MSU LIBRARIES .—c_. RETURNING MATERIALS: P1ace in book drop to remove this checkout from your record. FINES wi11 be charged if book is returned after the date stamped below. THE ISOLATION AND CHARACTERIZATION OF ERYTHRDID-EXPRESSED CLONES FROM A CHICKEN RETICULOCYTE CDNA LIBRARY BY Mark J. Federspiei A DISSERTATION Submitted to Michigan State University in partiaI fquiITment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1987 ABSTRACT THE ISOLATION AND CHARACTERIZATION OF ERYTHROID-EXPRESSED CLONES FROM A CHICKEN RETICULOCYTE cDNA LIBRARY BY Mark J. Federspiel In an effort to expand the set of isolated erythroid-expressed genes, erythroid-specific cDNA clones from a chicken reticulocyte cDNA library were isolated and several identified. Further study of the expression of this set of genes might make possible the identification of new tissue-specific control elements and/or other important regulatory DNA sequences. A chicken reticulocyte cDNA library was constructed in xgth after optimizing the efficiency of each enzymatic step in the cDNA cloning protocol. The library was screened with a cDNA probe enriched for non-globin, reticulocyte-expressed messages. A hybridization-selection procedure was employed to subtract globin and erythroblast sequences from 32 P-labelled single strand reticulocyte cDNA. The isolated clones were characterized by restriction enzyme mapping, Northern and Southern blot analysis, and nucleotide sequencing. A carbonic anhydrase II clone, which is maximally expressed in red blood cells, was identified from restriction maps and comparison of its nucleotide sequence, to that of a previously isolated clone. Two clones, coding for ferritin heavy chain and ubiquitin, were identified by comparing putative amino acid sequences, deduced from the clones' nucleotide sequences, to a protein database. Ferritin heavy chain is an iron storage protein found in blood, liver, spleen and bone marrow. Ubiquitin is a highly conserved 76 amino acid protein found in all tissues studied, and it has been implicated in a variety of cellular functions. A Charon 4A chicken genomic library was screened with the ubiquitin clone. The genomic organization of the two ubiquitin loci was deduced by analyzing the isolated Charon 4A clones by restriction enzyme analysis, ubiquitin fragment hybridization, and nucleotide sequencing. Our results are compared to recently reported studies of the chicken ubiquitin loci. To Mom and Dad iv ACKNOWLEDGEMENTS I foremost thank Dr. Jerry Dodgson for his patient scientific advice and long financial support. I thank Dr. Edward Fritsch for the scientific experiences of the Cold Spring Harbor Cloning Course and his lab. Finally, I also acknowledge the scientific influences and support provided by Dr. Hsing Jien Kung and Dr. Wynne Lewis. TABLE OF CONTENTS LIST OF TABLES ........................... ix LIST OF FIGURES ........................... x INTRODUCTION ............................ 1 References ......... . ................. 13 CHAPTER I. RETICULOCYTE cDNA LIBRARY CONSTRUCTIONS ......... 16 Experimental Procedures .................... 21 Materials ......................... 21 Reticulocyte RNA Isolation ................ 21 Poly(A)+RNA Separation .................. 22 cDNA Synthesis ...................... 23 First Strand Synthesis ................ 23 Second Strand Synthesis ............... 24 SI Nuclease Digestion ................ 24 pBR322 cDNA Library Cosntruction ............. 24 Dligodeoxycytidine Addition ............. 24 Annealing and Transformation Reactions ........ 25 Screening the cDNA Library .............. 25 Agth cDNA Library Construction .............. 26 EcoRI Linker Addition ................ 26 Vector:cDNA Ligation and In Vitro Packaging ..... 26 Amplification .................... 27 Results and Discussion ..................... 28 Isolation of Reticulocyte Poly(A)+ Cytoplasmic RNA . . . . 28 cDNA Synthesis ...................... 28 vi cDNA Library Constructions ................ 33 References ........................ 42 CHAPTER II. THE ISOLATION AND CHARACTERIZATION OF ERYTHRDID-EXPRESSED cDNA CLONES ................... 44 Experimental Procedures ................... 50 Materials ......................... 50 RNA Isolation ....................... 50 Reticulocyte-Enriched Probe ................ 51 cDNA Synthesis .................... 51 Globin Subtraction .................. 52 Erythroblast Subtraction ............... 53 Screening the xgth Reticulocyte cDNA Library . . . . 53 Clone Characterization .................. 54 Results and Discussion ..................... 56 Reticulocyte-Enriched Probe ................ 56 Globin Subtraction .................. 57 Erythroblast Subtraction ............... 59 Screening the Agth Reticulocyte cDNA Library . . . . 60 Clone Characterization .................. 64 References ........................... 81 CHAPTER III. THE GENOMIC ORGANIZATION OF CHICKEN UBIQUITIN ..... 83 Experimental Procedures .................... 88 Genomic Library Screening ................. 88 Results and Discussion ..................... 91 References ........................... 104 vii APPENDIX I. ISOLATION OF THE CHICKEN CARBONIC ANYHDRASE II GENE . .106 APPENDIX II. ISOLATION OF RECOMBINANT cDNAS ENCODING CHICKEN ERYTHROID G-AMINOLEVULINATE SYNTHASE ................ 109 viii INTRODUCTION CHAPTER I TABLE 1 1 LIST OF TABLES PAGE Chicken globin genes expressed in primitive and definitive erythroid cellsa ......... 6 Distribution of erythroid cell types of various temperature sensitive avian erythroblastosis (tsAEV) strains and cell lines grown at 36° and 42°a’b .................... 6 A summary of the cDNA synthesis protocol efficiencies ................... 32 Agth vector characteristics and ligation efficiencies ................... 37 ix INTRODUCTION CHAPTER I CHAPTER II FIGURE 1 LIST OF FIGURES PAGE A schematic representation of the chicken erythroid differentiation pathway ...... 4 The effect of avian erythroblastosis virus (AEV) on chicken erythropoiesis. . . . 8 Overall scheme for the isolation of chicken erythroid-specific genes ...... 11 A schematic of the overall cDNA synthesis protocol ...... . ...... 17 A schematic of cDNA library construction by homopolymer tailing . . . ........ 34 A schematic of cDNA library construction by linker addition ............. 37 Overall scheme for the isolation of chicken erythroid-specific genes ...... 45 A schematic of the preparation of a reticulocyte-enriched cDNA probe ...... 47 Restriction enzyme maps of the SP6-globin clones ........ . .......... 58 Restriction enzyme maps of chicken aD- globin and carbonic anhydrase 11 cDNA clones . . . . . . . . . . . . . ...... 61 Northern blot analysis of clones 11 and 59 (A), 37 (B), CAII-1.2 (C), and 22 (1350 bp) (D) ................ 63 X CHAPTER III 10 11 12 13 14 The nucleotide sequence of clone 11 ..... 66 The nucleotide sequence of clone 59 ..... 68 Restriction enzyme maps of clones 4 (A), 136 (B), 104 (C), and 200 (D) ........ 70 Northern blot analysis of clones 4 (A), 104 (B), and 200 (C) ............ 71 Chicken genomic Southerns of clones 4 (A), 136 (B), 104 (C), and 200 (D) ........ 72 Nucleotide sequence of clone 4 (unidentified) ............... 74 Nucleotide sequence of clone 136 (unidentified) ............... 75 Nucleotide and amino acid sequences of clone 104 .................. 77 A comparison of the amino acid sequences of clone 104 and human ferritin heavy chain .................... 78 Nucleotide and amino acid sequences of clone 200 .................. 89 A comparison of the nucleotide sequences of clone 200's ubiquitin coding repeats. . .90 A comparison of the nucleotide sequences of two ubiquitin cDNA clones ........ 92 A comparison of the nucleotide sequences of clone 200 and the ubiquitin I genomic locus sequenced by Bond and Schlesinger (15) .................... 94 xi Restriction enzyme maps of chicken genomic clones containing ubiquitin sequences. . . .96 A comparison of the ubiquitin containing restriction fragments of clones 2 (A), 5 (B), and 6 (C) .............. 98 Differential hybridization of chicken genomic clones containing the two ubiquitin genomic loci .......... 100 Southern blot analysis of the chicken genomic ubiquitin regions ......... 102 xii INTRODUCTION The basic structure of a typical eucaryotic gene has been elucidated by analyzing genes with a wide variety of differential and developmental fates. Many of these genes have been isolated by taking advantage of the fact that they are transcribed to high levels during some stage of development (1). For example, the chicken globin genes were initially isolated using a cDNA probe from reticulocytes, a late cell type in the erythroid differentiation pathway in which globin sequences constitute 90% of the total cellular poly(A)+ RNA (2-4). The study of various gene families has elucidated precise programs of specific expression of certain members of such families during tissue differentiation and/or animal development. Chicken globin gene expression begins specifically at the erythroblast stage during erythropoiesis. and different sets of globin genes, embryonic and adult, are expressed at different times in chicken development (5-7). DNA sequence elements which appear to regulate transcription of adjacent genes in gig have been identified through the analysis of the expression of mutant genes, either those identified in vivo or those constructed in vitro (8-13). Although the expression of several eucaryotic genes has been examined in detail, the mechanisms by which a differentiating cell concomitantly regulates as many as a hundred or more genes remain obscure. Generally, only a few of the genes whose expression is 1 regulated in any given pathway of cellular differentiation have been isolated and studied in detail. The aim of this thesis research was the isolation and possible identification of genes which would expand the pool of available genes known to be expressed in chicken erythroid cells. Several such genes have been identified including erythroid-specific and "housekeeping" genes. Housekeeping genes code for proteins constitutively expressed in many or all tissues and include common metabolic enzymes and structural proteins. We primarily hoped to expand the set of isolated genes which are specifically expressed late in erythropoiesis as opposed to housekeeping genes. Further study of the expression of this set of genes might make possible the identification of new tissue-specific control elements and/or other important regulatory DNA sequences. Chicken erythropoiesis offers many advantages as an experimental system in which to study the coordinate expression of tissue-specific genes: 1.) The stages of avian erythroid differentiation and development have been extensively studied. 2.) Erythroid cell lines generated by transformation with avian erythroblastosis virus (AEV) provide a system in which to conduct jn_vitro experiments. These cell lines also enable certain stage-specific cell populations to be isolated in quantities suitable for biochemical analysis. 3.) Chicken reticulocytes have 1/10 the mRNA sequence complexity of comparable mammalian cells (14). 4.) Many proteins found in red cells have been isolated and examined in some detail which may assist in the identification of the function of the clones we intend to isolate. Normal avian erythropoiesis proceeds through a sequence of precursor cell types that are morphologically, biochemically, antigenically and kinetically distinguishable (6, 15-24) (Figure 1). The earliest identifiable erythroid-specific precursor is the colony forming unit-marrow (CFU-M). Nhen CFU-M cells were injected into the bone marrow of irradiated chickens, macroscopic erythrocytic clones (of cells) developed that exhibited the self-renewal properties characteristic of immature stem cells (17, 18). Two distinct classes of later erythroid progenitors were identified according to their respective outcomes when placed in an in 313:9 culture system dependent on anemic chicken serum. These are the burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) (19). Bursts are large aggregates of 3-20 clusters with each cluster containing 8-60 erythroid cells while colonies are a single compact cluster of 8-150 cells. Both cell types can also be distinguished by their differences in antigen expression and growth factor sensitivity. Im antigen expression (see Figure 1), a characteristic antigen of immature erythroid cells only becomes fully detectable by the CFU-E stage (18). Erythroid potentiating activity (EPA), produced in T cells, has been shown to stimulate the growth and cell division of early mouse erythroid precursor cells (25). Both BFU-E and CFU-E cells are stimulated by EPA. However, only CFU-E cells show an absolute requirement for erythropoietin, a growth factor produced in kidney cells, for continued erythroid differentiation to erythroblasts (21, 26). Erythroblasts are a late erythroid cell type characterized by the onset of hemoglobin production, and they are the last erythroid cell stage capable of cell division. Morphological differences are primarily used to distinguish the final erythroid stages - early, middle and late polychromatic erythrocytes, reticulocytes, and mature Pluripotent a... Stem cell-”PF"-M 1” 31+ “- O O I . CF . E 4—— crythropoiettn eon-1:1“ O O 0 1.0 1.- lr- Ir- Erythrofiast—aErythrogyte 0 cell divisions: ‘ :3-14 ' 4 5 Figure 1. A schematic representation of the chicken erythroid differentiation pathway. The figure information is primarily from Gazzolo et al. (20). Abbreviations: colony forming unit-marrow (CFU-M), burst forming unit-erythroid (BFU-E), colony forming unit-erythroid, Im antigen (Im), brain antigen (Br), and hemoglobin (Hb). erythrocytes. In these non-dividing stages, the overall cell size gradually decreases. The cell nucleus also compacts in part due to the replacement of histone H1 with the erythroid-specific histone H5 (27, 28). Avian erythroid cells retain their nuclei, but the mature erythrocyte has no detectable transcriptional or translational activities. Mathematical analysis of kinetic growth experiments estimates 17-19 cell generations are required for erythrocyte differentiation from the CFU-M stage, with the last 4-5 cell divisions occurring at the erythroblast stage (20). Approximately three generations of differentiating erythroid cells arise during avian embryonic development. As development progresses, the site of erythroid cell production moves from the blood islands, to the yolk sac, and finally to the adult erythropoietic organ, the bone marrow (6, 29). At approximately 120 hr of embryo development, a characteristic switch from the production of primitive erythroid cells to definitive erythroid cells occurs. This switch can be observed morphologically and can also be observed by analyzing globin gene expression (5, 6) (Table 1). The embryonic globin genes n, p and a. are only expressed in the primitive or first erythroid generation. The adult globin gene 8, is only expressed in the second and successive generation of definitive erythroid cells. Two alpha genes, aA andch, are expressed in all erythroid generations but their level of expression in primitive cells is only a fraction of that in definitive cells. Several labs have developed continuous erythroid tissue culture cell lines from chicken bone marrow cells transformed with avian erythroblastosis virus (AEV) (19, 20). The target cells of AEV 6 Table 1. Chicken globin genes expressed in primitive and definitive erythroid cellsa. PRIMITIVE DEFINITIVE (Embryonic ; 5 Days) (Adult) b a -type 2 3 -type 2b a -type 2b 3 -type 2b W 70 0 7O 0 A 70 8 100 a A 20 6 30 a D 30 a D 10 a from Brown and Ingram (5). percentage of total globin type. Table 2. Distribution of erythroid cell types of various temperature- sensitive avian erythroblastosis (tsAEV) strains and cell lines grown at 360 and 42° a,b. macarwummuae’Cacrc 38'6 42'C Coils Eb! ER LR Evy Ebl ER Evy meson. c2 100 o o o 99 o 0.5 0.5 macaw»: so i o ' o 93 o 1 o 634 clone 4 I7 3 O 0 IO 23 34 24 834 clone. 33 1.5 0 o ‘2 33 16 9 ms? done 1 100 o. o o 25 is a? 25 mar done a 100 o o o s 29 32 3! ms: clam a 99 1 O o i 3 3: as me: cm. s so 2 o o o o a _ 97 no: you. 32 09 i 0 o _.32 38 22 a no: W41 99 i O O .21 32 29 IO a from Beug et al. (23). b 42° cells were grown_in differentiation medium containg anemic chicken serum. _ A . ‘-- infection are the BFU-E, but the cells continue to differentiate to approximately the CFU-E stage (21) (Figure 2). The AEV transforming genes, v-erb-A and v-grb-B, immortalize the cells and arrest differentiation. AEV temperature-sensitive mutants (tsAEV) have also been isolated and, in cells transformed with these viruses, the differentiation block can be released at the non-permissive temperature (42°) in the presence of anemic chicken serum (23, 24). As seen in Table 2, tsAEV-infected erythroid cells are nearly 100% erythroblasts when grown at the permissive temperature (36°) (23). In this case, the term erythroblast is used to indicate all precursor cells prior to the hemoglobin-producing stage. Different tsAEV viruses show variable abilities in the extent to which the differentiation block is released at 42°. Differentiation following the temperature shift has an absolute requirement for anemic chicken serum, probably as a source of erythropoietin. Three highly enriched cell subpopulations, erythroblasts, reticulocytes and erythrocytes, can be isolated by fractionation of temperature-induced tsAEV cells by Percoll density gradient centrifugation (30). Adkins et al. compared the pattern of protein synthesis within these three subpopulations by two dimensional 35S-methionine labelled cell lysates (31). gel electrophoresis of Twenty-eight major protein synthesis changes were observed as differentiation proceeded. Nine proteins increased in abundance in erythrocytes relative to erythroblasts, fifteen decreased, and four were found gg’ggyg in erythrocytes. Cell populations from several precursor cell stages of chicken red cell differentiation can be isolated. The lg vitro AEV-transformed erythroid cell system offers a relatively homogeneous erythroblast (all Pluripotent Stem Cell—‘cru' M BFU-E I m rum czus "’5 OF -E“ Diff:{°e::iation/ Ery hroblast—eErythrocyte Figure 2. The effect of avian erythroblastosis virus (AEV) on chicken erythropoiesis. The target cells of AEV infection are the BFU-E which continue to differentiate to the CFU-E stage. The CFU-E cells are transformed (**) by the AEV transforming genes v-grb-A and v-grb-B. or mostly CFU-E type) population at the permissive temperature. Non-homogeneous but highly enriched populations of erythroblasts, reticulocytes and erythrocytes, can also be fractionated from temperature-induced cells as mentioned above. A relatively homogeneous jn_yixg population of reticulocytes can be isolated from chickens with anemia induced by phenylhydrazine or bleeding (32). Reticulocytes are postmitotic and differ from most other eucaryotic cells studied in that their transcriptional repertoire is severely restricted. Lasky et al., in a kinetic analysis of the hybridization of reticulocyte poly(A)+RNA to cDNA made to such RNA, estimated that globin mRNA accounts for 90% of the total mRNA pool in these cells (14). Analysis of the hybridization of enriched nonglobin cDNA to total reticulocyte mRNA indicated that approximately 100 nonglobin mRNA species comprise the remaining 10% with the concentration of each RNA species at about 1/200 the globin concentration (33). It is likely, however, that these studies led to a somewhat over-generalized picture of reticulocyte mRNA complexity due to the limitations of solution hybridization of bulk mRNAchNA populations. Because of the ease of isolating erythroid cells and their relatively simple gene expression pattern, many erythroid cell proteins have been characterized. The globin genes were among the first genes cloned due to their abundant expression in reticulocytes. Since then, several genes expressed in erythroid cells, both erythroid-specific and housekeeping genes, have been cloned and characterized. Carbonic anhydrase II (34, 35), histone H5 (27, 28) erythroid antigens and other membrane proteins (18, 36-38), red cell cytoskeletal proteins (e.g., spectrin) (39, 40), and enzymes involved in heme synthesis (41, 42) and 10 other aspects of erythrocyte metabolism are among the known red cell proteins for which the corresponding genes have been isolated, at least in some species. To achieve the goal of isolating other erythroid-specific genes, many of the advantages of chicken erythroid cells just discussed were utilized. The availability of relatively homogeneous cell populations of reticulocytes and erythroblasts allowed a reticulocyte-specific probe to be produced. This was done by employing a hybridization selection procedure refined by Davis et al. (43). Figure 3 shows a schematic of the overall procedure. Two gene sequence populations were subtracted from total reticulocyte cDNA: globin sequences and erythroblast sequences. This resulted in an enrichment of reticulocyte-specific, non-globin gene sequences. This enriched probe was then used to screen a previously-constructed reticulocyte cDNA library. The hybridizing clones were then characterized by recombinant DNA techniques. In this thesis the research will be described in three parts. In Chapter I, the cDNA synthesis and library construction techniques and results will be discussed. Chapter II will describe the production of the erythroid-specific probe, the screening of the reticulocyte cDNA library, and the characterization of the isolated clones. The further characterization of one clone - the analysis of the genome organization of chicken ubiquitin will be described in Chapter III. Two publications are included in appendices. Appendix I contains the publication that describes the isolation and identification of the first chicken carbonic anhydrase 11 cDNA clone, pPE5-0.3 (34). This clone was isolated from the pBR322 reticulocyte cDNA library described 11 . POLY-M «encumm: m1 non meme cmcrcus men SPECIFIC A nvm cDNA cwms , SINGLE 51mm 3 P-cDNA GLOBIN SUBTRACTION ERYTHROBLAST SUBTRACTION RETICULOCYTE cDNA LIBRARY 325,-qu PROBE cunxcutn FOR 'NONGLOBIN" RETICULOCYTE ncssmcs \LSCREEN cDNA LIBRARY cDNA SUBLIBRARY ENRICHED FOR RETICULOCYTE SPECIFIC CLONES t GLOBIN SCREEN RESTRICTION ENZYHE ANALYSIS NONGLOBIN RETICULOCYTE ENRICHED CLONES NORTHERN ANALYSIS SOUTHERN ANALYSIS DNA SEOUENCING ¢IPROTEIN DATA BASE SEARCH CLONE CHARACTERIZATION AND POSSIBLE GENE IDENTIFICATION» Figure 3. Overall scheme for the isolation of chicken erythroid-specific genes. 12 in Chapter 1. Appendix II contains the publication describing the isolation of chicken erythroid a-aminolevulinate synthase cDNA clones (42). Co-workers transferred the reticulocyte cDNA library constructed in Agth (described in Chapter I) into a Afusion/expression vector xgtll. They then screened the resulting expression library to identify clones coding for 6-aminolevulinate synthase, the first enzyme of the heme biosynthesis pathway. 10. 11. 12. 13. 14. 15. 16. 17. 18. 13 REFERENCES Lewin, B. 1980. pp. 771-776. In Gene Expression 2. John Wiley and Sons, New York. Engel, 0.0. and 0.8. Dodgson. 1978. 0. Biol. Chem. 253:8239-8246. Dodgson, 0.8., 0. Strommer and 0.D. Engel. 1979. Cell. 17:879-887. Engel, 0.D. and 0.8. Dodgson. 1980. Proc. Natl. Acad. Sci. U.S.A. 77:2596-2600. Brown, 0.L. and V.M. Ingram. 1974. 0. Biol. Chem. 249:3960-3972. Keane, R.N. and U.K. Abbott. 1980. Develop. Biol. 75:442-453. Harrison, P.R. 1976. Nature. 262:353-356. McKnight, S.L. and R. Kingsbury. 1982. Science. 217:316-324. Davidson, E.H., H.T. Jacobs and R.J. Britten. 1983. Nature. 301:468-470. Treisman, R., M.R. Green and T. Maniatis. 1983. Proc. Natl. Acad. Sci. U.S.A. 80:7428-7432. Meyers, R.M., N. Lumelsky, L.S. Leuman and T. Maniatis. 1985. Nature. 313:495-498. Kollias, G., N. Hrighton, 0. Hurst and F. Grosveld. 1986. Cell. 46:89-94. Choi, O.-R. and 0.D. Engel. 1986. Nature. 323:731-734. Lasky, L., N.D. Nozick and A.0. Tobin. 1978. Develop. Biol. 67:23-39. Cline, M.0. and D.w. Golde. 1979. Nature. 277:177—181. Harrison, P.R. 1976. Nature. 262:353-356. Samarut, 0., 0.P. Jurdic and V. Nigon. 1979. 0. Embryol. Exp. Morph. 50:1-20. Samarut, 0., 0.P. Blanchet and V. Nigon. 1979. Develop. Biol. 72:155-166. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 14 Samarut, 0. and M. Bouabdelli. 1980. 0. Cell. Physio. 105:553-563. Gazzolo, L., 0. Samarut, M. Bouabdelli and 0.P. Blanchet. 1980. Cell. 22:683-691. Samarut, 0. and L. Gazzolo. 1982. Cell. 28:921-929. Graf, T., N. Ade and H. Beug. 1978. Nature. 275:496-501. Beug, H., S. Palmieri, C. Freudenstein, H. Zentgraf and T. Graf. 1982. Cell. 28:907-919. Beug, H., G. Dorderlein, C. Freundenstein and T. Graf. 1982. 0. Cell. Physio. Supplement 1:195-207. Gasson, 0.C. et al. 1985. 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Sanders. 1982. Develop. Biol. 91:389-396. Neise, M.J. and L-N. L. Chan. 1978. J. Biol. Chem. 253:1892-1897. 38. 39. 40. 41. 42. 43. 15 Nelson, C.H., 0.P. Allison, K. Kline and 8.6. Sanders. 1982. Cancer Res. 42:4625-4630. Lazarides, E. and N.0. Nelson. 1982. Cell. 31:505-508. Elgsaeter, A., B.T. Stokke, A. Mikkelsen and D. Branton. 1986. Science. 234:1217-1223. Granick, S. and 8.1. Beale. 1978. Advan. Embryol. 46:33-203. Yamamoto, M., N.S. Yew, M. Federspiel, 0.8. Dodgson, N. Hayashi and 0.0. Engel. 1985. Proc. Natl. Acad. Sci. U.S.A. 82:3702-3706. Davis, M.M., D.I. Cohen, E.A. Nielson, M. Steinmetz, R.E. Paul and L. Hood. 1984. Proc. Natl. Acad. Sci. U.S.A. 81:2194-2198. CHAPTER I RETICULOCYTE cDNA LIBRARY CONSTRUCTIONS In 1981, when this work was begun, cDNA cloning techniques varied widely in efficiency and cDNA cloning vectors were limited mainly to 3-104 cDNA clones per microgram plasmids such as pBR322 (1-3). Only 10 cDNA were commonly obtained upon transformation into E. 2911 but often only nanogram quantities of clonable cDNA are available. Thus, the first priority in cDNA library construction was the establishment of reliable, high yield cDNA synthesis procedures. New cloning vectors (4, 5) and transformation procedures (6) have since been established employing different strategies that greatly increase the yield of cDNA clones. This chapter will describe the cDNA synthesis techniques employed in the construction of two reticulocyte cDNA libraries. The overall procedure developed for cDNA synthesis was a combination of new and published strategies (1-6). The methodology developed is basically similar to that of "Molecular Cloning. A Laboratory Manual" by Maniatis et al. (6). The overall cDNA synthesis scheme is outlined in Figure 1. Briefly, the first cDNA strand is synthesized by reverse transcriptase which initiates from an oligo (dT) primer hybridized to the polyadenylated portion of mRNA. Upon removal of the RNA template, a 3'-hairpin loop is created by the secondary structure of the cDNA strand which is sufficient to prime DNA Polymerase I - Klenow fragment- 16 17 AAHAA POLY(A)+RNA REVERSE TRANSCRIPTASE OLIGOIDTI12_18 ‘..‘..‘..‘.A T,,,, MRNA:CDNA HYBRID DEGRADE RNA 3'-HAIRPIN FORMATION TITII SINGLE STRAND cDNA KLENON FRAGHENT REVERSE TRANSCRIPTASE DOUBLE STRANDED cDNA 81 NUCLEASE <——<———-—c——<——— DOUBLE STRANDED cDNA Figure 1. A schematic of the overall cDNA synthesis protocol. 18 catalyzed second strand synthesis. To maximize the length of the cDNA second strand, reverse transcriptase is also employed in this step. Finally, 51, a single strand-specific nuclease, digests the hairpin loop leaving double stranded cDNA ready for cloning. The experimental details of these steps, which are described in Methods, are the final result of many quantitative and qualitative experiments. Effort was concentrated on testing aspects of several published methods (1—3) to establish routine conditions and procedures for each step, titrating all enzymes involved and improving overall product recovery (described below). The first reticulocyte cDNA library was inserted into pBR322 using a homopolymer tailing procedure (6-8). Terminal deoxynucleotide transferase synthesizes a single stand deoxynucleotide tail from 3'-OH-ends. Synthesized cDNA was tailed with deoxycytidine (dC) by terminal transferase and annealed to deoxyguanine (dG) - tailed vector. The vector was PstI-digested pBR322. Cutting at the PstI site interrupts the ampicillin (amp) resistance gene but leaves the tetracycline (tet) resistance gene intact. Annealed cDNA:vector clones were transformed into E. 2911. The phenotype of transformants in which cDNA was successfully inserted into plasmid DNA should be tetracycline resistant (tetR) and ampicillin sensitive (amps). Problems occurred with screening large numbers of plasmid transformants as well as with clone viability in storage, so the decision was made to construct another cDNA library in a lambda vector. The pBR322 library was used to screen for the chicken carbonic anhydrase II (CAII) gene using a homologous mouse CAII probe (9). This also allowed us to test the 19 reliability of the overall cDNA synthesis procedure and how well the RNA pool was represented. The second reticulocyte cDNA library was constructed in gth using a linker addition procedure (6). Synthetic EcoRI linkers were ligated onto synthesized cDNA creating flanking EcoRI sites. The cDNA could then be ligated into the single EcoRI Site of xgt10 which is within the A repressor (CI) gene. The two pathways of lambda growth, lytic and lysogenic, are controlled by an intricate balance of host and lambda gene products and are employed here as a selection for cDNA transformants (5, 6). Central to this control is the CI gene product which is a repressor of lambda early transcription and consequently blocks late gene expression. High repressor concentrations plus the jag gene product lead to the insertion of the lambda DNA into the genome of the bacterial host - the lysogenic pathway. Low repressor concentrations release the inhibition on the maintenance promotor, Pm, allowing lambda to enter the lytic cycle which results in a many fold replication of lambda DNA and eventual lysis of the host cell. The product of a lysogenic infection is a cloudy plaque because those bacteria in which lambda is integrated into the host genome will be resistant to further infection and produce the turbidity within the cleared area resulting from the lytic cycle infection. A purely lytic infection, though, produces a clear plaque morphology. The small percentage of lytically growing lambda that give rise to the cloudy plaque can be further reduced by infecting an E. £911 gfl (high frequency lysogenization) mutant (5). Deletion of the host Ejlfi gene enhances the stability of several lambda factors resulting in a stimulation of CI synthesis and a high frequency of lysogenization. So 20 on an hi1 host, a CI+ phage, e.g., intact Agth, will give a faint, almost invisible plaque, but a CI' phage will still give a clear plaque since no A repressor is made. The insertion of cDNA into the EcoRI site of Agth interrupts the CI gene resulting in a lytic infection with a clear plaque morphology. By amplifying the primary cDNA-vector ligations on an E. £911 5:15 mutant, the resulting high titer phage stock will be 98-99% CI'. The CI' phage must have resulted from insertion of cDNA (or linker DNA) into the EcoRI site or be naturally occurring CI' mutants. 21 EXPERIMENTAL PROCEDURES Materials Phenylhydrazine was purchased from Aldrich. Heparin, HEPES, and proteinase K were purchased from Sigma. Oligo (dT)-cellulose type 775 and oligo (dT)12_18 were purchased from Collaborative Research. RNasin, terminal deoxynucleotide transferase, and deoxynucleotides were purchased from P.L. Biochemicals. Reverse transcriptase was purchased from Life Sciences. EcoRI - 10 mar synthetic linkers, DNA polymerase I - Klenow fragment, Sl nuclease, T4 DNA ligase, T4 polynucleotide kinase, and all restriction enzymes were purchased from either Bethesda Research Labs, New England Biolabs, or Boehringer Mannheim. [a-32P]dCTP, [3H]dCTP, and PstI-digested, dG-tailed pBR322 were purchased from New England Nuclear. Reticulocyte RNA Isolation The method employed was a modification of the procedure of Longacre and Rutter (10). Hens were injected with 2.5% (w/v) phenylhydrazine, pH 7, on six consecutive days according to the following schedule: 0.7, 0.7, 0.4, 0.4, 0.5, and 0.6 ml. 0n the seventh day, the chickens were bled by heart puncture and the blood added to four volumes NKM solution (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 0.1% heparin). The cells were recovered by centrifugation at 1500 x g removing the supernatant and buffy coat. The cells were then 22 washed three times with cold NKM and recovered as before. Two volumes of lysis buffer (2 mM MgCl 2 mM dithiothreitol, 10 mm Tris-HCl, pH 2. 7.5, and 10 mM iodoacetate) were used to resuspend the final cell pellet and the mixture was incubated at 0° for 30 min. The nuclei and cell debris were then removed by centrifugation at 16,000 x 9, 4°. The supernatant was made to 1.0% SDS, 10 mm EDTA, and 100 ug/ml proteinase K and incubated at 37° for 60 min. One tenth volume of 3 M NaOAc, pH 5.0 was added, mixed, and the solution extracted with one half volume phenol (equilibrated with 0.1 M Tris-HCl pH 7.0) and one half volume chloroform. The phases were separated by centrifugation at 1500 x 9, 25°. The aqueous phase was extracted with one volume chloroform and separated as above. The final aqueous phase was ethanol precipitated with 2.2 volumes ethanol at -20° for 18 hr. The RNA was recovered by centrifugation at 12,000 x g for 20 min at 0°; the pellet dried under vacuum, and resuspended in ddH20. The RNA recovery was determined from A260 measurement (40 ug/0.D.). All steps were done with ribonuclease free technique (6). Poly(A)+RNA Separation All steps were carried out under ribonuclease free conditions. Dligo (dT)-cellulose type 775 was pretreated with 0.1 N NaOH at 25° for 20 min. The cellulose was then washed with water until the pH reached neutral, packed into a suitable column, and washed with 1 volume high salt buffer (500 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.05% SDS, 1 mM EDTA). Total RNA in elution buffer (10 mM Tris-HCl pH 7.5, 0.05% SDS, lmM EDTA), was heated at 55° for 5 min, cooled and made 500 mM in NaCl. The solution was added to the cellulose column, eluted, and the column 23 washed with high salt buffer until the eluate A260 was near 0. The column was then washed with several volumes of low salt buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.05% SDS, 1 mM EDTA). The poly(A)+RNA was then eluted with elution buffer, ethanol precipitated as before, and resuspended in ddH20. cDNA Synthesis First Strand Synthesis. The reaction mixture (100 pl) contained the following: 100 mM Tris-HCl pH 8.3, 140 mM KCl, 10 mM MgCL 100 2. ug/ml oligo(dT)12_18, 30 mM B-mercaptoethanol, 1.1 mM each of dGTP, dCTP, dTTP and dATP, 60-100 Ci [a-3ZPJdCTP, 50 units RNasin, 98 units reverse transcriptase, and 5-10 ug poly(A)+RNA. All first strand solutions, equipment, and techniques were ribonuclease free. All components, except the RNA, were combined and incubated at 25° for 10 min. Synthesis was initiated with the addition of the RNA, incubated at 42° for 2 hr, and terminated with EDTA to 25 mM. An aliquot of the cDNA synthesized, with [a-32P]dCTP incorporated, was quantitated by TCA precipitation (6). The reaction was then extracted with one half volume phenol, one half volume chloroform; and the aqueous phase passed over a one ml Sephadex G-50 column (6). The excluded volume was made 0.16 N in NaOH, incubated at 68° for 10 min, neutralized, ethanol precipitated, and resuspended in 10 mM Tris-HCl pH 7.5. Second Strand Synthesis. The reaction mixture contained the following: 10 mM HEPES pH 6.9, 80 mM KCl, 3 mM MgCl 2.3 mM 2’ dithiothreitol, 1 mM each of dGTP, dCTP, dTTP, and dATP, and 40-50 units of DNA polymerase I - Klenow fragment per microgram of first strand cDNA synthesized. The reaction was incubated at 15° for 15-20 24 hr and terminated with EDTA to 25 mM. The mixture was phenol:choloroform extracted, ethanol precipitated and resuspended in the following: 100 mM Tris pH 8.3, 140 MM KCl, 10 mM MgCl 30 mM 2. B-mercaptoethanol, and 1.1 mM each of dGTP, dCTP, dTTP, and dATP. The Klenow synthesized second strand cDNA was lengthened by incubating the mixture at 42° for 2 hr with 90 units of reverse transcriptase. The terminated reaction was extracted, ethanol precipitated, and the double stranded cDNA resuspended in S1 nuclease buffer (200 mM NaCl, 50 mM NaOAc pH 4.5, and 1 mM ZnSO4). Sl Nuclease Digestion. A set of small scale pilot reactions were carried out to determine the optimal concentration of S1 required to digest only the hairpin loop connecting the two cDNA strands. To 25,000 cpm aliquots of cDNA; 10, 5, 2.5 and 1.25 units of SI nuclease were added. Incubations proceeded at 37° for 30 min and were terminated with EDTA to 25 mM. The digestion products were analyzed on alkaline agarose gels (6), which were 1.4% agarose and run with 30 mM NaOH and 2 mM EDTA as electrophoresis buffer. The full scale S1 digestion was an exact scale up of the optimal $1 pilot digestion conditions. The completed digest was then phenol:chloroform extracted, ethanol precipitated, and the SI-digested double stranded cDNA resuspended in ddHZO. pBR322 cDNA Library Construction Dligodeoxycytidine Addition. The reaction mixture contained 1.4 mM B-mercaptoethanol, 7.5 mM dCTP, 400 mM potassium cacodylate, pH 6.9, 1 mM CoClz, and 500 units/ml terminal deoxynucleotide transferase. The reaction was initiated by the addition of the double stranded cDNA, 25 incubated at 37°, and terminated with EDTA to 25 mM. The optimal incubation time to add 20-25 deoxycytidine residues varied with each enzyme lot and was determined by a pilot time course reaction using [3H]dCTP. Following termination, the reaction was phenol:chloroform extracted and passed over a one ml Sephadex G-50 column previously equilibrated with annealing buffer (100 mM NaCl, 10 mM Tris-CHl pH 7.5, 1 mM EDTA). Annealing and Transformation Reactions. PstI - digested, oligodeoxyguanine-tailed pBR322 was combined with the deoxycytidine- tailed cDNA in a 8:1 molar ratio and diluted with annealing buffer to a final DNA concentration of 0.8 ug/ml. The mixture was incubated at 68° for 10 min, then 42° for 2 hr, cooled to 20°, and transformed into E. £911 strain H8101 by a modification of Hanahan's x1776 procedure (11). The completed transformation reaction was plated on L8 plates with tetracycline (lSlJQ/MI). Screening the cDNA Library. Background ampicillin resistant colonies were screened out by duplicate plating on tetracycline and ampicillin (50 ug/ml) plates. Ampicillin sensitive and tetracycline resistant colonies were screened for carbonic anhydrase II (CAII) by a modification of the method of Grunstein and Hogness (12). In short, colonies were grown on two replica nitrocellulose filters, lysed with alkali, and neutralized. The released DNA was bound to the filters by baking at 80° for 2 hr under vacuum, and probed by hybridization with a 32P-nick translated labelled CAII probe. A 300 bp PstI fragment from the coding region of a mouse CAII cDNA clone obtained from Peter Curtis, was isolated by the method of Girvitz (13) and labelled with 32F by nick translation (6). Hybridization was carried out with 30% 26 formamide at 42° for 30 hr (6). The filters were then washed first with 2 X SSC (0.3 M NaCl, 0.03 M sodium citrate pH 7.0) at 25° then washed with 0.2 X SSC at 55°. The filters were exposed to Kodak X-Omat R film. xgtlo cDNA Library Construction EcoRI Linker Addition. The ends of the synthesized cDNA were repaired by DNA polymerase I - Klenow fragment (in second strand buffer) to create blunt ends prior to linker addition (6). Synthetic EcoRI - 10 mar linkers were phosphorylated by T4 polynucleotide kinase in the following reaction mixture: 1 pg linkers, 66 mM Tris-HCl, pH 7.6, 1 mM ATP, 1 mM spermidine, 10 mM MgCl 15 mM dithiothreitol, 2. 2 ug/ml BSA, and 2 units T4 polynucleotide kinase. The mixture was incubated at 37° for 1 hr, terminated by heating at 68° for 10 min, and then added to the repaired cDNA. Six units of T4 DNA ligase were added and the mixture incubated at 15° for 18 hr. The linkers were then extensively digested with EcoRI and the digest electrophoresed on a 1% agarose gel run in TAE (40 mM Tris-0H, 20 mM acetic acid, 2 mM EDTA). Two size fractions of linker added cDNA, 400 bp-800 bp and 800 bp-3 kb, were isolated by the method of Girvitz (13). VectorchNA Ligation and In Vitro Packaging. A set of trial ligations was used to determine the optimal EcoRI digested Agth (vector):cDNA ratio giving the highest plaque forming units (pfu) per microgram of vector yield. The completed ligations were jg vitro packaged by the method of Hohn (14), and grown on two E. coli strains: C600 and C600 fljl (5). The full scale ligation, with the remainder of the cDNA, was carried out under the determined optimal conditions and 27 1g vitro packaged. A separate library was constructed with each of the isolated cDNA size fractions. Amplification. 380,000 pfu from each library were plated separately, on 18 150 mm L8 plates with C600 3:1 and grown for 10 hr at 37°. The plates were harvested by scraping the nearly confluent plaques into a tube, and adding an equal volume of SM (100 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM M9504, 0.01% gelatin) and 0.01% chloroform. After mixing extensively the agar was pelleted by centrifugation at 4000 x g for 10 min at 4°. This xgtIO-cDNA stock was titered and stored at 4°. 28 RESULTS AND DISCUSSION Isolation of Reticulocyte Poly(A)+ Cyt0plasmic RNA Reticulocytes were isolated from the circulating blood of chickens with induced anemia. The phenylhydrazine treatment schedule of Longacre and Rutter (10) induces the highest anemia with the lowest mortality. The total cytoplasmic RNA yield by this method averaged 60 mg RNA per 100 ml of blood. The RNA yield was quantitated by A260 measurement using 25 A260 0.0. units equalling 1 mg RNA as a conversion. Approximately 2% of the total cyt0plasmic RNA was separated by oligo (dT)-cellulose chromatography as poly(A)+RNA which agrees with the estimate of Lasky et al. (15) for reticulocytes. Agarose gel analysis of the selected poly(A)+RNA showed the predicted 95 globin RNA band along with very minor contamination with 18S ribosomal RNA and transfer RNA (results not shown). Agarose gel 32P-labelled first strand cDNA synthesized from the analysis of poly(A)+RNA showed a smear ranging in size from 200 bases to 3 kb with a 600 bases band corresponding to globin (results not shown). cDNA Synthesis The overall cDNA synthesis scheme is outlined in Figure 1. Through incorporation of 32P into the first strand cDNA, the first and second strand synthesis products as well as the SI nuclease test digestions could be analyzed by autoradiography of alkaline agarose 29 gels. Under the denaturing conditions, the double stranded molecules denature but the first and second strand cDNA strands are connected by the 3'-hairpin loop. Thus the size of the double stranded cDNA population should be approximately twice that of the first strand. After 51 nuclease digestion, however, the two strands are no longer connected, and therefore the size of the labelled cDNA should revert to approximately that of the first strand cDNA. Also, by incorporating 32F into the first strand cDNA, the yield of cDNA synthesized, the efficiency of each enzymatic reaction, and the overall recoveries could be quantitated by TCA precipitation. In reviewing the established cDNA synthesis protocol, several critical areas determined the overall success. Reverse transciptase quality and reaction conditions were shown to be critical for efficient, full length first strand cDNA synthesis. A high molar excess of enzyme to template (20 fold), the optimal pH of 8.3, the optimal monovalent cation concentration (140 mM KCl) for strand elongation, the required divalent cation (10 mM Mg++), and a high concentration of all four deoxynucleotides (1 mM) were shown to dramatically increase first stand synthesis (results not shown). RNA quality was another critical factor. To prevent RNA degradation, ribonuclease free techniques as well as a potent ribonuclease inhibitor, RNasin, were used. Under these conditions approximately 20% of the initial mRNA was copied to generate first strand cDNA. Theoretically, all of the mRNA could template first strand cDNA synthesis by reverse transcriptase. In reality, only 10-30% conversion is typically as reported in the literature (1-6). 30 An absolute requirement for efficient second strand cDNA synthesis was the complete degradation of the RNA template that primed the first strand. Published degradation conditions were either so harsh that the synthesized cDNA and RNA were both degraded, or inadequate, leaving a large proportion of the RNA intact. Incomplete RNA hydrolysis significantly inhibited enzyme initiation and second strand elongation. The conditions developed for treating the completed first strand reaction, 0.16N NaOH at 68° for 10 min, optimally degraded the RNA yet left the cDNA strand intact. Two enzymes were used to synthesize the second cDNA strand primed off of the 3'-hairpin loop formed naturally by the first strand cDNA. Initially, the second strand was synthesized by DNA polymerase I - Klenow fragment. This enzyme's optimal reaction conditions included a pH optimum of 6.9, a 15° incubation temperature to minimize snapback DNA, and a long incubation time (18 hr) to allow the enzyme time to initiate off of the unstable hairpin loop. An additional enzymatic synthesis step was added to further lengthen the second strand and thereby increase the percentage of full length double-stranded cDNA. It has been observed that there are several positions along a DNA single strand, perhaps relating to its intrinsic secondary structure, that result in a pause or st0p in replication (6). It is thought that each enzyme has its own specific types of strong-stops, that are not necessarily recognized by other enzymes. In this procedure, by continuing second strand synthesis with reverse transcriptase, cDNA second strand length increased an average of 10% as estimated from an alkaline agarose gel. The average product recovery, after the total 31 second strand synthesis, was 33% of the 32 P incorporated into the first strand cDNA synthesized. The optimal Sl nuclease conditions must be determined for each preparation of cDNA due to 51's ability to digest even double stranded DNA at high enzyme concentrations. It was also imperative that the full scale 51 reaction, with the remaining double stranded cDNA, be scaled up exactly to the conditions of the appropriate S1 pilot reaction. Approximately 75% of the double stranded cDNA molecules were resistant to SI nuclease digestion. A plateau of 80% resistance has been reported in the literature (2). The recovery of the completed double stranded, Sl-digested cDNA averaged 25% of the original first strand synthesized (Table 1). A crucial factor in the overall success of the cDNA synthesis involved methods of handling the various reactants and reaction mixtures. The frequent extractions, precipitations, buffer changes, and other manipulations of the cDNA initially resulted in substantial product loss. Considerable time, therefore, was spent in minimizing the number of transitional clean-up steps to maximize product recovery without sacrificing enzyme efficiency. Silanized tubes and micropipets substantially increased product yields by reducing adhesion of cDNA and RNA to surfaces. Another valuable technique was the use of Sephadex G-50 (1 ml) spin columns (6) to remove reactants and change buffer systems in a minimum volume. The most severe loss of product occurred during second strand synthesis despite all improvements. The large- reaction volumes needed and two synthesis reactions resulted in an average 63% loss. Table 1 summarizes the enzymatic yields and recovery 32 Table l. A summary of the cDNA synthesis protocol efficiences a. Reaction b DNA Recovery (ug) Overall Reaction Step Recovery (Z) SSc Recovery (Z)e 1.) First Strand --- 2.1 -—- 100 -Processingf 89 1.9 --- 89 2.) Second Strand -K1enow Fragment of 75 1.4 2:8 66 DNA Polymerase I -Reverse Transcriptase ’ 50 0.7 1.4 33 3.) SI Nuclease 85 0.6 1.2 28 data is from one experiment. efficiency of each step (recovered product divided by initial reactant cocentration). only the first cDNA strand was 32P-labelled (SS-single strand). double strand (DS) DNA concentration was estimated by doubling the 88 concentration. recovery relative to the first strand. includes RNA degradation, extraction, Sephadex G-50 exclusion, and ethanol precipitation. 33 efficiencies of a representative cDNA preparation used to construct the Agth reticulocyte library. cDNA Library Constructions One of the main advantages of studying chicken reticulocytes is the relatively small number of expressed genes. Lasky et al. (15) estimate only about 100 mRNA species comprise the nonglobin 10% of the mRNA of reticulocytes. The number of clones required for a cDNA library with a given probability that every such mRNA would be represented can be estimated by the following equation: In (l-P) N: —— 1n (1-n) where N = the number of clones required, P = probability desired (usually 0.99), and n = fractional proportion of the total mRNA population that a Single type of low abundance mRNA represents (6). To construct a cDNA library with a 99% probability that each of the 100 nonglobin mRNA species are represented (P = 0.99, n = 0.001) only 4600 clones are required. The isolation of a sequence present at even a ten fold lower abundance would only increase the number of clones needed to 46,000, a number easily within the efficiency of the described cDNA cloning procedures. The first reticulocyte cDNA library was constructed in a plasmid vector, pBR322, by the homopolymer tailing method (Figure 2). By adding a deoxycytidine single strand "tail" to the synthesized cDNA, and a deoxyguanine "tail" to PstI-digested pBR322 with terminal deoxynucleotide transferase, the two DNA pieces could be annealed. The regenerated plasmid was transformed into E. coli strain H8101. The 34 terminal "micron (G + dGTP cTGC‘ 6:67“ 6:408“ term transferase + dCTP (Cln (Cln l J A annealing transformation cnxfimamc-———————cqamnmuh qumnmmcc———Efiz—bamwcflmn: (— a... ) IF“! A c7 “In—”MTG“ 6 ACGTICln-———c)n G ‘c t recovered frequent ”Rm (CDNAI Figure 2. A schematic of cDNA library construction by homopolymer tailing.This figure is taken, in part, from Maniatis et al. (6). See text for details. 35 terminal transferase reaction, however, was exceedingly sensitive to inhibitors. Tris, EDTA, and ethanol in even trace amounts are all potent inhibitors and repeated extractions and precipitations must be done to remove these common reactants. The terminal transferase reaction employed a large excess of enzyme to ensure that the number of residues added was essentially independent of cDNA concentration. For each lot of enzyme, a pilot reaction, using [3H]dCTP, was done to establish a residue addition time course standard curve. The reaction time was determined that added 20-25 deoxycytidine residues to the cDNA, which in previously published experiments (16) was shown to be optimal for dC-dG annealing as measured by the yield of transformants. The transformation procedure, a modified Hanahan x 1776 procedure (11), 8 routinely produced 107-10 colonies per microgram of intact plasmid. The annealed cDNA:vector mixture, in a 1:8 molar ration, produced 6.7 x 5 10 transformants per microgram vector - an expected 100 fold decrease compared to intact plasmid. Ligation and transformation of the vector 2 alone gave 5 x 10 colonies per microgram vector. 10,692 transformants were screened for background tetR ampS colonies yielding 1296 (12%) plasmids with no inserted sequences. 49% of the 9396 tetR ampS transformants hybridized to a globin probe consisting of the three adult globin clone fragments 32P-labelled by nick translation. Approximately 70% of the resulting 4800 "non-globin“ clones were lost due to the laborious manual screening procedures used. During the long storage periods needed to perform the various screening procedures used, many of the individual colonies (stored on plates) that made up the original library lost viability. This problem might have been obviated by storing mini-glycerol stocks of the isolated 36 individual colonies. The remaining 1300 viable clones were screened for chicken cDNAS homologous to the mouse carbonic anhydrase II gene (CAII) to assess the representation of CAII cDNAS in the library. The probe was a nick-translated, 32 P-labelled 300 bp fragment from the coding portion of a mouse CAII cDNA clone provided by Peter Curtis (Histar Institute). Three positive autoradiographic signals, as well as nine other clones, were examined. The average insert size was 300 bp ranging from 75-500 bp. Nucleotide sequences were determined for two of the CAII-positive clones, with insert sizes of 370 bp and 300 bp. Open reading frames were identified and possible amino acid sequences deduced. The amino acid sequences corresponding to the inserts of both cDNA clones were 60% homologous with the known CA 11 sequences (Appendix 1). Thus, we identified two CAII clones in approximately 780 non-globin reticulocyte cDNA clones screened. (From the results above, we expect 40% of the 1300 clones to be globin clones.) This is in close agreement with the estimate that CAII mRNA comprises approximately 0.1% of the reticulocyte poly(A)+ cellular RNA (see above). Due to the small insert sizes and to viability problems in maintaining large numbers of colonies, the pBR322 library was abandoned. At this time the phage cDNA cloning systems Agth and Agtll became available, and it was felt that this would be a more suitable vector for this project. The second reticulocyte cDNA library was constructed in xgt10 using a linker addition protocol (Figure 3). The gt10 vector offers the advantages of easily screening large numbers of clones, high yield cDNA cloning, and convenient storage at 4° with little loss in viability. Plasmid clone libraries can be stored for long time periods 37 WFMUENT in nu LIGASE EcoRI Links I-) LI 1 I] I _Lr1 TLTfl EcoRI DIGEST AGAROSE GEL FRACTIONATE l l a... a... l 1 dI\\\Y\AX\‘]r—7 + £05 1r" El cos Keno l InmumE “’5 IHI \ \ \ \K \TPI cos Figure 3. A schematic of cDNA library construction by linker addition. See text for details. Methylated EcoRI sites are denoted by (*). Abbreviation: cohesive ends (COS). 38 in bulk (e.g., as a pooled glycerol stock). However, we were afraid that differences in growth rates of different clones would significantly alter the representation of the plasmid library, so we attempted to store it as individual colonies on plates. As described above, this led to seriously reduced clone viability. As shown with the CAII screening of the pBR322 library, non-globin genes were represented but with small insert sizes. The cDNA synthesis procedure was altered slightly to help increase the size of the synthesized cDNA ready for cloning. The extent of the SI digestion was reduced to digest less of the double stranded cDNA. When using a linker addition protocol, one usually methylates the restriction sites of the synthesized cDNA corresponding to the enzyme to be used to cut the linkers. In our case, treating the cDNA with EcoRI methylase would prevent any cDNA EcoRI site from being digested when the linkers were cut to expose the EcoRI ends. Due to problems with the methylation reaction, adequate methylation was never achieved. Because full length cDNA clones were not required for the isolation protocol described in Chapter II, we did not feel that the small number of cDNA clones containing EcoRI sites would cause problems. After EcoRI linkers were added and extensively digested, the cDNA was size fractionated on agarose gels, isolating the 400-800 bp and 800 bp - 3 kb cDNA size ranges. Each size range was inserted into xgt10, creating two separate cDNA libraries. The two critical areas that determined the success of cloning into Agth both deal with clone selection. In Agt10, the cDNA inserts are cloned into the single EcoRI site in the repressor (CI) gene of Agth. Thus phage with inserts are CI' and grow lytically producing clear 39 plaques. Lambda without inserts grow lysogenically, producing cloudy plaques or virtually no plaques at all, if grown on an E. £211 Ejl (high frequency lysogen) mutant. The first critical area is the isolation of a igt10 stock with a very low spontaneous CI" background, i.e., less than 0.25% (17). Upon 19 11339 packaging, the vector DNA stock should give a titer of 108 pfu/pg DNA. Infectivity of the DNA should be reduced at least three logs after EcoRI digestion prior to cloning, and upon ligation of vector DNA clone, one should recover 5-30% of its original infectivity with little or no increase in the CI' background (Table 2). The second critical area is the complete removal of all digested EcoRI linkers. A synthetic linker can interrupt the CI gene coding sequence producing clear plaques. Thus it is imperative to size fractionate an extensively digested cDNA-EcoRI linker ligation to purify the cDNA away from the excess linkers. After testing several phage isolates, a Agt10 vector stock that fulfilled all the requirements listed above was found.- However, upon test ligations of this vector DNA with cDNA the CI" percentage could only be increased two fold over the no cDNA insert control. Normally one would like a 5-10 fold increase (Table 2). Consequently, only one half of the CI' pfu of the constructed libraries are expected to have cDNA inserts. This was judged not to be a major problem since large numbers of cDNA transformants were available compared to the relatively small number of clones needed for a "complete" library. The two libraries constructed were a 400 bp - 800 bp library yielding 2.8 x 106 independent CI" pfu and a 800 bp - 3 kb library yielding 1.7 x 106 CI' pfu. 380,000 CI" pfu from each library were amplified resulting in amplified bulk library stocks for storage of 180 ml at 5.6 x 109 CI- 40 Table 2.1Agt10 vector characteristics and ligation efficiencies. ‘- E_xpected >43th DNA Actual Agtlo Vector Characteristics Characteristics total infectivity 2 of original CI-ZC (per ug vector) infectivity Total 108 pfu/ug vector 6 X 107 pfu 100 0.2 infectivity Infectivity after Greater than 3 logs 1 x 103 pfu 0.0017 -- EcoRI digestion less infectivity No insert EcoRI 5-302 of original 5.1 x 106 pfu 8.5 0.5 th10 ligation infectivity soon AgtIo with 5-3o: of original 1.0 x 10; pfu; 16.7 1.2 insert ligation infectivity 1.0 x 10 pfu 16.7 1.6 . inserts were the 400 - BOObp fraction of reticulocyte cDNA. b inserts were the BOObp - 3th fraction of reticulocyte cDNA. c spontaneous background C - plaques should be less than 0.251 in the vector stock. I 41 pfu/ml (400-800 bp) and 190 ml at 5.3 x 109 CI- pfu/ml (800 bp - 3 kb). The size fractions were chosen to clone most of the globin messages (550-600 bp) in the library with smaller inserts in order to aid in screening for non-globin clones (see Chapter 11). Both reticulocyte cDNA libraries were transferred into the xfusion/expression vector xgt11 by another laboratory as described in Appendix II. They then screened the gt11 cDNA library with a heterologous antibody for o-aminolevulinate (ALA) synthase. Several cDNA clones encoding ALA synthase were isolated and characterized. ALA synthase is the first enzyme in the heme biosynthesis pathway. These results, along with those to be described in Chapter II, demonstrate that a variety of non-globin, reticulocyte messages are represented within these cDNA libraries. 10. 11. 12. 13. 14. 15. 16. 42 REFERENCES Efstratiadis, A. and L. Villa-Komaroff. 1979. pp. 15. lg Genetic Engineering. Vol I. 0.K. Stelow and A. Holla, eds., Plenum Press, New York. Wickens, M.P., G.N. 8uell and R.T. Schimke. 1978. 0. Biol. Chem. 253:2483-2495. Kurtz, D.T. and C.F. Nicodemus. 1981. Gene. 13:145-152. Okayama, H. and P. Berg. 1982. Mol. Cell. Biol. 2:161-170. Huynh, T.V., R.A. Young and R.W. Davis. 1985. pp. 49-78. In DNA Cloning: A Practical Approach. Vol I. D.M. Glover, ed., IRL—Press, Washington, D.C. Maniatis, T., E.F. Fritsch and 0. Sambrook. 1982. In Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Ldboratory, New York. Villa-Komaroff, L., A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S.P. Naker, W.L. Chick and W. Gilbert. 1978. Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731. Peacock, S.L., C.M. McIver and 0.0. Monohan. 1981. Biochem. Biophys. Acta. 655:243. Yoshihara, C.M., M. Federspiel and 0.8. Dodgson. 1983. Annals of the New York Academy of Science - Biology and Chemistry of the Carbonic Anhydrases. 429:332-334. Longacre, 5.5. and W.0. Rutter. 1977. 0. Biol. Chem. 252:2742-2752. Hanahan, D. 1985. pp. 109-136. lg DNA Cloning: A Practical Approach. Vol I. D.M. Glover, ed., IRL Press, Washington, D.C. Grunstein, M. and D. Hogness. 1975. Proc. Natl. Acad. Sci. U.S.A. 72:3961-3964. Girvitz, S.C., S. Bacchetti, A.0. Rainbow and F.L. Graham. 1980. Anal. Biochem. 106:492. Hohn, 8. and K. Murray. 1977. Proc. Natl. Acad. Sci. U.S.A. 74:3259. Lasky, L., N.D. Nozick and A.0. Tobin. 1978. Develop. Biol. 67:23-39. Deng, G. and R. Wu. 1981. Nucleic Acids Res. 9:4173-4188. 43 17. St. John, T. personal communication. 44 CHAPTER II ISOLATION AND CHARACTERIZATION OF ERYTHROID EXPRESSED cDNA CLONES The specific goal of this research was the isolation and characterization of genes expressed during erythroid differentiation as described above. To do this we used two homogeneous erythroid precursor populations that can be isolated in large quantities. An 1_ 3119 population consisting almost entirely of reticulocytes, a late erythroid precursor which is still active in transcription, can be isolated from the blood of anemic chickens (1). Another homogeneous erythroid precursor population can be isolated from an jg 313:9 cell line derived from bone marrow cells transformed with avian erythroblastosis virus (AEV) (2). Erythroid differentiation is arrested by AEV at approximately the CFU-E stage. For simplicity, this population will be called erythroblasts. Temperature sensitive AEV mutants (tsAEV) can be induced to terminally differentiate by growth at the permissive temperature (42°) in medium supplemented with anemic chicken serum (2). Three highly enriched erythroid cell populations, erythroblasts, reticulocytes and erythrocytes, can be fractionated by Percoll density centrifugation from induced tsAEV cells (3). In a study comparing protein synthesis in these three populations, twenty-eight major protein synthesis changes were observed by two 35 dimensional gel electrophoresis of S-methionine labelled cell lysates 45 , POLY-M RETICULOCYTE RNA _ FRON ANEHIC CHICKENS * HIGH SPECIFIC ACTIVITY cDNA CLONING , SINGLE STRAND 32F-cDNA GLOBIN SUBTRACTION ERYTHROBLAST SUBTRACTION 32R-tDNA PROBE ENRICHED FOR f 'NONGLDBIN' RETICULOCYTE NESSAOES \LSCREEN cDNA LIBRARY RETICULOCYTE cDNA LIBRARY EDNA SUBLIBRARY ENRICHED FOR V RETICULOCYTE SPECIFIC CLONES t GLOBIN SCREEN RESTRICTION ENZYNE ANALYSIS NONGLOBIN RETICULOCYTE ENRICHED CLONES NORTHERN ANALYSIS SOUTHERN ANALYSIS DNA SEOUENCING VPROTEIN DATA BASE SEARCH CLONE CHARACTERIZATION AND POSSIBLE GENE IDENTIFICATION Figure 1. Overall scheme for the isolation of chicken erythroid-specific genes. 46 (4). It was therefore hoped that these visible differences in the proteins that these cells were making would be correlated with significant differences in the poly(A)+RNA present in the reticulocytes versus that of the erythroblast cells. Of course, the most obvious change in erythroblast versus reticulocyte RNA is the presence of very high levels of each of three adult globin mRNAs in the latter. Since these three genes have already been studied in some detail, we attempted to minimize their representation in our cloning protocol. The three adult globin genes were subcloned into Promega Biotec's SP6 transcription vectors (5). The SP6 vectors have a specific promotor site, recognized by the bacterial SP6 polymerase, upstream of a polylinker cloning site. Run-off RNAS are transcribed from the promotor ig.yiggg, through the cloned DNA, to a site of restriction enzyme digestion at the 3' end of the region which one desires to copy. Coding or non-coding strand RNA can be transcribed depending on the direction of the insert relative to the vector. Figure 1 outlines the experimental approach for isolating erythroid-expressed genes employing the refined hybridization-subtraction procedure of Davis et al. (6). To enrich for non-globin reticulocyte gene sequences, two RNA populations, globin and erythroblast, were subtracted from the cDNA made to the reticulocyte 32P-labelled first strand cDNA poly(A)+RNA. High specific activity, was transcribed from poly(A)+ reticulocyte RNA isolated from anemic chickens. Globin cDNAs were subtracted by the hybridization of SP6-transcribed globin RNA to the reticulocyte 32P-cDNA by the subtraction procedure outlined in Figure 2. Hydroxylapatite A. IEIl£NLO£IIE.£III£HEI_IIOIE 'ETICIIMTE HIV-A9 IA m TRANSCRIPTASE ACTIWCIN D SIACLE Sim ”rm NITN 10' criws SPECIFIC ACTIVITY GLDIII SUITAACTION -7Nx NYssIDS ~25! CDNA ~95! OVERALL RECOVERY Cm - ”Ir WT ”TRACT!“ -ass NYsaIss ~51: EDNA -95! sscavssv emu - 'GLIIIIWT' USE DIRECTLY TO ME film CM LIBRARY Hum EIICIED CMA SIILIIRARY NITN 515 CLUES 47 SNIIIACIIOI_IIOID§OL NICN SFECIFIC ACTIVITY 32Ii-cDIIA TARA IO-so FOLD was ID at NYBAIDIzAIION VOLDNE 0.5 A HasnIATE BUFFER 63°C FDA Iona-2000 Cat cDNA AND cDNA/Am NYBAIDS NYDImIYWATIIE CIIAOIIAIOCAAFNY 0.12 N museum BUFFER mm "mm 60°C - was NYBAIDS cw IN ELUATE Figure 2. A schematic of the preparation of a reticulocyte-enriched cDNA probe. The overall procedure, including results, of producing an enriched reticulocyte cDNA population is shown in (A). A detailed summary of the subtraction procedure is outlined in (8). 48 chromatography separated the cDNA:SP6-RNA hybrids (double stranded molecules) from the unhybridized cDNA (Single strand molecules). Hydroxylapatite, under the appropriate conditions, specifically binds double stranded nucleic acids. With an increase in temperature or phosphate concentration, the double stranded molecules can also be eluted. Sequences which were common to both erythroblasts and reticulocytes were subtracted by the hybridization of the single-stranded cDNA off the first column to erythroblast poly(A)+RNA isolated from the AEV-erythroid cell line LSCC H03 (7). The resulting reticulocyte-enriched cDNA probe was used to screen the reticulocyte xgth cDNA library (Chapter 1) leading to the isolation of a reticulocyte-enriched cDNA sublibrary. This chapter describes the production of the reticulocyte-enriched cDNA probe, the screening of the reticulocyte Agth cDNA library, and the characterization of some members of the enriched sublibrary. Since the globin subtraction was not 100% efficient, the members of the cDNA sublibrary initially chosen for study were found to contain contaminating globin sequences. Further characterization led to the identification of eight non-globin clones which were analyzed in some detail. The size and tissue specificity of the mRNA homologous to each clone were determined by analysis of Northern blots. Fine-structure restriction enzyme maps were generated, and the nucleotide sequence of the cloned cDNA inserts were determined. The functions of several clones were identified. A clone specifically expressed in reticulocytes, clone 37, was identified as coding for carbonic anhydrase II (CAII) by comparing its restriction map and partial nucleotide sequence to that of a chicken CAII cDNA clone which I 49 previously had isolated (Appendix 1). Two other clones, 104 and 200, were identified by the comparison of their corresponding amino acid sequences, as deduced from an open reading frame in the nucleotide sequence, to the Protein Sequence Database using the program FASTP (8). Clone 104 was identified as coding for ferritin heavy chain, an iron storage protein found mainly in the blood, liver, spleen, and bone marrow. Clone 200 was identified as ubiquitin, a small 76 amino acid protein found in all tissues. Ubiquitin has several known and putative functional roles and is the subject of Chapter III. The other potential erythroid Specific clones have not been identified. 50 EXPERIMENTAL PROCEDURES Materials The SP6 transcription system including the vectors pSP64 and pSP65, and the SP6 polymerase were purchased from Promega Biotec. Hydroxylapatite was purchased from Bio-Rad. Collodial tubes were purchased from Schleicher and Schuel. The LSCC H03 cell line was a gift from H.J. Kung (Case Western Reserve). Dulbecco's Modified Eagle's Medium (4500 mg/ml glucose), chicken serum, and fetal bovine serum were purchased from Gibco. HEPES and proteinase K were purchased from Sigma. All restriction enzymes were purchased from either New England Biolabs, Boehringer Mannheim, or Bethesda Research Labs. RNA Isolation Reticulocyte poly(A)+RNA was isolated from chickens with induced anemia as described in Chapter I. Erythroblast poly(A)+RNA was isolated from the AEV-transformed erythroid cell line LSCC H03. This continuous cell line was grown on Dulbecco's Modified Eagle Medium (4500 mg/ml glucose) with 8% fetal bovine serum, 2% chicken serum, and 10 mM HEPES pH 7.3. A 500 ml ( 107 cells/ml) culture, grown in a spinner flask, was centrifuged at 1500 x g for 10 min. The cells were washed in phosphate buffered saline, recentrifuged, and the cells resuspended in two volumes of lysis buffer (0.5 M NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% SDS, 200 ug/ml 51 proteinase K) and incubated at 37° for 1 hr. The mixture was sonicated using a large probe in 15 sec bursts at 4° until the solution lost its viscosity. 200 ug/ml of fresh proteinase K was added to the solution followed by further incubation for 1 hr at 37°. The poly(A)+RNA was isolated by two rounds of oligo(dT)-cellulose chromatography as described in Chapter I. RNA was synthesized from the three adult globin genes subcloned into SP6 vector plasmids. The transcription reaction mixture contained the following: 40 mM Tris-HCl, pH 7.5, 6 mM MgClZ, 2 mM spermidine, 500 NM each of ATP, UTP, GTP and CTP, 10 mM dithiothreitol, 1000 units/ml RNasin, 40 ug/ml SP6 linear plasmid DNA, 50 uCi [a-32PJUTP, 5-10 units/pg DNA SP6 polymerase. The reaction was carried out at 40° for 2 hr and then digested with 30 ug/ml ribonuclease-free DNase I at 37° for 10 min. The solution was made 3.5 M in NH40Ac, and tRNA was added to 10 pg/ml followed by phenol:chloroform extraction and ethanol precipitation. The RNA pellet was resuspended in 2.5 M NH40AC, ethanol precipitated, and resuspended in water. All steps were carried out using ribunuclease free technique. The RNA synthesized was quantitated by TCA precipitation and the transcript size evaluated by agarose gel electrophoresis. Reticulocyte-Enriched Probe cDNA Synthesis. The first strand cDNA reaction mixture contained the following: 100 mM Tris-HCl, pH 8.7, 140 mM KCl, 10 mM MgCl2, 100 ug/ml oligo(dT)12_18, 30 mM B-mercaptoethanol, 1.1. mM each of dGTP, dATP and dTTP, 36 DM dCTP, 500 units/ml RNasin, 1 mCi [o-32P]dCTP, 98 units reverse transcriptase, 100 ug/ml actinomycin D, 52 and 10119 poly(A)+ reticulocyte RNA. The reaction was carried out at 42° for 2 hr and terminated with EDTA addition to 25 mM. Ribonuclease free technique was used until completion of the cDNA synthesis. The solution was then made 0.16 N in NaOH, and incubated at 68° for 10 min. Following neutralization with HCl and phenol:chloroform extraction, and acqueous phase was passed over a 1 ml Sephadex G-50 column. The excluded cDNA was made 200 pg/ml in tRNA and ethanol precipitated. Globin Subtraction. A 10-20 fold sequence excess of the three SP6 globin synthesized RNAs was combined with the high specific activity first-strand cDNA. These nucleic acids were then precipitated and resuspended in 5-10 ul TE. The RNA and cDNA were hybridized in 0.5 M sodium phosphate buffer, pH 6.8, 0.5% SDS and 1 mM EDTA (total volume less than 20 ul) in a sealed glass micropipet. The solution was heated at 90° for 2 min, then incubated at 68° for 18 hr. The hybridized solution was diluted with 1 ml HAP buffer (0.12 M sodium phosphate buffer pH 6.8, 0.1% SDS) and loaded onto a water-jacketed, 1 ml hydroxylapatite column previously equilibrated with 0.12 M phosphated buffer, 0.1% SDS at 60°. The column was washed with HAP buffer at 60°, and 1 ml fractions were collected. One microliter aliquots of each fraction were counted in a scintillation counter. The peak fractions of single strand cDNA were combined and dialyzed in a collodial tube against 50 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA for 6 hr at 25°. The double-stranded molecules were eluted by washing the column with HAP buffer at 98° for purposes of quantitation. The dialyzed single strand cDNA molecules were extracted with sec-butanol to reduce the total volume to less than 0.5 ml, followed by phenol:chloroform extraction and ethanol precipitation. 53 Erythroblast Subtraction. The single stranded cDNA fraction from the globin subtraction was combined with a 10-50 fold sequence excess of poly(A)+ erythroblast RNA isolated from LSCC H03 cells (see above) and precipitated with ethanol. The hybridization reaction and hydroxylapatite chromatography were performed as before. The combined single-strand cDNA peak fractions constituted the reticulocyte-enriched probe. Screening the ygt10 Reticulocyte cDNA Library. 40,000 pfu from each size fraction Agt10 reticulocyte library (see Chapter 1) were plated with C600 hf] onto five 150 mm LBM plates (8,000 pfu/plate). The plates were incubated at 37° for 7 hr and stored at 4°. Duplicate nitrocellulose filter replicas were made of each plate. These were denatured, neutralized, washed and baked at 80° for 2 hr as described by Benton and Davis (9). The baked filters were then prewashed in 50 mM Tris-HCl, pH 8.0, 1 M NaCl, 1 mM EDTA, at 42° for 2 hr in a rotating water bath. The filters were prehybridized in 50% formamide, 0.5% Ficoll, 0.5% polyvinylpyrrolidine, 0.5% bovine serum albumin, 5 X SSC (1 X SSC = 150 mM NaCl, 15 mM Sodium Citrate, pH 7.0), 0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl, 100 ug/ml denatured salmon sperm DNA, 10 ug/ml yeast tRNA, 10 ug/ml poly(rC) and 10 ug/ml poly(rA), at 42° for 18 hr with gentle Shaking. Hybridization was initiated by adding the reticulocyte-enriched probe directly to the prehybridization mixture. Incubation continued at 42° for 60 hr with gentle shaking. The filters were then washed first with 2 X SSC at 25° and then with 0.2 X SSC at 68°. They were exposed to Kodak X-Omat film. All plaques showing hybridization to both duplicate filters were picked and 54 together they constitute the two reticulocyte-enriched cDNA sublibraries, one sublibrary for each original size fraction library screened. Clone Characterization An initial screen for contaminating globin clones was done by spotting 1 ul of each cDNA plaque lysate stock in a grid pattern on an E. coli lawn, making filter replicas, and hybridizing the replicas to a 32P-labelled globin probe (containing the three adult 32 nick-translated, globin sequences) and a P-reticulocyte cDNA probe. Small DNA stocks of non-globin, reticulocyte-positive clones were made by the plate lysate method of Fritsch (10). Clone DNA was digested with EcoRI, electorphoresed on 1.2% agarose gels, Southern blotted and rescreened with the probes described above. The inserted cDNA of clones which continued to hybridize to the reticulocyte probe but not to the globin probe was isolated by the method of Girvitz (11). These DNA fragments were subcloned into the EcoRI site of the plasmid pUC8 (12) grown in the E. 9911 strain 0M107. 32P-labelled subclone DNA was hybridized to Nick-translated, Northern blots of poly(A)+ reticulocyte, erythroblast and pancreas RNAs (2—12 ug of each) electrophoresed in formaldehyde gels (10). 1.2% agarose formaldehyde gels were run in 20 mM MOPS, pH 7.0, 1 mM EDTA, 5 mM sodium acetate, and 2.2 M formaldehyde. RNA samples were ethanol precipitated, resuspended in sample buffer (20 mM MOPS, pH 7.0, 1 mM EDTA, 5 mM sodium acetate, 50% formamide, 2.2 M formaldehyde), and heated at 60° for 10 min before loading. After electrophoresis the gel was soaked in a large volume of 20 X SSC at 25° for 30 min. The RNA 55 was transferred to nitrocellulose with 20 X SSC and the filter was baked at 80° for 2 hr under vacuum. Hybridizations were carried out as described above at 42° for 15-20 hr. Restriction enzyme maps were generated by single and double enzyme digestions (10). Appropriate DNA fragments were treated with calf alkaline phosphatase and labelled with T4 polynucleotide kinase (Chapter 1). The 32 P-labelled DNA fragment was sequenced by the method of Maxam and Gilbert (13) as modified by Smith and Calvo (14). Possible amino acid sequences, as deduced from nucleotide sequence open reading frames, were compared to the Protein Sequence Database by the computer program FASTP (8). The Protein Sequence Database is part of the Protein Identification Resource release of August 6, 1985 by The National Biomedical Research Foundation, Georgetown University Medical Center. The database contains 738,997 residues in 3309 sequences. 56 RESULTS AND DISCUSSION Reticulocyte-Enriched Probe Figure 2A outlines the procedure employed in producing a reticulocyte-enriched probe. For use as a probe, several modifications in the first strand cDNA synthesis procedure described in Chapter I were added. High Specific activity cDNA (>108/ug) needed to be synthesized, rather than the cDNA with a nominal 32 P-incorporation used to quantitate reactions. Reverse transcriptase can tolerate dCTP concentrations as low as 36 uM (dATP, dTTP and dGTP at 1.0 mM) and still transcribe full length CDNA copies with reasonable yields. This condition routinely yielded a specific activity of 2-3 x 108 cpm/pg and copied 10-15% of the poly(A)+RNA to cDNA. Another modification was the addition of actinomycin D to reduce the 3'-hairpin loop that can spontaneously form during the synthesis of the cDNA probe (6). The hairpin loop has enough double-stranded character to increase the "single-stranded" cDNA's affinity for hydroxylapatite (see below). The three adult globin gene sequences and the erythroid housekeeping message sequences were subtracted from the 32P-labelled, reticulocyte cDNA by two rounds of hybridization-subtraction. The key features of the subtraction protocol are shown in Figure 28.. A large sequence excess (10-50 fold) of the RNA was hybridized to labelled cDNA in a small volume (10-20 ul) to drive the hybridization to completion in an overnight incubation. Most RNA:cDNA hybridization reactions near 57 completion at a Cot of 500-2000. The Speed of the procedure was important due to the rapid breakdown of the high specific activity cDNA. The unhybridized cDNA was separated from the cDNAzRNA hybrids by hydroxylapatite chromatography. Double stranded nucleic acids bind to hydroxylapatite at 60° in 0.12 M phosphate buffer, while single stranded molecules elute. The peak single strand fractions were pooled, dialysed to remove the phosphate buffer, and precipitated. The double strand hybrid molecules were eluted at 98° in 0.12 M phosphate buffer to determine the recoveries. The overall recovery off the hydroxylapatite column (comparing the recovered double-strand hybrid CPM plus the precipitated single-strand cDNA cpm to the total cpm loaded) was 95%. Globin Subtraction. Globin gene sequences were subtracted from the reticulocyte cDNA by using RNA transcribed from the coding strand of subcloned globin chromosomal DNAs in the SP6 transciption system. The two adult alpha globin SP6 subclones were a gift from 0.0. Engel (Northwestern University). The A subclone, pSP aA(+), contains the 1.7 kb 8amHI/EcoRI fragment from pBRa7-1.7 (15) in pSP64 (Figure 3). The 00 subclone, pSP aD(+), contains the 1.1 kb BEE/EcoRI fragment from TSVaD (16) in pSp64 (Figure 3). The adult beta gene (8), on a 2.5 kb EcoRI/SacI fragment of p818R15 (17), was subcloned into pSP65 and named pSP B(+) (Figure 3). Upon run-off transcription by SP6 polymerase, the SP6 subclones yielded 1-2 ug of RNA per ug of linear plasmid template. In the subtraction, 1.65 ug of 32P-labelled reticulocyte cDNA was hybridized to 16 pg of oA, 8 ug of OD and 15 pg of BRNA, and the products were separated on hydroxylapatite. Subtraction of 74% of the cDNA was accomplished using the SP6 globin RNA. 58 1.7 as INSERT CONYAININC tut-BA CLosIN sews sisal!) Pu ii HID] I” he Mechanisms hues cut-m luneslla 1.1 as INSERT CONTAININC TNECLD sLosIN GENE 3 JIIII...IIIIIIIIIL l = E v a 0 VI Q ~ ] Is I- S 1 I— “A“ See! he! 3‘” be! Ball Pstl lies 2.5 II INSERT MAIN!" MIMI. sews Figure 3. Restriction enzyme maps of the SP6-globin clones. The SP6 vector plasmids, pSP64 and pSP65, have an ampicillan resistance gene, an origin of replication and an SP6 promotor site just 5' of a M13 polylinker cloning region. The SP6 promotor/polylinker regions of both vectors are shown at the top of the figure. The three adult globin genes C(A,0~O:'f§M AAAAAAAAAAAAAAAAAAA A Figure 3. A comparison of the nucleotide sequences of two ubiquitin cDNA clones. The nucleotide sequences of clone 200, isolated from a reticulocyte cDNA library, and clone 7 (CEFU), isolated from a heat Shocked chicken embryo fibroblast cDNA library and sequenced by Bond and Schlessinger (14), are compared over their overlapping 600 bp. Sequence differences are noted (*). The 519's, the stop codon (IAE), and the polyadenylation signal (AATAAA) are marked. 93 polyadenylation signal but does end in a poly(A) run. Bond and Schlesinger have recently reported the nucleotide sequence of ubiquitin I and showed that clone 7 corresponds to a part of the ubiquitin I gene message (15). A comparison of the nucleotide sequences of clone 200 and the ubiquitin I genomic locus shows that their 3'-untranslated regions are virtually identical (1 bp difference) E (Figure 4). There are seventeen nucleotide differences in the 854 bp overlap between clone 200 and the ubiquitin I ubiquitin coding regions; only one of these differences corresponds to a nucelotide difference noted between Clones 200 an 7. One of the nucleotide differences A between ubiquitin I and 200 eliminates a characteristic XhoI site, however, leaving only one XhoI site 274 bp 5' of the HindIII site. Since virtually all the nucleotide differences between clones 200 and 7 were not reproduced when comparing clone 200 and ubiquitin I, the sequence differences remaining may be attributable to DNA sequencing errors and/or allelic variations. The two clones (clone 7 and ubiquitin I) sequenced by Bond and Schlesinger differ more from one another than either of them does from clone 200. Also, the 3'-untranslated sequence they report for clone 7 is clearly incorrect. Consequently, we presume that much of the variation involved is due to sequencing errors on their part. Errors in our data, allelic variation between the sources of chicken nucleic acids used, and sequence artifacts due to cloning may also contribute to these differences. Clone 200 most likely is a cDNA clone corresponding to the ubiquitin I gene message since Bond and Schlesinger reported only a 20% homology between the 3'-untranslated sequences of ubiquitin I and II. The large divergence between the 94 1 III M D ID Wmmmmmmnmnamumnmmm 19 Ill asurnwcrccmaswcmunummumwammmmmmammmm a qwmmmmaaummamxmmhwmnmmmmmamua mmmmmdmmamm«mummfimunmmmmmmmma m m «mummmmamnamnmummmmmmmnmwmmma «mammmmnhnamumn a dammnmwmmma m a mmmwmmamwmmmmnmmmmmmmmmudaamm ummw“mammmmmmnmmmnmummmddaamm m ummmmmwmnmmmfimamumamwmmmmnmua amummfiwmammmmhmmmnamwmmmmnmna_ R an “KIATCKICI'TWGTTWGI‘TBCKIMHWUEKCCKmmfiflflaMTUJCTW a an .osoennssltmsnsicucosmmwasmcarnsmacmxhrosmasmamail masmmmcmcrcecnsmmmmamuxmmmmmmmmmmnmm m m mmmmmmmafiwmmmmummamumnmmmmqmu mmmamnaammmmmmnmnumnmmmmmmamu a nmmqmmummmmmakmmmwmwummmnummm nmmumuammfimmaammmwmwummmmammm m m dwamnmnawumwmaunmwmmmamaaawmm amaanmmummmmmmnnmmmmmammmammm m m WWWWW WWW WWW m m HindIII . a “WWW Figure 4. A comparison of the nucleotide sequences of clone 200 and the ubiquitin I genomic locus sequenced by Bond and Schlesinger (15). The sequence differences are noted (*). 95 3'-untranslated regions of clones 200 and 7 may be due to a cDNA Cloning anomaly, or an error in reassembling the sequence information. With the ubiquitin sequences of clone 200 as a probe, eleven genomic clones were isolated from screening approximately five genomic equivalents of the Dodgson et al. Charon 4A chicken genomic library (19). Nine of these clones were purified and DNA stocks prepared. Initial restriction enzyme mapping divided the nine clones into two major groups (1, 2, 4, 5, 6, 7, 9 and 3, 10). Clone 1, 2, 4, 7 and 9 gave identical restriction fragments, while clones 5 and 6 were very similar (data not shown). Clones 3 and 10 yielded completely different restriction fragments compared to the other seven clones (data not shown). Further restriction mapping was done on clones 2, 3, 5, 6 and 10. The restriction maps of these clones are shown in Figure 5 along with the maps of the genomic clones (UbI and UbII) reported by Bond and Schlesinger. A detailed comparison between our clones and the ubiquitin loci is hampered by the relatively few restriction sites mapped by Bond and Schlesinger. It should be noted that Bond and Schlesinger used the identical genomic library to ours, so we would expect that some of our ubiquitin clones would be identical to theirs. The restriction maps of clone UbI and our clone 2 are identical with respect to the EcoRI, KpnI and SmaI sites (Figure 5A). This suggests, in fact, they are the same clone, since both clones were isolated from the same genomic library. Bond and Schlesinger also reported a detailed restriction map of the ubiquitin protein coding region (PCR) of U61 and nearby flanking regions. Their detailed UbI map was constructed by combining the restriction maps of two subclones. They propose that the ubiquitin I gene is transcribed from left to right 96 A. Oluauhhr I»: "" rLuh Tao-NI P A "KEELQ, Niall L T 5 T i Y VNNIII AKpal IRAN Chm! “CI ‘— Tsui ‘47 v crflui a 1;} .LXNsI 1' l A T L a t g n—KL T v ilr TO .1 1.1 a T l Class-1 I L M l I. “Sn * Q 1 i 9 i L P Y I ,__L__' L ilr 9 ill ill ill 9 \I! v T 9 9 S 5 6 NF 5 d I.____£Iaael l l file-s.“1 I Figure 5. Restriction enzyme maps of chicken genomic clones containing ubiquitin sequences . Clones maps from two chicken genomic loci are Shown, ubiquitin I (A) and ubiquitin II (B) (as defined by Bond and Schle singer, 15). In (A), the predicted ubiquitin protein coding regions (PCR) of the ubiquitin I locus clones are marked, with the arrow indicating the direction of transcription. The small restrition map above phage UbI, indicates restriction sites mapped from two overlapping subclones, but not directly compared to the UbI phage clone by the authors (15). In (B), the restriction fragments hybridizing to ubiquitin are marked OJET). 97 with respect to the UbI phage restriction map (Figure 5A), and that it is located to the right of the KpnI site (on the 5.0 kb KpnI/ECORI fragment). We conclude from our data that Bond and Schlesinger inverted the map of the subcloned ubiquitin I region around the KpnI site with respect to the UbI phage map. Consequently, we propose that the ubiquitin I gene is transcribed from right to left relative to Figure 5A, and that it is located to the left of the KpnI site on the 6.7 kb EcoRI/KpnI fragment. Two facts support our conclusions. First, the 0.7 kb HindIII fragment, which contains part of the 3'-untranslated region of the ubiquitin I gene, maps 2.2 kb to the left of the KpnI site. Second, the XhoI sites, which are found in the ubiquitin coding regions, also map to the left of the KpnI site and 274 bp to the right of the 0.7 kb HindIII fragment (Figure 5A). The locations of the HindIII and XhoI sites are shown in the nucleotide sequences of clones 200 and U61 (Figure 4). Clones 5 and 6 have very similar restriction maps to clones 2 and UbI (Figure 5A). Clones 5 and 6, however, differ in that they contain an additional EcoRI site and SmaI site that flank the PCR of clones 5 and 6, as well as a third XhoI site. AS described below, these individual restriction site differences between clones 5 and 6 and clone 2 are likely due to allelic variation in the chicken genomic library. In Figure 6 the ubiquitin-hybridizing XhoI, XhoI/HindIII, and HindIII restriction fragments of clones 2, 5, and 6 are shown. The restriction digests were run on an agarose gel and a Southern blot was prepared. The filter was probed with 32 P-labelled ubiquitin sequences of clone 200. The characteristic 456 bp XhoI fragment and 274 bp XhoI/HindIII fragment hybridize to ubiquitin (clone 200) in clone 2. 98 Figure 6. A comparison of the ubiquitin containing restriction fragments of clones 2 (A), 5 (B), and 6 (C). Clone DNA was digested with HindIII (H), HindIII + XhoI (HX) and XhoI (X), run on a 0.7% agarose gel, and a Southern blot was prepared. The filter was probed with the ubiquitin sequences from clone 200, 32P-labelled by nick translation. Only the small restriction fragments found within the ubiquitin coding regions are shown. 99 These characteristic fragments also hybridize in clones 5 and 6 along with a new 228 bp XhoI fragment. Since the 228 bp XhoI fragment hybridizes to ubiquitin, this extra XhoI site is probably located within a ubiquitin repeat directly 5' to the 456 bp XhoI fragment. The ubiquitin repeats are 228 bp units. In clone 200, the two XhoI sites are located within the second and fourth repeats resulting in 456 bp fragment (2 x 228). Remember Clone 200 does not extend completely through the first ubiquitin repeat. Consequently, we do not know if another XhoI site is in this first repeat in the cDNA clone. Therefore, we do not know if the cDNA clone originated from a two XhoI site ubiquitin 1 allele (e.g., clone 2) or a three XhoI site ubiquitin I allele (e.g., clone 5 and 6), or a third type of allele. The DNA of clones 5 and 6 was sequenced from the HindIII site of the 3'-untranslated region toward the PCR. Identical nucleotide sequences were obtained from Clones 5 and 6. Their sequence corresponded exactly with the ubiquitin nucleotide sequences of the 3'-regions of clones 200 and UbI. We therefore conclude that our Clone 2 and UbI of Bond and Schlesinger represent clones containing one allele of the ubiquitin I locus while clones 5 and 6 represent clones containing the other allele. The library used by both their group and ours was obtained from a single chicken (bred for heterozygosity for a restiction site polymorphism at the ovalbumin locus), and thus only two alleles of any given locus should be present therein. Clones 3 and 10 are overlapping genomic clones whose restriction maps are similar to the UbII clone isolated by Bond and Schlesinger, at least near the ubiquitin hybridizing region (Figure 5B). A detailed comparison is again hampered by the fact that they report only a few lOO Figure 7. Differential hybridization of chicken genomic clones containing the two ubiquitin genomic loci. Clone DNA was digested, run on 0.7% agarose gels, and Southern blots were prepared. The filters were probed with ubiquitin sequences from clone 200 (homologous to the UbI locus, Figure 4). 32 P-labelled by nick translation. Clones 2 (A), 5 (C), and 6 (D) are homologous to the ubiquitin I locus while clones 3 (B) and 10 (E) are homologous to the ubiquitin II locus (see also Figure 5A and 8). 101 flanking restriction sites. Both clone 3 and UbII contain a ubiquitin homologous region on a 4.3 kb SmaI fragment with a characteristic SacI site near the gene (Figure 58). The ubiquitin-containing restriction fragments of clones 3 and 10 always hybridized weakly compared to the hybridization signals of clones 2, 5 and 6 (Figure 7). This difference was considerably greater than would be expected from the existence of four ubiquitin repeats in ubiquitin I and only three repeats in ubiquitin II. This may be attributable to a significant difference in the ubiquitin nucleotide sequence between ubiquitin I and ubiquitin II. Bond and Schlesinger reported that the last (3') ubiquitin protein coding repeats of clones UbI and UbII were 93% homologous in the nucleotide sequence. The restriction map of UbII has several differences with the maps of clones 3 and 10, especially in the order of EcoRI fragments. Clones 3 and 10 also have two KPNI sites and two SmaI sites rather than the one KPNI and three SmaI sites reported for UbII. Even with these mapping differences, we feel that clone 3 and 10 contain the same ubiquitin II locus as UbII as defined by Bond and Schlesinger, because of the similarity of restriction sites near the ubiquitin homologous region. The mapping results of clones 2, 3, 5, 6, and 10 compare accurately with the results of a chicken genomic Southern probed with clone 200 (Figure 8). Chicken chromosomal DNA was digested with EcoRI, HindIII, and 8amHI, singlely and in pairs, run on an agarose gel and a 32P-labelled Southern blot was prepared. The filter was probed with libiquitin sequences of clone 200. The different hybridization intensities of the ubiquitin I and II loci, noted above, are reflected in the results of all three single digest lanes. For example, a high 102 wwomd Figure 8. Southern blot analysis of the chicken genomic regions. Chicken chromosomal DNA (10,Ag/lane) was digested with EcoRI (lane 1), EcoRI + HindIII (lane 2), EcoRI + BamHI (lane 3), HindIII (lane 4), HindIII + BamHI (lane 5), and BamHI (lane 6), run on a 0.8% agarose gel, and a Southern blot was prepared. The filter was probed with the ubiquitin sequences of clone 200, 32P-labelled by nick translation. Faint hybridizing fragments are noted (0). 103 intensity 5.5 kb fragment and a weak intensity 5.0 kb fragment are produced in a HindIII digest. The ubiquitin-hybridizing regions of clones 2, 5, and 6 (high intensity, ubiquitin I) are contained on a 5.5 kb HindIII fragment while the ubiquitin regions of clones 3 and 10 (low intensity, ubiquitin II) are contained on a 5.0 kb HindIII fragment. A complete, one-to-one comparison of the clone mapping data with the genomic Southern cannot be made for all lanes for two reasons. First, at least one of the flanking sites of the larger DNA fragments is often not present within the cloned region of chicken chromosomal DNA. Second, the Southern blotting procedure does not always transfer different size DNA fragments equally. All the ubiquitin-hybridizing restriction fragments of known size correctly correspond to the appropriate chromosomal fragment on the genomic Southern (Figure 8). Our data supports the results of Bond and Schlesinger, in that we found two different genomic loci in chickens containing sequences homologous to ubiquitin. We feel we have isolated clones from both alleles containing the ubiquitin I locus which are present in our library; clones 2 and UbI of Bond and Schlesinger contain one allele and clones 5 and 6 contain the other allele. Clones 3 and 10 correspond to the ubiquitin II locus, although Bond and Schlesinger did not report enough data on restriction sites flanking the ubiquitin II coding locus in clone UbII for a complete comparison of our clones to theirs to be made. The differences between their results and ours in the nucleotide sequence and clone map data can probably be resolved by a more detailed analysis of the ubiquitin loci and direct comparison of the clones isolated by the two labs. 10. 11. 12. 13. 14. 15. 16. 104 REFERENCES Goldstein, G., M. Scheid, U. Hammerling, E.A. Bayse, D.H. Schlesinger and G. Goldstein. 1975. Proc. Natl. Acad. Sci. U.S.A. 72:11-15. Ciechanover, A., D. Finley and A. Varshavsky. 1984. J. Cell i Biochem. 24:27-53. Finley, D. and A. Varshavsky. 1985. Trends Biochem. Sci. 10:343-347. Goldknopf, F.L. and H. Busch. 1977. Proc. Natl. Acad. Sci. U.S.A. 74:864-868. West, M.H. and W.M. Bonner. 1980. Nucleic Acids Res. 8:4671-4680. Levinger, L. and A. Varshavsky. 1982. Cell. 28:375-385. Matsui, S.I., B.K. Sron and A. Snadberg. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:6386-6390. Ciechanover, A., D. Finley and A. Varshovsky. 1984. Cell. 37:57-66. Gallatin, M., T.P. St. John, M. Siegelman, R. Reichert, E.C. Butcher and I.L. Weissman. 1986. Cell. 44:673-680. Siegelman, M., M.W. Bond, W.M. Gallatin, T. St. John, H.T. Smith, V.A. Fried and I.L. Weissman. 1986. Science. 231:823-829. St. John, T., W.M. Gallatin, M. Siegelman, H.T. Smith, V.A. Fried and I.L. Weissman. 1986. Science. 231:823-829. Wiborg, 0., M.S. Pedersen, A. Wind, L.E. Berglund, K.A. Marcher and 0. Vunst. 1985. EMBO J. 4:755-759. Lund, P.K., B.M. Moats-Staats, 0.6. Simmons, E. Hoyt, A.0. D'Escole, F. Martin and 0.0. VanWyte. 1985. J. Biol. Chem. 260:7609-7613. Bond, U. and M.J. Schlesinger. 1985. Mol. Cell. Biol. 5:949-956. Bond, U. and M.0. Schlesinger. 1986. Mol. Cell. Biol. 6:4602-4610. Dworkin-Rastl, E., A. Shrutkowski and M.B. Dworkin. 1984. 6:4602-4610. 17. 18. 19. 20. 21. 105 Izquierdo, M., C. Aribas, J. Galceran, J. Burke and V.M. Cabrera. 1984. Biochem. Biophys. Acta. 783:114-121. Ozkaynak, E., D. Finley and A. Varshavsky. 1984. Nature. 312:663-666. Dodgson, 0.8., J. Strommer and 0.0. Engel. 1979. Cell. 17:879-887. Benton, W.D. and R.W. Davis. 1977. Science. 196:180-182. Maniatis, T., E.F. Fritsch and 0. Sambrook. 1982. In Molecular Cloning. A Laboratory Manual. Cold Spring Harbor E3boratory, New York. APPENDIX I ISOLATION OF THE CHICKEN CARBONIC ANHYDRASE II GENE 106 Isolation of the Chicken Carbonic Anhydrase II Gene CORINNE M. YOSHIHARA, MARK FEDERSPIEL. and JERRY B. DODGSON Department of Microbiology and Public Health Michigan State University East Lansing. Michigan 48824-1101 The carbonic anhydrase (CA) gene family displays considerable variation in its eXpression pattern. In amniotes (birds, reptiles, and mammals) the three genetic loci that have been identified that encode isozymes CA I. CA II. and CA III, vary in their expression between classes of amniotes and in tissues within a class. The genes for CA I and CA II are both expressed in most mammalian red blood cells (RBC), for example, while in avians only the CA II gene is expressed in the RBC. In mammals. at least, the genes that encode CA I and CA II enzymes appear to be linked. ' In order to study the relationship between the structure and organization of CA genes and CA gene expression. it is necessary to first isolate the genes. We describe here the initial steps toward isolation of the chicken CA II gene. A chicken RBC cDNA library was prepared in the plasmid pBR322 with poly- (A)+ chicken anemic red cell cytoplasmic RNA by dG-dC tailing into the Pst I site.‘ Bacterial colonies that were tet"amps were transferred to nitrocellulose filters, lysed, and the liberated DNA fixed to the filters. The filters were first screened for those colonies containing recombinant plasmids with globin DNA inserts by hybridizing the filters with a and B globin-specific probes. The nonglo- bin colonies were selected and screened with ”P—labeled mouse CA [I cDNA.’ The three colonies whose DNA gave a positive autoradiographic result were isolated. DNA sequence analysis demonstrated that one of the three clones iso- lated whose insert was approximately 300 bp was a bona fide chicken CA II cDNA clone. The restriction map of the chicken CA ll clone is shown in FIGURE I along with the strategy for sequence analysis. The amino acid sequence predicted from the nucleotide sequence shows extensive homology with the known amino acid sequences of human (65%). rabbit‘ (63%), and mouse" (60%) CA ll (FIG. 2). The ”$5-03ij Y1 ° 6 9‘: I A '3 I}: Am a W ‘ Me I ‘ we. I an I v f as. I Nine n Y Isa I ‘ Iii-«H $00.” +Sesl f soon a 9 Nu u T 1.. , A No. it 9 In! messes: FIGURE 1. cDNA clone of carbonic anhydrase ll. Restriction map of pPES-O.3. Arrows indicate the direction and extent of the sequence determined by Maxam and Gilbert se- quence analysis. 332 107 YOSIIIIIARA et al.: CHICKEN CA ll GENE ”3 mmacmaaarmcmcmrxmmmax 10 20 onwarrmyacntsmmymmMsuernuMmem main -lqs-— - G1u—-ttisl.yskp- mean -I.ya—---Glu——Hialoy_aAQ—- mmrrwmnnu—aum-unmup—— maxmmcmurmmarmarmxru 30 01169101! ne Ala Ann 61y Glu Arr; Gln Set Pro Ile Ala me Set 1hr marr——tys—~~———Vatup—up— mantra-~upu-—————up—Mp— Insecan—--—-a-p-—--—Va1Asp—Asp— mmcomarmmcarmccmmcmcrcamm to cumuzrmmnaammupmmlfilmmtm'wm manmm—m—--s«r¢u———-vn mncarrup——wstus—-S¢rrm—-—amvu mallmm~fliam——~mcln--uune METEGATGIGCRGSAMGIMCGICMAICGB 50 ’ 60 (111008011 Sermupmctymanmmnevummmy WCAII— —GlnAl.a—SerleuArg-uu——— MIT CAII - — Glu His Pro ne Set Arq An; - ne - — - mmII———Lyanana5er-Ser-———- acmmmmcmcmmcocmrmacmm 70 anamaxrnusermmmmumnpnpmwnpmsc main -—A1a ——-—----Gln -A1a main--—-----——-Ius——- mmrr.—-————————mn—mm mmmnmmmmum so anamonmifilmnmymymmnpmysu main -neuLys—-Pru---1‘hr mean ~mloyaclu—Pm—Glu—‘Ihr main -uul.ys--Prc—Sarhq>-— FIGURE 2. Nucleotide sequence of pPES-0.3. The predicted amino acid sequence of chicken carbonic anhydrase ll is compared with homologous amino acid sequences of human. rabbit. and mouse carbonic anhydrase II. The amino acid sequence predicted by the nucleotide sequence is given below the coding regions along with its numbering. Y refers to C or T. Only those amino acids of the human. rabbit. and mouse CA ll proteins that differ from the predicted chicken CA ll sequence are shown. See reference (rand citations therein for the human CA ll sequence. 108 336 ANNALS NEW YORK ACADEMY OF SCIENCES chicken cDNA clone contains sequence from the coding region at the 5'-end of the CA ll mRNA from amino acids 7 to 86. A comparison of amino acids 7 to 86 of chicken to the corresponding amino acids of mammalian CA I. II. III' indicates that chicken CA II is identical to l of the l2 invariant and unique residues of CA I; I of the l5 for CA Ill; and 5 of the 9 for CA ll (residues 7. 26. 66. 68. and 75). Chicken CA II is identical with all of the 6 residues that are located in the active site regions of the CA isozymes (residues 28. 60. 63. 64, 66. and 68). The chicken cDNA clone was used to isolate phage from a a Charon 4A chicken genomic library.9 The phage that hybridized to the cDNA clone presum--. ably contain the chicken CA II gene. These recombinant clones are presently being characterized by restriction enzyme analysis. Future experiments will in- volve detailed restriction enzyme analysis, subcloning. and DNA sequence analy- sis (particularly the 5' flanking region) of the clones and will provide preliminary data for the study of the chicken CA gene family. ACKNOWLEDGMENTS We are grateful to Dr. Peter 1. Curtis for providing the mouse carbonic anhydrase cDNA clone. We also thank Dr. Richard E. Tashian and Dr. David Hewett- Emmett for their advice and encouragement. REFERENCES I. Caann. N.D. I972. Carbonic anhydrase isozymes in Cavia porcellus. Carvia apera and their hybrids. Comp. Biochem. Physiol. 43B: 743-747. 2. DeSmone. 1.. M. LINDE & R. E. Tasman. I973. Evidence for linkage of carbonic anhydrase isozyme genes in the pig-tailed macaque. Macaca neinestrina. Nature (New Biol.) 242: 55-56. 3. EICIIER. E. M., R. H. Srean. .I. E. Wouacx. M. T. Davrsson. T. H. Ronerucx & S. C. REYNOLDS. I976. Evolution of mammalian carbonic anhydrase loci by tandem duplication: Close linkage of Car-I and Car-2 to the centromere region of chromo- some 3 of the mouse. Biochem. Genet. Id: 6SI-660. Manrarts. T., E. F. Fnlrscn 8: J. Samaoox. I982. Molecular cloning manual. Cold Spring Harbor Laboratory. Cold Spring Harbor. N.Y. Cunns. P. 1. I983. Cloning of mouse carbonic anhydrase mRNA and its induction in mouse erythroleukemia cells. J. Biol. Chem. 258: 4459—4463. Peanut. R. E., S. K. Sraour. R. J. Tanrs & R. E. Tasman. I978. Amino acid sequence of rabbit carbonic anhydrase ll. Biochim. Biophys. Acta 533: l-l I. Cuarrs. P. .l.. E. Wrrnens. D. DEMUTH. R. Wart. P. .I. Venra & R. E. Tasman. I984. The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase ll. Gene. In press. 8. Tasman. R. E., D. HEWE‘IT-EMMEI'T & M. Gooouan. I983. On the evolution and genetics of carbonic anhydrase I, II. and Ill. lsozymes: Current Topics in Biological and Medical Research 7: 79-I00. 9. Dawson. 1.. J. Snowmen a J. D. Enact. I979. The organization of chicken globin genes. Cell 17: 879-887. 99‘5“? APPENDIX II ISOLATION OF RECOMBINANT cDNAS ENCODING CHICKEN ERYTHROID <5 -AMINOLEVULINATE SYNTHASE 109 Isolation of recombinant cDNAs encoding chicken erythroid 8-aminolevulinate synthase mytawm/raddwmfl Masavuru Yarrasro'ro'. Necson S. Yew’. Manx Feoeasrrec'. leaav B. Doooson'. Noaro Havasrttl. ano lanes DOUGLAS ENGEI.‘ malt-enemyJaalsalw IialagyaaaCal mm emunmsm. January )7. I”, ABSTRACT We report 0e habtlaa achNA clan. an- S-aatlnalevallaate synthase (ALA synthase; EC 2.3.IJ7l.thelb'Iauymehthehe-ebhayathetlepathwayh tut hetraaaa'lbedfra-afamlyefgeaesthdareclaaelyrahtad handeatIdeaeqneace-dleeachregiatedhadevalap- maualypdlcmmc. tAminolevuIinate synthase (ALA synthase: EC 2. 3. l. 37). woduct(heme)andcanbeinducedbyavarietyofchemical effectorsofhepaticporphyria. notably 3.3-dicarbethoxy-I. 4- Ghydrbcollidinc and allylisopropylacetamide. Furthermore. Minductionappearstoberegulatedatboththetranscrip- tionalandtranslationallevels(3-II).Intheliver.hemealso mearstod'a'ectlyinterferewiththetranslocationofthe peenzymefromthecytoplasmictothemitochondrialcorn- partments.causinabnormalincreasesinthecytoplasmic kvelscftheprunzymetlz. I3). Incontrasttotheseobservationsinhepatictissue.the aythrbidformoftheenzymeisunreaponsivetoehemicals thunormauyinduceporphynmandthisisozymedoesnot upeartoaccunmlateinthecytoplasmoferytll'ocyteson tremmentofarn'asalswiththe sameporphyrogenie agents ‘l'hapfihcarsnacansdthsutieleweredafiayedimbymch-p mfl'Us-nchmnthmafarebaheby-tad “W imam-bulllnc IINMUWHM. Univ-say. EWILMI. 'Dapartaraatsat Iincha-atry. Wham. EaatImam. III”. .0er M. Tobaka Microbialagyand Umvera'aySchaoIdIladicias. Send- I" 04-18). Instead. the biosynthesis of ALA synthase in erythroidcellsappearstobestronglystimulatedbychemical agents normally used for induction of anemia [e. g.. phenylhydrazine (G. Kikuchi and M. Hasegawa. personal communication”. Thus the enzyme activity appears to be regulated in a cellospccific manner. In keeping with the findings that there exist differentially regulated ALA syn- these enzymes. it has been reported that the liver and erythroid counterparts of the enzyme also differ in size. both as preertzymes and as mature proteins (19). We me interested irt studying the regulatory mechanisms whereby ALA synthase becomes activated in chicken erythroid and liver cells in a developmentally specific man- aer. Furthermore. one report has claimed that ALA synthase is one of the earliest erythroid genes that is transcriptionally activated alter dimethyl sulfoxide treatment of Friend mia cells. thus suggesting the intriguing possibil- ity that ALA synthase induction might serve as an early temporal marker for erythrocyte maturation (20). This served as an additional incentive to examine the regulation of this particular erythroid-specific gene in detail. MATERIALS AND METHODS Iaclarlaphge mi Ilau Strms. Bacteriophage I vectors gth and gtll and lysogenic and lytic Eshen'clu’a coli host strains mass. “090) were obtained from Tom St. John (Department of Pathology. Stanford University Medical School) (21. 22). and strain 88337 (23) was obtained from Ed Fritsch (Genetics Institute). WcDNALDr'yhepuadaaandSueenm.cDNA was prepared as described (ref. 24. pp. 230—238). and EcoRI inkers (Bethesda Research Laboratories) were ligated tothe mixture of erythroid cDNAs. The linkers were then cleaved with EcoRI to reveal the cohesive ends. and the cDNAs were hactionatedbygelelectrophoresis intopopulationsthatwere greaterthanandlessthanlllbasepairst‘bp). The twopools were individually collected artd separately ligated to a gtlo DNA treated with EcoRI. The ligated “"srrtall (i.e.. (Ill-bp) and large" (>m-bpl cDNA pools were then packaged in vitro (ref. 24. pp. 291-292) and plated on All host 35137. Since the packaged phage that contain recombinant cDNAs willnot integrateinthetdlstraintthescoltl inserts interrupt the a cl gene. whose expression is essential for Iysogeny) only recombinant bacteriophage efficiently produce plaques. Theu'tersofthetwolibrarieswere l. 7 x Io‘plaqueeforming units (pfu) for the large inserts (ca. 10‘ pfu of (1' revertants) andca. 2. 8 x Io‘pfuforthesmallfragmenth’brary. Thelarge hsertlibrarywasamplifredat 2 x Io‘pfu/ISO-mmdishfor 4hrat42‘ConYlm;theampIiftedphagcwereeatracted froratopagarose aspreviously described (25. 26). The phage W: ALAsthaae; ”Want”: bp. base aa'atsltafa. 110 Developmental Biology: Yamamoto er al. centrifugation; DNA was isolated fromthe phqe by addition of sodium dodecyl sulfate and proteinase K as described (ref. 24. P- 85). The purified a gtIO library was transferred into I gtll by treatment ofthe a gth DNA cohesive ends with nuclease Sol 31 until in vitro packaging efficiency of the gtll) DNA had droppedbyafacrorofat least IO‘ (T. St. lohn.l. Rosen.and I'I. Gershenfeld‘. personal communication). The exonuclease- treated gtlo phage pool was then dipsted with EcoRI and added to ligated a gtll arms that had been digested with EcoRI and dephosphorylated by using column-purified calf intestine alkaline phosphatase (27). The phage were ligated at a roughly 2. I weight ratio“ gtlo library to x gtll arms) and packaged In vitro Packqiu efficiency was usually about 10‘ pfu/pg of total DNA added. resulting in ”—40% recornbi nants as judged by clear plaque forntation on S-chloro-4— bromo-3-indolyl B-o-galactoside lX-Gal) plates. We usually chose ligation ratios that gave about 20% recornbinants. to avoid multiple cDNA inserts. a gtll recombinants were plated for screening in top agarose at ca. 3 x lo‘ pfu per ISO-mm Petri dish on host strain YIO90 (21). The plates were incubated for 4 hr at 42°C and then overlaid with nitrocellulose filters that had been soaked in 10 mM isopropyl p-o-thiogalactoside and dried. The overiays were then incubated for 4-12 hr more. The filters were then removed into a blocking solution (3% nortfat dry milk plus 0.1% Nonidet P40 in Trisobuffered saline [20 mM Tris-HCl. pH 7.3/0.5 M NaCl/0.02% sodium azide (3% milk solution” and placed on a rotator to agitate for 6—12 hr at 4'C iii the presence ofa sonic lysate ofa gtll-infected YIO90 cells (all subsequent treatment of the filters was also performed at 4‘0. Anti-ALA synthase antibody was then added to the filters at a final concentration of 5 rig/ml. and the filters were allowed to bind to the antibody for an additional 6 hr. The filters were then removed from the antibody solution. washed four times for 30 min each in 3% milk solution. and then moved to a second dish. containing "’Habeled goat antibod- ies to rabbit lgG (5 x 10‘ cpm/ml). The filters were bound to the second antibody for 2—3 hr and again washed four times for 30 min in 3% milk solution. The filters were then dried and exposed for autoradioyaphy at -70'C for 12-18 hr. Recom- binant plaques were purified as previously described (26). Antibodies. The antibody recognizing chicken liver ALA synthase was raised in rabbits; the specificity of this antibody has been previously characterized by demonstration that dilute antibody is able to specifically inhibit the enzymatic condensation reaction when presented with partially purified ALA synthase from either chicken liver or red blood cells (I9). The antibody was ptrrifred by binding to. and elution from. staphylococcal protein A-Sepharose CL-4B (Phar- rnacia): we found this to be the minimal purification neces- sary to reduce background adequately for successful anti- body screening. Prior to their use in screening. antibodies were treated by preabsorption with a a gtll-infected Yl090 sonic lysate for a minimum of 12 hr at 4‘C (22). Iodinated. affinity-purified. goat antibody to rabbit IgG was the gener- ous gift of Susan K. Pierce (Northwestern University). Mar MeOads. Positive hybrid selection and in vitro translation of the released mRNA was performed by modi- fication (28lofthe original method (29). adapted tothe useof single-stranded RNA bound to filters. Rabbit reticulocyte lysate used for in vitro translation reactions was from Promega Biotec (Madison. WI) and was used according to the supplier‘s specifications. Subcloning in Spb vector 95:65 (30) was as previously described. as was the preparation of DNA template for the Sp6 in vitro transcription reactions; RNA blots were also perforated as previously described (31). lsotopically labeled precursor amino acids and nucleotides were purchased from Amersham: labeled and unlabeled Proc. Natl. Acad. Sci. USA 82 (INS) 3703 protein standards for gel electrophoresis were purchased from Amersham and Sigma. respectively. Protein blotting was performed as specified (32). with the exceptions that the filters were first blocked in 3% milk solution (above) and then washesafterthef'trst and secondantibody bindinreactions were in Tris-buffered saline/0.5% Triton X-llll. RESULTS W of the Anti-ALA Syflh-e annuity. To ensure that the antibody raised against the chicken liver form of ALA synthase would be successful for isolation d the erythroid ALA synthase gerte in antibody screening. two experiments were undertaken to test the affinity of the antibody preparation to erythroid ALA synthase. In the first experiment. ALA synthase from in vitro translated anemic chicken red blood cell poly(Al‘ RNA was immunoprecipi- rated by using the antibody and protein A Sepharose. The r n r“ “‘ [”SImethionine- labeled erythroid preenzyme was recovered in high yield from an in vitro translation mixture of total reticulocyte poly(A)‘ mRNA. as expecteleig. I;ref. 19). While it is clear thaterythroid ALA synthase is the only protein recognized in immature erythroid cells. a clearly different molecular weight species of ALA synthase is immunoprecipitated from committed erythroid progenitor cells (Fig. 1. lane C). Whether this implies that these virally transformed cells exhibit ambiguous develop- mental properties in their pattern of gene expression or are developmentally “switched" in the type of ALA synthase isozyme expressed during erythroid maturation is currently unknown (see Discussion). A second. more salient. experiment was performed to ask 3 ALA synthase would associate with the antibody after the enzyme was immobilized on nitrocellulose filter blots (32). The second experiment successfully demonstrated that the erythroid ALA synthase strongly associates with the anti- body raised against the liver isozyme when the antigen is fixed to filters (Fig. 2). ABCD kDa 69-0 - - ALA synthase 4.. . “-0 Pro I. Inmrnoprecrprtanond AMsynthaae from In vitro translationofch'ckearedcelIRNA. Chickearedcelpnlysomestos Auunkaachtisolatedfront Ildaye-bryonictlaneAlorman'c adult (lane B)erythroidcellsweretranslated'ntthepreseaced (”Shnethionine. PolytAl° RNA from H06 cells (tsAEV-trans formedearty lineageprecursorcellszref ”ludic- aaemicadultchickenredbloodeellsflanesCuIdD D.Iespeetively) wastrartslatedinvitro.alsointhepreaenceofradiolabeledmcth'o nine. All four samples were then imnnrnoprecipitated from the translatiooreactionatixttnesbyusingrabbnlgcanubodiesto chicken liver ALA synthasetl9landprote'atA- .These precipitates were then electrophoresed on I0! polyaeryl- amide/llJisodiurndodecyIsulfategelslMl. fixcdandbathed‘m EN’IIANCE (Amersham). dried. and exposed for My. Siaesweredetivedbyconmarisonoftheeleetrophoretiemobihtyd “C-labeledproteinstandardsrun'mawalellanedthesarnegel. 111 3704 Developmental Biology: Yamamoto et al. mAICDEF . 116-7 97- ,. ALAsyathase _- (33) " - 8 29_. FIG. 2. Imrnuaolop’cal detection J ALA synthase p-galac- tosidase fusion proteins it h gtll recombinants. Recombinant bacteriophage that were repeatedly positive on plaque purification were inteu'ated into a lysogenic host suain (Ylm: ref 21). The lysogenic cells weregrowntoOD..- -.o 3st 32'C. at whiehtime lytic replication was induced by shifting the temperature to 42‘C for 13 minzaflerculturingforartadditionaIIhrat 38‘C.thecellswere harvested mid immediately lysed in sodium dodecyl sulfate sample buffer (30 mM Tris~l~lCl. pH 6.8/1.3% sodium dodecyl sulfate/30 atM dithiothreitol/4 M urea). Identical amounts of protein synthe- sized by induced a lysogens (either treated (lanes C and E) or not treated (lanes D and F) by the addition ofisopropyl thiogalactoside to 1 mM final concentration at the time of heat induction) were electrophoresed on 7.3% polyacrylamide/o.1% sodium dodecyl sulfate gels ( 34). The protein was then electrophoretically transferred to nitrocellulose as described (32). The nitrocellulose transfer was then blocked with 3% milk solution. and bound protein was allowed to react with IgG antibody to chicken liver ALA synthase: the filter was washed. exposed to affinity-purified goat l”I-Iabeled antibody to rabbit lgG. washed. and exposed for autoradiography. Lane A is a parallel lane on the protein blot stained with india ink (35) to determine the position of commercial markers. Lane B contained total mitochondrial matrix proteins from anemic chicken red blood cells.boundtoantibodyatthesametirneaswerethelytoa¢nic bacterial proteins. IsohtlonafALA SyflaecDNARecamblrmts. lnourfirst successful screen of the a gtll expression library. we isolated 14 positive plaques (from ca. 2 X 10‘ total pfu screened: 28% recombinants) of which 6 were positive on repeated plaque purification. These 6 were grown as minipreparations for phage DNA isolation. and. while all 6 yielded isopropyl t" "‘ ‘ ' ‘ “ fusion proteins larger than osidase. only 4 contained inserts that were released by digestion with EcoRI. The 2 of these that gave the strongest hybridization signal in protein blots were character- ized further. The two putative ALA synthase recombinants chosen for subsequent analysis are designated A4 and A14; the size of the inserts in these two recombinants are ca. 530 and 1” bp. respectively. [The fact that these recombinants contain cDNA sequertces that are unexpectedly small (since the cDNAs were initially selected to contain inserts >80) bp) anaeststhatthesiaefractionationofthecDNApopuIation wasnotacctrrate. IAsaninitialeontrol. wefirstshowedthat sidasecontrol. asexpectedofanALAsynthase hybrid fusion protein.andthattheresultantfusionproductwaslargerthan nativep-gahctosidase. AsshowninFig. 2. theantibody binds proteins that are higltly 'mduced after ueatment of recombinant A4 and A14 lysogens with isopropyl thi- ogalactosideflanesC—F). andbothfusion proteinsarelarger th-Ithenativep-galactosidasernmkerlllbkna. IaneA) Proc. Natl. Acad. Sci. USA 82 “ml Furthermore. as stated in the previous section. the 33-kDa erythroid ALA synthase enzyme (from whole red blood cell mitochondrial matrix) is the only protein recognized in a parallellaneonthesameblotlFig.2.laneBl. Roenmhhnnt A4 Encodes Erythrold ALA Syn“. To prove that the isolated recombinants encode the red cell- specific ALA synthase protein. two complementary experi- ments were performed. First. d’ the two recombinants that gave strong antibody signals in protein blots (Fig. 2) both actually code for different or overlapping seynents of the same structural gene. three criteria should be met for the corresponding reticulocyte mRNA encoding ALA synthase: recombinants A4 and A14 should hybridize to art RNA of the same size in RNA blotting analysis. hybridization to those red cell mRNAs should be strand specific. and finally. the mRNA that hybridizes to the strandospecific probes must be large enough to encode the erythroid ALA synthase preenzyrne of SSokDa (corresponding to a minimum mRNA size of 13m nucleotides: ref. 19). The second experimern to verify the identification of these recombinants would be that ALA synthase mRNA should be selectable. in a strand- specific manner. from a population of total red blood cell poly(A)‘ RNAs. and that the in vitro translation product of that mRNA filter selection should be precipitable with the anti-ALA synthase antibody and should correspond in size to the precursor form of the enzyme found in red cells. The results of RNA blot analysis are shown in Fig. 3. in which strand-specific radiolabeled probes have been hybrid. ized to identical lanes of anemic adult chicken red blood cell poly(A)‘ RNAs. Strand-specific probes were created by subcloning the recombinant EcoRI inserts from the A4 and A14 phage in the vector pSpoS (30) in both orientations. The subclones were individually cleaved distal to the Sp6 pro- moter and recombinant segments of the ALA synthase gene (inserted at the EcoRI site of pSp65) and then transcribed by using Sp6 RNA polymerase and radiolabeled nucleotide precursors as previously described (31). The four individual A B C D 285- ' . p-ActiL Its — . In t— :' fi-Globin ‘ I FIG. 3. BlotanalysisoferythroidALAsyntlaaeaIRNAwith strandcspecif‘rc probes. One microgram of poly(A)’ RNA 'nolated fromthecireulatingredcellsofanemicadulthenswas resedonidenticallanesofaverticall .2%formaldebydeaureaegels (24). the RNA was transferred to nitrocellulose. baked. prehybridizedasdescribed(31).‘l'heinsertsfromrecodrinnt bacteriophage A4andAl4wereexcisedwith£eoRlandstdrcloned 'mpSprlJOIinbothfrnmentorientations.Bothorientationsdboth neonsbinaflcDNMwemmsaMinthemd la-"PIGTP and Spb polymerase (31). These transcripts leand Al4e (laneAI. Al4fflaneBI. A4b(laneCl.ltdA41(|aneDIlwerethea hybridized to individual lanes of red cell poly(A)’ RNA u SS'C. washedirtthepresenceofRNaseAl31. 36Iat4ug/ml. andeaposed forautoradiography. Exposure times were 13 hrflanes AaadClor ShrllanesBandD). Identicallaneswerehybridizedtthdlla chicken ribosomal gene recombinant (37)).pBIBR13 laduh ch'cken a-globingenornic subclonel27)l.andap-actincDNAclone(381Ior interndsizestandardieationtdatanotshownl Developmental Biology: Yamamoto er of. transcriptswerethenhybridizedtosepnratelanescontaining redbloodcellmRNAzthe blotswerethen treatedwithRNase (31)and exposed for autoradiography. As shown in Fig. 3. the two recombinants both recognize an mRNA of the same size ('m only one of the two strand-specific probe orientations). The mRNA recognized by the two different recombinants is easily large enotuh (approximately 2000 nucleotides) to encode a protein of 55 kDa. Additional proof that A4 encodes erythroid ALA synthase is provided by the hybrid selection/translation experiment shown m Fig. 4. Since we demonstrated above that the transcript produced by Sph subclone A41 was complemen- tary to the putative ALA synthase mRNA. this implies that I unlabeled strud specific transcript of that recombinant. when fixed to filters. should be able to preferentle select an mRNA whose product in an in vitro translation reaction should be immunoprecipitable with the anti-chicken liver ALA synthase antibody. Fig. 4 shows the results of such an experiment. in which the mRNA for the preenzyme was 'mdeed selected from total red cell poly(A)’ RNA with the synthetic Sp6 transcript from clone A41 but was not selected with the synthetic Sp6 RNA transcript from the complemen- tary strand (prepared from subclone A4b). Thus. recombi- nant A4 does contain at least part of the erythroid ALA synthase gene. ThaneSpeellcltyofALASynthaseGeneExpr-emlan.To determine whether or not we could distinguish between the various tissueospecific forms of ALA synthase at the mRNA level. further RNA blot analyses were performed. It was anticipated that. on the basis of these experiments. we could gain some insight as to whether or not the ALA synthase mRNAs transcribed in various chicken tissue and cell types were the same or different sizes [correlating with the different sizes of the proteins (19)] and that we might be able to rrtake preliminary armaments regarding the possibility that the tissue-specific ALA synthase enzymes were transcribed from the same. or different genets). We again made use ofthe strand-specific Sp6 transcript complementary to erythroid ALA synthase mRNA. One might expect that if the red cell and liver (and perhaps other) forms of ALA synthase were encoded by different genes. they might have divernd suf- ficiently in nucleic acid sequence that a probe synthesized fromonegenewouldhaveonly partialhornologytoanyorall of the heterologous genets). On the other hand. i various tissue-specific forms of ALA synthase mRNA were found to be highly homologous to a single conserved (coding se« quence) probe. that would leave open the possibility that the tissue specificity of ALA synthase arises by differential cellapecificprocessingoftranscr'mtsproducedfromthe same genetic locus. p-Actin tss : 112 Proc. Natl. Acad. Sci. USA 82 (I985) 3705 AICDEFGH ” - n .0 O I F 0*. m (a C FIG 4 Hybrid-releaser’avirrotranslationandimmunoprectp- 'nation with anti-ALA synthase antibody. Ten microgrus d red blood cell poly(A)‘ RNA (isolated from anemic adult hens) was hybridized to strand-specific Sph transcripts of recombinant subclone A4 fixed on nitrocellulose filters. The hybridized mRNA wasreleasedflomthefiltersbybriefboilingandthentranslatedin vitro (28. 29). The in vitro translation reactions are shown diet the follow'mg :laneA.nofIIterselection(totalredbloodcell mRNA): lane 3. hybridization of total red bloodcell mRNA and release from filterbound 8pc transcript A4b: and lane C. hybridiza- tion and release from Spb transcript A41 (lane D shows the mobility of the "C-Iabeled protein markers on this 12. 5‘! polyacryl- addelo. 1% sodium dodecyl sulfate gel). Final lanes depict the pitation reaction of total red cell (”Simethiom'ne- hbeled protein after treatment of the translation reaction mixtures with either anti-ALA synthase antibody and protein A-Sepharose flaneHIorpIoteinA-Sepharosealoneflane E). LnnesFandGshow theresultsofimmunoprecipitationofthetranslationproductsof hneslandC. respectively. withanti-ALA synthaseantibodyand prote'm AoSepharoae. The results of RNA blot analysis of various poly(A)‘ mRNAs isolated from cell lines and various chicken tissues me shown' In Fig. 5. Fig. 5A shows the hybridization pattern ofradiolabeled Sp6 A41 transcript to the mRNAs in a standard' high- -stringency' ‘wash solution (equivalent to 10 mil monovalent cation. ”'0 and Fig. 58 shows the same blot washed in a solution containing RNase A at 30’C. [We have previously established that the latter condition favors thereleaseofallbutspecificallyboundRNAprobesInsuch blotting experiments (31)]. As is readily apparent from the data presented in Fig. 5. the RNA (probelRNA (mRNA) hybrids from different tissues are clearly distinguishable (A). Mbermore. the size of the mRNAs homologous to the erytlaoid-specdie ”obevariesaccordingtotissueandcell type. Thus. the largest cellular RNA that is detectable in FIG. 5. Deve specificity of ALA syn- the mRNA synthesis. RNA was isolated from vari- ouschickentissuesandcelllinesbybanr'hgof Mum thiocyanate-isolated RNA and poly(A)‘ RNAwasisolatedbytwocyclesofoligdd‘n-celhrlose My (31). These RNAs were electropho .... reaedflblotted andhybridiaedtoradiolabeledsubclone MlSpbtranscripHseelegendtoFig 3). Chicken ulslarRNAsampleswereisolatedfromthe following 2 samees:hne1.hfSB-1eells(39);.hne2ll-day _ . cmcken embryo fibroblasts; lane 3. 4.3-day whole ~" -. chckenembryos:he4.11