bovine thyroid cDNA library with several degenerate oligonucleotide probes. Among them, two independent clones #46M34 and #47M34 (#43M34) showed cross- hybridization with other oligonucleotide probes. Rescreening the library with the whole insert of clone #47M34 generated three additional clones r2-1a, r8-1, and r20-2. Except for clone r2-1a, the other four clones started at the same 5’ end with a cleaved EcoRI site. Sequencing and PCR analysis demonstrated that there was sequence homology between these clones. Clones #46M34, #47M34, r20-2, and r2-1a have an incomplete 3’ region. However, clone I47M34 contained approximately 450 bp of different sequence at its 3' end, while clone r2-1a possesses additional sequence in both ends which appeared to be gene cloning artifacts. Besides clones #46M34 and #47M34, clone #17M348 was also identified from the original screening but showed no cross-hybridization with other oligonucleotide probes. This clone was proved later to be B—mannosidase cDNA and was located adjacent to the 5' ends of other clones. The remaining 5' region of the bovine B- 129 mannosidase cDNA was cloned by using 5' RACE technique. The whole cDNA contains 3852 base pairs, which includes a 74-bp 5' non-coding region, a 2637-bp coding region encoding 879 amino acids, a 1141-bp 3' non-coding region and a 13-bp poly (A) tail. A 17-residue sequence after the predicted initiation codon contains the characteristics of a signal peptide sequence. Six possible glycosylation sites were predicted. All ten peptide sequences determined by amino acid sequencing were found in the predicted amino acid sequence from the bovine cDNA, comprising a total of 105 amino acids. A few mismatches were present. A striking sequence similarity was observed between the B-mannosidase cDNA and a human expression tag sequence to an unknown gene. Northern blot analysis demonstrated a single transcript of 4.2 kb in bovine and caprine tissues. The size of the transcript in the affected animal did not change, but the yield was reduced. Southern blot analysis indicated that there were no gross gene rearrangements in animals with B- mannosidosis. The B-mannosidase gene was mapped on human chromosome 4 by Southern hybridization of DNAs from 24 rodent/human somatic cell hybrids. 130 3.4 BEYOND THE CLONING OE fl-NANNOBIDABE CDNL The availability of B-mannosidase cDNA enabled immediate initiation of several studies on which there has already been significant progress made in this laboratory: (1) mutation analysis; (2) fluorescence in situ hybridization (FISH) analysis; (3) Cloning and sequencing analysis of the normal caprine B-mannosidase cDNA; and (4) isolation of human B-mannosidase cDNA (a separate project undertaken by a colleague). For mutation analysis, cDNA was prepared by reverse transcription of affected bovine or caprine total RNA. PCR was performed using internal primers designed from the cloned bovine B-mannosidase cDNA. Overlapping PCR products were generated to cover the whole encoding region and directly sequenced. Eleven overlapping PCR fragments covering the whole encoding region and the partial 3' non-coding region of a bovine affected RNA were analyzed. No mutation, except for a single bp change at 2573 (g/a), was found. This g/a change introduced a stop codon 22 residues before the normal stop codon (Fig. 3.13). Preliminary PCR analysis of genomic DNAs indicated that affected B-mannosidase calves were homozygous for this nonsense mutation and B-mannosidase carriers were heterozygous, which was consistent with the autosomal recessive inheritance pattern for B-mannosidosis. Samples from more Salers cattle with B-mannosidosis will be 131 analyzed. The identification of this mutation in samples from B-mannosidosis calves further supports the conclusion that the isolated cDNA encodes B-mannosidase. Mutation analysis of affected goats is still underway. Approximately 2 kb cDNAs from both normal and affected goats were already amplified and sequenced. A missense mutation (A/D) was observed (Fig. 3.14), which introduced an Alfl III restriction site at this position. PCR analysis of genomic DNA will be performed to verify this A/D change. The partial normal cDNA for caprine B-mannosidase showed a stop codon at the same position as bovine cDNA and 95% sequence homologies with the bovine counterpart at both DNA and amino acid levels, reflecting the close evolutionary relationship between the two species. The FISH technique was developed more than a decade ago (Rudkin and Stollar, 1977). Its applications include prenatal diagnosis, tumor biology, and gene mapping (Poggensee and Lucas, 1992). A FISH analysis system was used to verify the B-mannosidase gene mapping determined by Southern analysis and to generate a regional assignment. The preliminary studies using a random primed labeled B- mannosidase cDNA probe produced a high background. A probe labeled by the nick translation method would be more s98uitable due to its small size. Hybridization and washing 7conditions should be analyzed further. Human B-mannosidase clones would be better probes for use in FISH analysis 132 OFTTTACCCT1EOGGAAA A TTTTMCCTTGAAAG‘ . ”fie-— M kA A: n.t ttttacccttggaaa n.t ttttacccttgaaaa a.a F Y P W K a.a F Y P * A Normal sequence Mutant sequence Fig. 3.13 A nonsense mutation in fl-mannosidosis calves. PCR products generated from normal and affected animals were directly sequenced by the dye terminator method. n.t, nucleotide; a.a amino acid; *, stop codon. The single base change is marked by an arrow. 133 — Bovine B-mannosidase cDNA gnmj96. fragment (~900 bp) -------------- gamj96. consensus A (“900 bp) gnmj89. fragment (~800 bp) ----- gamj122. consensus (~450 bp) -——————gnmj94.con (~soo bP) Fig. 3. 14 Summary of PCR analyses of cDNAs from normal and affected fl-mannosidosis goats. Dash lines represent sequences from an affected goat. Lines represent normal caprine sequences obtained. Solid bar represents the coding region of bovine B-mannosidase cDNA. All caprine sequences were obtained by PCR amplifications of normal or affected caprine cDNAs using bovine primers derived from the cloned bovine B-mannosidase cDNA. A possible missense mutation (A/D change) is marked with A. 134 because high stringency conditions can be used. There is a significant contrast in the clinical manifestations and biochemical perturbations between human and ruminant fi-mannosidoses. Isolation and characterization of human B-mannosidase cDNA will help define the genetic basis of these differences. The successful isolation and characterization of bovine B-mannosidase cDNA enabled us to pursue cloning of the human counterpart. Although screening a human cDNA library using bovine B-mannosidase cDNA probes did not produce positive clones, an alternative screening approach using a PCR product generated by a bovine B- mannosidase internal primer and a human primer from the human tag sequence has identified several putative clones. Further studies are underway. Besides the three studies described above, isolation of a genomic clone encoding the B-mannosidase gene and expression of the bovine cDNA in COS cells can be done because of the successful cloning and sequencing of B- mannosidase cDNA. However, a full-length construct has to be made before initiating in vitro expression. Cloning of a genomic DNA for B-mannosidase will provide the information regarding the structural organization, regulation, and evolution of the B-mannosidase gene. 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