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Z M S degree in oology flan/{2244 Major professor ,— 0-7 639 J MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE ll RETURN BOX to roman-thin checkout from your record. TO AVOID FINES mum on or balm dd. duo. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Ema! Opportunity Initiation Wan-w CLONING AND CHARACTERIZATION OF A GENOMIC ACTIN OF NAEGLERIA FUWLERI BY Suzanne M. Gorospe A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE DEPARTMENT OF ZOOLOGY 1995 ABSTRACT CLONING AND CHARACTERIZATION OF A GENOMIC ACTIN OF NAEGLERIA FOWLERI BY Suzanne M. Gorospe The purpose of this study was to isolate the 5' and 3’ regulatory regions of a Naegleria fowleri actin gene in order to construct a transformation vector. A transformation vector could be utilized to analyze putative virulence genes in vivo. A 250 bp cDNA insert was obtained by screening a partial cDNA library with an actin oligonucleotide. A northern blot probed with a 250 bp cDNA insert revealed that actin was expressed without variation between amoebae cultured; Axenically, with bacteria, and after a third passage through a mouse brain. The northern blot revealed that actin was expressed without variation between three strains of Naegleria fowleri; LEE, Hb-l, and L;L. A Naegleria fowleri genomic library was constructed and screened by colony hybridization utilizing the 250 bp cDNA insert as a probe. Sequence analysis of two genomic clones revealed a TATA-like sequence, ATAATTAA, located -65 bases upstream of the +1 A of the ATG and a conserved polyadenylation signal, AATAAAA, located 36 bases downstream of the stop codon TAA. The actin genomic sequence did not contain introns and consisted of 1125 nucleotides encoding a 375 amino acid protein. ACKNOWLEDGEMENTS I would like to thank.my major professor, Dr. R.N. Band, for his tremendous support and encouragement throughout my master's thesis project. I am very grateful to Dr. R.N. Band for giving me the opportunity to complete my master’s degree under his guidance. I would like to thank Dr. Will Kopachik for his support, for his instruction in molecular biology techniques, allowing me to use laboratory space and equipment, and his critical analysis of my research. I would like to thank Dr. Jim Smith for his guidance and help during the construction and analysis of the phylogenetic trees. I would also like to thank my other committee members: Dr. Ralph Pax and Dr. Robert.Brubaker for their guidance and assistancerwith the completion of my thesis. I would like to thank my family and friends for their patience and understanding during my study. A special thanks to Robert Marzano for his continuous support throughout my master's project. iii TABLE OF CONTENTS Page LIST OF FIGURES ....................................... iv LIST OF TABLES ......................................... v INTRODUCTION ........................................... 1 MATERIAL AND METHODS.. ...... . ........... . .............. 8 Culture and Genomic DNA Isolation ....... ... ...................... 8 Plasmid DNA Isolation .................... 9 Dot Blot ................................ 10 Genomic Library Construction and screening 0 O O O O O O O O O O O O O ............. l 1 Southern Blot.. .................... .....12 Northern Blot...... .......... . .......... 13 Sequencing .............................. 13 Actin phylogenetic analysis ............. 14 RESULTS ......OOOOOOOO ..... 0 OOOOOOOOOOOOOOOO 000......15 Dot Blot. O O ........ O O O O 0 O O O O O ...... O O O O O 15 Northern Blot Analysis ................ 15 Southern Blot Analysis . . . ........... . . . 16 Sequence Analysis......................19 Actin Protein Parsimony Analysis ........ 20 DISCUSSION AND SUMMARY ................................ 22 LITERATURE CITED ............................... . ..... .33 iv Figure Figure Figure Figure Figure Figure Figure Figure Figure 8a 8b LIST OF FIGURES Page Autoradiograph of Dot Blot of cDNA clones screened with an actin oligonucleotide consensus sequence........42 Autoradiograph of Northern Blot of RNA from amoebae cultured axenically, with bacteria and after third passage through a mouse brain; and of RNA from amoebae from different strains of Naegleria, L.L., LEE, and Hb-l... ..... ........ ....... 44 Autoradiograph of Southern Blot of genomic clones probed with an oligonucleotide specific to the 5’ end of actin..................................46 Autoradiograph of Southern Blot of genomic clones probed with an oligonucleotide specific to the 3’ end of actin...........48 Autoradiograph of Southern Blot of genomic clones probed with an oligonucleotide specific to the 5’end of actin............50 Nucleotide and deduced amino acid sequence genomic actin sequence of Naegleria fow16r10000000 ....... .....OOOOOOOOOOOOOOOOSI Partial 3’ nucleotide and deduced amino acid sequence of clone ACT I of Naegleria leeriOOOOO0.0.000000000000000053 Parsimony analysis of actin protein sequences, one of the three most parsimonious trees reconstructed using MacClade..................................55 Parsimony analysis of actin protein sequences, modification of the tree in Figure 8a....0.......0.00.0000...0.00.00.0055 Table 1 LIST OF TABLES Actin protein sequences from 25 taxa used for phylogenetic analysis ..... . ........ 56 vi INTRODUCTION Naegleria fowleri, free-living ameboflagellates, are opportunistic pathogens to man, and the primary cause of the disease, primary amebic meningoencephalitis (PAM). Primary amebic meningoencephalitis was first detected in 1965 in Australia (Fowler et al., 1965). As of 1990, approximately 149 cases have been reported in humans worldwide (Ma et al., 1990). Naegleria fowleri have been isolated in lakes, streams, ponds, sewage and sludge samples, soil and even chlorinated swimming pools (John, 1982) (Marciano-Cabral, 1988). The majority of patients who contracted the disease were healthy children and adults who had recently been swimming in ponds, lakes or pools (Marciano-Cabral, 1988). The route of invasion appears to occur by the introduction of water, contaminated with the amoebae, into the nasal cavities of man. Once in the nasal passages, the amoebae penetrate the cribriform plate and pass into the CNS by travelling along the olfactory nerves to the brain. (John, 1982). Once the amoeba are in the brain, hemorrhaging and necrotizing meningoencephalitis occurs (Marciano-Cabral, 1988). Patients with PAM exhibit severe headaches, nausea, severe fever and anorexia (Duma et al., 1971). The patients usually die within 72 hours after the symptoms first appear (Duma et al., 1971). As of 1990 effective treatments were still unavailable (Ma et al., 1990). 2 The life cycle of Naegleria fowleri consists of three stages: 1) trophozoite (amoeba with pseudopods) 2) flagellate and 3) cyst. (Schuster et al., 1979) Trophozoites are capable of,transforming into flagellates or cysts. However, flagellates cannot transform into cysts, and cysts cannot transform into flagellates. (Schuster et al., 1979) The genus Naegleria consists of six species: N. fowleri, N. australiensis, N. lovaniensis, N. gruberi, N. jadini and N. thorntoni (Marciano-Cabral, 1988). While N. australiensis, is pathogenic to mice, N. lovaniensis, N. gruberi and N. jadini are nonpathogenic species of Naegleria (Marciano-Cabral, 1988). The study of the genus Naegleria is important for a variety of reasons. Naegleria is utilized as a model for study because of the following reasons. 1) Determination of the regulatory elements responsible for Naeglerial transformation from amoeba to cyst, or amoeba to flagellate, could be significant in the field of developmental biology. 2) A treatment for PAN would be significant to the patients afflicted. 3) Determination of the mechanism of pathogenesis could be significant such that it could contribute to the elucidation of how other pathogenic organisms compromise their hosts and cause disease. The purpose of my study of Naegleria fowleri was to isolate an genomic actin, including the promoter and termination region. The promoter and termination regions 3 could be utilized for the construction of a transformation vector; thus cloned genes can be introduced into Naegleria fowleri and possibly change the phenotype. A transfection system can be utilized to analyze putative virulence genes in vivo. Actin was chosen because of its relative abundance, extensive characterization in this organism, as well as many other organisms, and relative ease of isolation because of the availability of oligonucleotide primers. Actin is a ubiquitous protein, highly conserved among eukaryote organisms. There are two major forms of actin protein: muscle and cytoplasmic. The differences in the two forms of actin appears to occur in the NH2 terminal region of the gene (Vandekerckhove and Weber, 1984). Invertebrates express the actin protein that is essentially homologous to the vertebrate cytoplasmic form (Vandekerckhove and Weber, 1984). The cytoplasmic actin protein plays a major role in many vital cellular activities. These cellular processes include: cytoskeletal structure (Schliwa et al., 1981), cell motility, cytokinesis (Rubenstein et al., 1990), endocytosis, exocytosis (Hightower and Neagher, 1986), cell growth and development (Pollard and Cooper, 1986) and chromosomal condensation and mitosis (Wesseling et al., 1988). Vertebrates express the cytoplasmic actin form, which is involved in the cellular activities listed above, as well 4 as the muscle actin form. In differentiated muscle tissue, actin is primarily involved in muscle contraction. Six different isoforms of actin have been characterized in mammals and birds. Four of the six isoforms of actin have been isolated from muscle tissue; two have been isolated from smooth muscle (alpha-5m and gamma-sm), and two from striated muscle (cardiac alpha-c and skeletal alpha-sk) (Hamelin et al., 1988). The four vertebrate muscle actins only differ by four to six amino acid replacements. Two cytoplasmic actin isoforms have also been characterized (gamma-cytoplasmic and beta-cytoplasmic). The two cytoplasmic isoforms of actin only differ by four amino acid replacements (Hamelin et al., 1988). Some higher plants express an actin protein with both muscle-specific and cytoplasmic-specific amino acid properties (Stranathan et al., 1989). Actin is typically present as a member of a multigene family in most organisms including Naegleria. Previous Southern data revealed that Naegleria fowleri express several actin genes (Jonghee Ahn master’s thesis, unpublished 1992). However, only a single copy actin gene has been found in the following organisms: VOlvox carteri (Cresnar et al., 1990) , Phytophthora negasperma (Dudler, 1990), Aspergillus nidulans (Fidel et al., 1988), Oxytricha nova (Greslin et al., 1988), Tetrahymena (Cupples et al., 1986), and Saccharomyces cerevisiae (Gallwitz and Sures, 5 1980). The actual number of actin genes in an organism can be quite variable. In vertebrates such as human, rat, chicken and Drosophila, approximately 20-30 actin genes have characterized, whereas lower eukaryotes such as Caenorhabditis algans (Krause et al., 1989), Acanthamoeba castellanii (Nellen and Gallowitz, 1982) and Strongylocentrotus purpuratus (Durica et al., 1988) contain between two to four actin genes. The actin multigene family of Dictyostelium discoideum consists of seventeen genes, although only one major gene product appears to be present (Vandekerckhove and Weber, 1980). All of the seventeen actin genes have been sequenced, and while there are relatively few differences in the amino acid sequence, the 5' and 3’ untranslated regions are highly variable (McKeown and Firtel, 1981). Actin genes are differentially expressed throughout the life cycle of Dictyostelium discoideum (McKeown and Firtel, 1981). The rate of actin synthesis in many organisms is developmentally regulated, as well as proven to be expressed in a tissue specific or cell-type specific manner. As mentioned earlier, vertebrates express six isoforms of actin, four of the actin forms have been isolated from specific differentiated muscle tissue (Vandekerckove and Weber, 1979). Two isoforms of actin have been isolated in the sea star, Pisaster ochraceus. The two forms of actin 6 have been proven to be differentially expressed in the life cycle as well as localized to specific cell types. The actin sequence with homology to the cytoplasmic form has been isolated in eggs and appears in early development. The sea star also expresses an actin sequence with homology to the muscle form which has been localized to the feet and testes (Kowbel and Smith, 1989). In Naegleria gruberi, actin mRNA appears to decrease during the transformation from amoebae to flagellate (Sussman et al., 1983). Actin is one of the most abundant proteins in eukaryotic organisms (Fletcher et al., 1994). Actin is one of the most conserved proteins among eukaryotes. Since actin is extremely evolutionarily conserved even among distant organisms, numerous studies have been performed using actin nucleotide sequence or amino acid sequence comparisons, to determine phylogenetic relationships among organisms (Fletcher et al., 1994) (Loomis and Smith, 1990). In regard to the genus Naegleria, phylogenetic relationships have been examined by; analysis of small rRNA nucleotide sequence divergence (Baverstock et al., 1989)(Clark and Cross, 1988), isoenzyme analysis to distinguish among strains of Naegleria (Nerad and Daggert, 1983) and restriction enzyme pattern analysis of mitochondrial DNA to estimate evolutionary divergence of strains of Naegleria (Milligan and Band, 1988). In this study, phylogenetic relationships with regard to the genus 7 Naegleria and six other protozoans, will be examined using protein sequence comparisons of actin. In summary, a Naegleria fowleri genomic library was constructed and screened with an actin cDNA insert. The goal was to obtain a genomic actin, including the 5' and 3' flanking regions. The 5' regulatory region and the 3' termination region of the actin genomic clone, will be utilized to construct a transformation vector. Methods a. Culturing and Genomic DNA Isolation The Lee strain of Naegleria fowleri was cultured axenically, in H4 media supplemented with hemin. (Band and Balamuth, 1974) Genomic DNA was isolated by the CsCl method according to (Nellen et al. 1987) with extensive modifications. Approximately 5x10” N. fowleri cells were harvested using a GSA rotor at 365 RCF for 5 min. The pellet was washed with LS and centrifuged in a table top centrifuge at full speed for 5 min. The pellet was resuspended in ice cold HMN (Nellen et al. 1987) buffer and the cells were lysed by pushing the suspension through a hand colloidal mill (A. H. Thomas Co.) homogenizer. The suspension was spun using the GSA at 1465 RCF for 5 min. to pellet the nuclei. The nuclei was dissolved in 25 ml of HNN buffer and the mixture was dripped into 30ml 0.1M EDTA (pH 8.2)/ 4% Sarcosyl. The mixture was incubated in a 65°C water bath for 5 min. Ethidium bromide was added to 0.4mg/ml and 0.9Sg/ml of CsCl was added to the suspension. The suspension was incubated at 55° C until all of the solid CsCl dissolved. The mixture was spun in a table top centrifuge at the maximum speed to get rid of the upper protein film. The sample was centrifuged using a Sorvall T865 rotor at 124,500 RCF at 15°C for 48 hours. The DNA 9 band was retrieved using a sterile syringe and transferred into dialysis tubing. The DNA solution was dialyzed against 4 liters of TE buffer for 2 days with 4 changes of TE buffer. The dialysate was ethanol precipitated with 0.1 vol of 3M NaAc and 2 volumes of 100% ethanol. The DNA appeared immediately as the tube was inverted. The DNA was . transferred to a sterile tube and dissolved in TE buffer. b. Plasmid DNA Isolation Plasmid DNA was isolated either by the alkaline lysis method (Sambrook et al. 1989) or by the Promega Wizard Miniprep system per the protocol recommended by the manufacturer with the following modifications. After addition of the neutralization buffer, mix and put on ice for 5 min. Spin at 19,000 RCF rpm using a SHT rotor for 15 min. Decant supernatant into a new microfuge tube and spin at room temp. at tOp speed in a microcentrifuge for 10 min. Decant supernatant to a new tube and proceed as per protocol. c. Dot Blot (Sambrook et al.,1989) Plasmid DNA (2ug) from 96 independent cDNA clones were dried to a pellet using a Speedyvac. The pellet was resuspended in 10ul of 0.3 N NaOH to denature the DNA. The 10 DNA was incubated at 80°C for 10 min. The DNA mixture was neutralized by the addition of 10u1 of neutralization buffer. The neutralization buffer was composed of 0.25M Tris pH 7.5, 0.25M HCl and 12.5X SSC. The DNA was spotted onto GeneScreen Hybridization Transfer membrane (Biotechnology System, E.I. du Pont de Nemours & Co.), dried under a heat lamp and baked at 80°C for 2 hours. The blot was prehybridized for 2 hours in hybridization buffer containing 6X SSC, 0.01 N NaPO4 pH 6.8, 1 mM EDTA pH 8.0, 0.5% SDS and 100ug/ml salmon sperm DNA. The probe was a 20mer actin consensus sequence 5'TAGAAGCATTTTCTGTGCAC 3', end-labelled with dCTP y-“P, with T4 polynucleotide kinase following the conditions recommended by the manufacturer (Boehringer Mannheim Biochemicals). The unincorporated nucleotides were separated from the radiolabelled DNA by using NucTrap Probe Purification columns (Stratagene) following the protocol recommended by the manufacturer. Approximately 1X10‘icpm of the end-labelled 20-mer was added to the hybridization buffer and allowed to hybridize at 52%:- overnight. After hybridization the blot was washed as follows: 2 times the blot was washed with 6X SSC for 5min at room temp, 2 times the blot was washed with 2X SSC/1% SDS at 50°C for 30 min. and 2 times the blot was washed with 1X SSC for 5 min at room temp. 11 d. Genomic Library Construction and Screening Approximately 10ug of genomic DNA was partially digested with Sau3A I and phosphatase treated as previously described (Naniatis et a1. 1989). Bluescript KS+ vector was digested with BamH I and treated with phosphatase. The genomic DNA was ligated to pBluescipt vector at a molar ratio of 10:1 with T4 ligase as per the protocol (Boehringer Mannheim). The ligation mixture was diluted 1:10 and lul was used to transform competent E. coli cells DH10B per protocol (Gibco, Life Technologies). Following transformation, the cells were plated on LB agar plates containing ampicillin. The genomic library was screened by colony hybridization using Colony/PlaqueScreen Hybridization Transfer Membranes essentially following the protocol of the manufacturer (Biotechnology System, E.I. du Pont de Nemours & Co). This method is a slightly modified version of the method originally published by Grunstein and Hogness. The membranes were screened with a 250 basepair cDNA Naegleria fowleri actin insert obtained from the dot blot (section C., see above). The actin insert was obtained by restriction digestion to cut the insert from the vector and electrophoretic size separation through a 1.5% agarose gel. The insert was purified by the Qiaex Gel Purification kit as per the protocol (Qiagen, Inc.). The insert was oligolabelled with dCTP a-"P, purified by Sephadex G-50 gel 12 filtration using chromatography and 1X10‘ cpm of the probe was added to the hybridization solution. The putative actin clones obtained from the first screening were diluted, replated and rescreened with the same probe to reduce false positives. The membranes were exposed to Kodak (XAR) X-OMAT x-ray film overnight. e. Southern Blot Plasmids from independent genomic clones (lug) were digested with restriction endonucleases and separated by size, electrophoretically on a 0.8% agarose gel. The gel was electroblotted onto GeneScreen Hybridization Transfer nitrocellulose membrane according to the manufacturer’s protocol (Biotechnology System, E.I. du Pont de Nemours & Co.). The hybridization and wash conditions when oligonucleotides were used as probes were as described in the Dot Blot section. The variable was the temperature at which hybridization occurred, this depended upon the length and G+C content of the oligonucleotide. The actin consensus sequence primer is the 3’specific oligonucleotide used to probe the southern blot in Fig. 4. The base sequence for the 3' specific sequence was given in section C. The base sequence for the 5' specific oligonucleotide is 5’CAGCCATGTATGTTGCC3', this oligonucleotide was used to probe the southern blots in Fig. 3 and Fig. 5. The 13 hybridization conditions for probes longer than 150 basepairs were as follows: the blot was hybridized at 65 C overnight in 1H NaCl, 1%SDS, 5X Denhardt’s, SOmM Tris-Cl pH 7.5, 100ug/ml denatured salmon sperm DNA with 1X10” cpm of the dCTP a-“P oligolabelled probe. The blots were washed: 2 times with 2X SSC at room temp for 5min., 2 times with 0.5x SSC/1% SDS at 65°C for 30 min. and 2 times with 0.1x SSC at room temp for 5min. f. Northern Blot A total of 10ug of total cellular RNA was separated by size, electrophoretically on a 1.3% agarose gel with formaldehyde as the denaturant (Maniatis et al. 1989). The gel was electroblotted onto GeneScreen Hybridization Transfer nitrocellulose membrane (Biotechnology System, E.I. du Pont de Nemours & Co.) according to the manufacturer’s protocol. The blot was hybridized at 42°C for 2 days in 50% formamide, 5x Denhardt's solution, 1% SDS, 10% Dextran Sulfate, 1M NaCl, SOmM Tris-Cl pH 6.8, 100ug/ml of denatured salmon sperm DNA and 1X10‘5 cpm/m1 of the probe. The washes were essentially the same as the southern blot washes. g. Sequencing and Sequence Analysis The plasmid genomic actin clones were purified for 14 double-stranded DNA sequencing by the Promega Wizard Miniprep system described earlier. A total of 5ug of double-stranded plasmid was denatured prior to sequencing with the addition of 0.4M NaOH. The DNA was neutralized with 0.1 volume of 3M Sodium Acetate pH 5.0, precipitated with 2 volumes of 100% ethanol and placed at -20%: overnight. The clones were sequenced by the dideoxy chain termination method (Sanger et al.,1977) using the Sequenase version 2.0 kit per protocol (United States Biochemicals). Labelling was performed with a-“S dATP for detection. Sequences were analyzed by the Wisconsin Genetics Computer Group software package version 8 (Genetics Computer Group). h. Actin phylogenetic analysis Table 1 is a list of the 25 species and accession numbers used for actin protein sequence comparisons. A heuristic search was performed utilizing PAUP 3.1 program (Swofford, 1993.) Protein parsimony analysis was performed utilizing the actin protein sequence from 25 taxa, the number of characters 377 amino acids. The sequences were aligned by eye and BESTFIT program from the Genetics Computer Group package version 8 (Genetics Computer Group). Results a. Dot Blot A total of 96 independent cDNA clones were screened with a 32P end-labelled 20 basepair actin oligonucleotide. The oligonucleotide sequence was determined by the comparison of actin nucleotide sequences from other organisms and generating an oligonucleotide primer utilizing Naegleria codon usage (Jonghee Ahn master's thesis,1992). One 250 basepair cDNA clone was detected (Fig. 1). The 250 basepair cDNA clone was used to screen a northern blot and to screen a Naegleria fowleri genomic library. b. Northern Blot A northern blot was performed using RNA isolated from amoebae cultured with different food sources and different strains of Naegleria fowleri. RNA was also isolated from virulent amoebae, amoebae that were obtained from the third passage through a mouse. A total of 10ug of total RNA from the sources listed above, were electrophoretically separated on an agarose gel containing formaldehyde. The 250 basepair cDNA insert was used to screen the blot. Fig. 2 reveals that actin is constitutively expressed without variation between strains; LEE, Hb-l or L.L. Fig. 2 reveals that actin is expressed without variation among the LEE, L.L. and Hb-l strains when the amoebae were cultured with different 15 16 food sources. The cells were cultured Axenically, nutrient broth supplemented with hemin (LEE:Ax), and the cells were cultured with bacteria as the food source, (LEE:Bact). Fig. 2 also reveals that actin is expressed without variation between nonvirulent amoebae, LEE-Bact and LEE—Axenic, versus virulent amoebae, LEE-Mp. There was no variation in expression of actin between LEE:Axenic, LEE:bacteria, or LEE:MP mouse brain passage. c. Colony Hybridization Approximately 10,000 genomic clones were screened by colony hybridization using an oligolabelled 250 basepair cDNA insert as a probe. The putative positive genomic colonies were immersed in a cluster of other colonies, thus in order to isolate true actin genomic clones, colony -clusters were serially diluted, replated and rescreened. Approximately 50 putative positives were isolated and analyzed further by Southern blot and sequencing techniques. (Data not shown) d. Southern Blot The 50 putative positives isolated by colony hybridization were restricted with endonucleases to release 17 the insert, and probed with an end-labelled oligonucleotide. Fig. 3 reveals that out of 23 genomic clones, 2 inserts: sizes 2.5kb(#208) and 1.9 kb(#lB), hybridized to the actin consensus oligonucleotide described in the material and methods. The genomic clone containing the 2.5kb insert (#20B), was renamed ACT8, and was analyzed further by sequencing. Another southern blot was performed with 27 different genomic clones. The blot was also probed with the actin consensus oligonucleotide. Four genomic clones hybridized to the actin consensus sequence: {12-6.0kb, #20- 6.0kb, 519-4.0kb and I15-3.5kb. (Data not shown) These clones were analyzed further before sequencing. The restriction fragments of these four clones were analyzed by southern blots in order to localize the 5' and 3' regions of the genomic actin clones. Fig. 4 reveals the restriction fragments from clones #12, #20 and #19 that hybridized to an end-labelled 3' specific oligonucleotide. Figure 4 reveals the following fragments hybridized to the 3' specific probe; Lane 1- a 2.0kb Hinc II fragment from clone #19, Lane 2- a 3.9kb Cla I fragment from clone #19, Lane 3- a 4.0kb Cla I/Bgl II fragment from clone #12, Lane 4- a 1.4kb EcoRV fragment from clone #12, Lane 5- a 4.0kb Bgl II/Cla I fragment from clone #20, Lane 6- a 1.8kb Cla I/Hinc II fragment from clone #20 and Lane 7- positive control- the 250 bp cDNA insert that was used for screening 18 the genomic library. There was no detectable hybridization in Lane 8. Lane 8- molecular size marker, restricted with EcoR I and Hind III. Fig. 5 reveals the restriction fragments from clones #19, #15, #12 and #20, that hybridized to an end-labelled 5' end specific oligonucleotide. Fig. 5 reveals the following fragments that hybridized to the 5' end specific probe; Lane 3- a 560bp Cla I/Hinc II fragment from clone #12, Lane 5- a 2.5kb EcoR V fragment from clone #12 and Lane 6- a 560bp Cla I/Hinc II fragment from clone #20. There was no detectable hybridization in Lanes 1, 4, 7 or 8. Lane 1- clone #19 restricted with Hinc II, Lane 4- #15 restricted with Cla I and Hinc II, Lane 7- cDNA insert used for screening the genomic library and Lane 8- molecular size marker- lambda restricted with EcoR I and Hind III. Clones #12 and #20, 6.0kb inserts, were confirmed to be the same clone since the clones exhibited the same restriction patterns and the 5' and 3' specific oligonucleotide probes hybridized to the same fragment sizes. The 560bp Cla I/Hinc II insert from clone #20 was subcloned and sequenced. The 1.4kb EcoR V fragment from clone #20 was subcloned and sequenced. Clone #20 was renamed ACTl. 19 e. Sequencing Figure 6 reveals partial sequencing of the 5’ flanking region and nucleotide sequence and deduced amino acid sequence of the coding region of clone ACT 8. The genomic sequence encompasses -346 bases upstream of the +1 of the A of ATG, and 1071 bases of coding region. The genomic clone ACT 8, lacked the last 75 bases of coding region and contained 1.4kb upstream, 5'flanking. Partial sequencing was performed with clone ACT 1. The coding region sequenced appeared to be identical to ACT 8, the partial sequence appears on Fig. 6 in lower case letters. Fig. 7 reveals the 3' and coding region, deduced amino acid sequence and 3' flanking noncoding region obtained from sequencing clone #20 or Actl. Since most of the coding sequence was identical to ACT 8, only partial sequencing was performed on ACT 1. The clone ACT 1, which was 6.0kb encompassed most of the coding region, but contained a cloning artifact or nonidentifiable sequence, after nucleotide +41 after the sequence GATC. The genomic library was constructed by partial digestion of genomic DNA with Sau3A I, and ligating the partially digested DNA to BamH I cut Bluescript. In this instance, a genomic fragment containing sticky ends ligated to another genomic fragment and then ligated to the vector. 20 f. Actin protein parsimony analysis A strict consensus tree was generated from three most parsimonious trees utilizing PAUP 3.1 program (Swofford, 1993) (trees not shown). The only difference in the three most parsimonious trees was the trees differed in the placement of the taxa of the higher metazoan clade. One of the three most parsimonious trees was reconstructed utilizing HacClade (Maddison and Maddison, 1992) Figure 8a. Figure 8b was created by modifying the tree in Figure 8a. The tree was modified by moving Naegleria fowleri to the root branch, the modification did not effect the tree length. Placing Naegleria in any other position increased the tree length. A total of 25 taxa were involved in the phylogenetic analysis, utilizing 377 total characters, including 121 informative characters. Both trees revealed a tree length of 488,consistency index (C.I.=0.65), and retention index (R.I.=0.63), excluding uninformative characters. Figures 8a and 8b reveal two different models of evolution with regard to Naegleria. Figure 8a reveals Naegleria shares a most recent common ancestor with the clade consisting of VOlvox, Zea and Arabidopsis up to the clade consisting of humans, Artemia, and Drosophila; including the clade consisting of the protists Entamoeba, Dictyostelium, and Acanthamoeba. ,Figure 8b reveals Naegleria shares a most common ancestor with the clade consisting of Plasmodium, Tetrahymena, Trypanomsoma, Achyla, 21 and Phytophthora. Both trees reveal a polyphyletic origin for the protists involved in the study, however the position of Naegleria remains unresolved based upon actin protein sequence comparison. Discussion and Summary The purpose of the current project was to clone the 5' regulatory region and 3' regulatory region of a Naegleria actin gene in order to construct a transformation vector. A transformation system for Naegleria is lacking at this time and such a vector could be utilized to analyze putative virulence genes in vivo. A Naegleria fowleri genomic library was constructed and screened using a 250 basepair cDNA insert as a probe. Sequence analysis was performed using two of the genomic actin clones. The actin sequence of Naegleria fowleri is 1125 nucleotides encoding a 375 amino acid protein. The 5' and 3' flanking regions were sequenced revealing a TATA-like sequence ATAATTATT beginning -65 relative to the +1 of the A of ATG, and a possible polyadenylation signal AATAAAA 36 bases downstream of the stop codon TAA. The N. fowleri genomic actin sequence does not contain introns, consistent with other protists that lack introns including: Dictyostelium, Tetrahymena, Entamoeba, and Plasmodium. The only protozoan presently known to contain an intron in the genomic actin sequence is Acanthamoeba castellanii beginning at nucleotide 315 (Nellen and Gallowitz, 1982). It is interesting to note that actin intron position appears to be conserved among related organisms. Actin intron position appears to be conserved between invertebrate and vertebrate organisms. Actin intron position also appears to be conserved between 22 23 different species of fungi (Wildeman, 1987). However, the position of introns in actin vary quite a bit between distantly related organisms (Wildeman, 1987). Further analysis of the actin promoter needs to be performed before a Naegleria transformation vector can be constructed. The actual start site of transcription should be determined for this clone by primer extension. The function of the promoter should be examined before designing a transformation vector for Naegleria fowleri. A transient expression assay utilizing the putative actin promoter could determine ability of the promoter to function in vivo. It would also be possible to pinpoint specific sequences that are necessary for promoter function by performing a series of sequence knockout experiments. A northern blot was performed with RNA isolated from amoebae cultured under different conditions; amoebae cultured axenically, cultured with bacteria as a food source, and subcultured from the third passage through a mouse brain. The northern blot was also performed with RNA isolated from different strains of Naegleria fowleri: LEE, Hb-l, and L.L. The northern blot was probed with the 250 basepair Naegleria fowleri cDNA insert. The purpose of the northern analysis was to reveal actin expression among amoebae that were cultured under different conditions and to examine actin expression of different strains of Naegleria. The northern blot revealed that actin was constitutively 24 expressed between the amoebae cultured axenically, cultured with bacteria as a food source, or subcultured after a third passage through a mouse. The results contradict a previous finding in which actin mRNA levels were higher in amoebae that were passed through a mouse brain than amoebae that were cultured axenically (Hu et al., 1992). Possible reasons for the discrepancy found by (Hu et al., 1992) compared to the present study could be: 1) in this study a 250 basepair cDNA insert was used as the probe whereas in the study by Hu et al., (1992) an actin oligonucleotide sequence was used to probe the northern blot. Possibly, the hybridization and post hybridization conditions were too stringent for the actin oligonucleotide in the Hu et al., (1992) northern blot experiment. 2) In the Hu et al. study, the LEE:Ax lane may have been underloaded. In this study, the LEE:Ax lane of the northern blot may have been underloaded, since there were no loading controls in this study. 3) The Naegleria LEE:Ax cells as well as the LEE:Bact and LEE:Mp cells may have changed over time. The cells have gone through numerous subcultures since the Hu et al. experiment. 4) The difference in mRNA expression of the LEE:Ax cells found in this study may be due to differences in the axenic media used in this study versus the axenic media used by Hu et al. Possibly the individual components used to make the H4 media may have been changed by the manufacturer. 5) Another plausible explanation could be 25 that in this study, the hybridization conditions and post hybridization washes may not have been stringent enough and thus the LEE:Ax band may actually be an artifact. Evidence for this explanation is that there appears to be hybridization of the cDNA probe to high molecular weight RNA in lanes 1, 4, 5, 6, and 7 of the northern blot (Fig. 2). The high molecular weight hybridization is probably the 265 rRNA, the 18s and 26s rRNA locations are designated on the figure. A total of 1596 nucleotides were sequenced by the dideoxy chain termination method, 1125 of the sequenced nucleotides encode a 375 amino acid protein. Inspection of the Naegleria fowleri genomic actin sequence revealed a fairly high A+T content in the untranslated and coding regions of the sequence. In the untranslated region, A+T = 67%; in the coding region, A+T=57%. The third codon position of the actin gene revealed, A+T=60%. The third codon position of Naegleria fowleri carboxypeptidase revealed a somewhat higher A+T content of 71% (Hu et al., 1992). The Naegleria fowleri % of A+T in the third codon position of actin is comparable to other protists; Dictyostelium A+T=65% (Warrick, 1987), and Tetrahymena A+T=51% (Martindale, 1989). Entamoeba (Edman et al.,1987) and Plasmodium (Wesseling et al., 1989) reveal a considerably higher A+T content in the third codon position of actin of, 78% and 85%, respectively. In contrast, 26 Acanthamoeba (Nellen and Gallowitz, 1982) revealed a much lower A+T in the third codon position of actin, A+T=15%. The frequency of an A+T in the third codon position in actin of Acanthamoeba is similar to Drosophila and humans of, 17% and 30%, respectively. Hu suggests that the third codon position frequency of Acanthamoeba and Drosophila infers that the two taxa diverged early in evolution (Hu et al., 1992). Codon usage data available from protozoans, Entamoeba histolytica and Plasmodium falciparum, reveal the A+T content of the known coding sequences to be approximately 67% (Tannich and Horstmann, 1992): (Saul and Battistutta, 1988). Entamoeba histolytica reveal a strong bias toward the usage of an A+T in the third codon position: an A+T has been found 84% of the time in the third codon position. The codon usage of actin appears to be representative of the total coding sequences known in Entamoeba, the third codon position of total known sequences, A+T=84%, versus A+T=78% third codon usage of actin. The A+T content of the 18s rRNA encoding DNA of Dictyostelium discoideum is fairly high. In the untranslated region of the 18s rRNA DNA, A+T=85%, in the coding region of the 18s rRNA DNA, A+T=57% (Loomis and Smith, 1990). The high A+T codon preference of the aforementioned amoebae can be compared to the A+T content of Escherichia coli which is 48% in the coding region of genes examined thus far (Wada et al., 1991). Codon usage 27 information for organisms is valuable for a variety of reasons. Codon usage information can help an investigator deduce noncoding and coding regions of new gene sequences, be used to design species specific oligonucleotide sequences (Tannich and Horstmann, 1992), and possibly play a role in the estimation of evolutionary divergence rates (Starner and Sullivan, 1989). It has been suggested that taking into consideration codon usage for specific organisms may be necessary in order to estimate nucleotide substitution rates and hence infer phylogenetic relationships (Starner and Sullivan, 1989). Previous studies on the taxonomic classification of protozoans have been based upon the morphological and physiological features of the organisms (Hilligan and Band, 1988). However, distinct genetic divergence and phylogenetic relationships cannot be drawn based solely upon morphological and physiological features of the organisms. One of the concerns with utilizing phenotypic traits as the basis for taxonomic classifications is that the traits under consideration may not be influenced by genetic factors, but environmental factors may have influenced phenotypic traits. Many studies use nucleotide and protein sequences for comparison to infer phylogenetic relationships between amoebae (BaverStock et al., 1989, Clark and Cross, 1988, Fletcher et al., 1994, Loomis and Smith, 1990). Loomis and 28 Smith (1990) suggest that protein sequence comparisons may be more reliable to infer phylogenetic relationships than comparison of untranslated nucleotide sequences when comparing organisms with relatively high A+T content in their genome. Loomis and Smith (1990) proposes that using untranslated sequences, such as 185 rRNA sequences, for sequence comparisons may be unreliable because there may be a selection in some organisms with high.A+T content, for an A+T in positions where it does not affect the function of the molecule. The function of the 18s rRNA is dependent upon the secondary structure and thus changes in the nucleotide sequence could be tolerated as long as the secondary structure is preserved. A preliminary investigation was performed to examine the phylogenetic relationships of seven protozoans including the genus Naegleria to other organisms using protein sequence comparisons of actin. A protein parsimony analysis was performed utilizing the actin protein sequence from 25 organisms, including seven protozoans. A heuristic search was performed using the PAUP 3.1 program (Swofford, 1993). Arbitrarily one of the three most parsimonious trees was chosen to be reconstructed using HacClade (Haddison and Haddison, 1992) Figure 8a. The only difference in the three most parsimonious trees was the trees differed in the placement with respect to the higher metazoan taxa. All three most parsimonious trees were the same with regard to 29 Naegleria and the other protists involved in the study. Figure 8a was modified by moving Naegleria to the root branch to create Figure 8b. Moving Naegleria to the root branch had no effect on the consistency index or retention index and did not increase the length of the tree. Both trees in Figures 8a and 8b have a tree length of 488. Placement of Naegleria in any other position increased the tree length. The results are shown in figures 8a and 8b as cladistic trees. The protozoan actin protein sequences infer a polyphyletic origin, since all seven protists lacked a most recent common ancestor. Figure 8a reveals Naegleria shares a most common ancestor with the clade consisting of VOlvox, Zea, Arabidopsis, and Oryza up to the clade consisting of Drosophila, Artemia, and humans including the clade consisting of the protozoans Dictyostelium, Acanthamoeba, and Entamoeba. Figure 8b reveals Naegleria shares a most common ancestor with the clade consisting of Plasmodium, Tetrahymena, and Trypanosoma. It is really unclear at this time which tree is accurate. The position of Naegleria remains unresolved based upon actin protein sequence analysis. The problem that was encountered was that it was unclear which organism to use to root the tree. Since bacteria lack actin, it was unclear from this study which organism contained the most primitive actin sequence. The results from this study seem to indicate that Haegleria may be the most primitive organism, although further 30 analysis needs to be performed to confirm this statement. Analysis of the protein actin sequences did not allow me to place Naegleria with any of the other sequences. A phylogenetic analysis performed using the 18s rRNA nucleotide sequence revealed that Naegleria was not monophyletic with Acanthamoeba or Dictyostelium (Baverstock et al., 1989, Clark and Cross, 1988). The actin protein parsimony analysis performed in this study revealed a polyphyletic origin for the protists, consistent with previous findings, since all seven protists did not appear in the same.clade. The actin protein parsimony analysis also revealed a polyphyletic origin for the fungi, which is also consistent with previous findings (Fletcher et al., 1994, Clark and Cross, 1988). The actin protein parsimony analysis inferred the protists Dictyostelium, Acanthamoeba, and Entamoeba share a most recent common ancestor. The parsimony analysis also revealed that the protists Plasmodium, Tetrahymena, and Trypanosoma share a most recent common ancestor. In summary, a 250 bp cDNA insert was isolated by screening of a partial cDNA library with an actin redundant oligonucleotide. A northern blot revealed that actin is constitutively expressed without variation between three different strains of Naegleria fowleri: LEE, Hb-l and L.L. The northern blot revealed that actin is expressed without variation among the three strains of Naegleria cultured with 31 different food sources; Axenic (Ax), bacteria (Bact), and passage through a mouse brain (Mp). The northern blot also revealed that actin is expressed without variation between nonvirulent versus virulent amoebae. The 250 bp cDNA insert was used to screen the northern blot. A genomic library was constructed and screened with a 250 bp actin cDNA insert by colony hybridization. Approximately 50 putative positive colonies were isolated and analyzed by southern blot technique. Sequence analysis was performed using two of the genomic clones. The Naegleria genomic actin sequence is 1125 bases encoding a 375 amino acid protein. The 5’ promoter region was analyzed and revealed a TATA-like sequence, ATAATTATT,-65 bases upstream of the +1 of the A of the putative start codon ATG. A polyadenylation signal was revealed, AATAAAA, 36 bases downstream of the TAA stop codon. Actin, a ubiquitous protein, is a highly conserved among organisms. The Naegleria protein actin sequence is homologous to the vertebrate cytoplasmic form of actin. A phylogenetic analysis was performed using the actin protein sequences from 25 organisms, including seven protists. The actin protein parsimony analysis inferred a polyphyletic origin for the protists, since all seven protists did not appear in the same clade. The phylogenetic analysis revealed that the protozoans Dictyostelium, Acanthamoeba, and Entamoeba form a monophyletic group. The actin protein parsimony 32 analysis also revealed that the protists Plasmodium, Tetrahymena, and Plasmodium form a monophyletic group. 10. 11. 12. References Adam, L., Laroche, A., Barden, A., Lemieux, C., and Pallotta, D. 1991. An unusual actin-encoding gene in Physarum polycephalum. Gene, 106:79-86. Ahn, J. 1992. Molecular Study of Actin. Master thesis. (unpublished) Band, R.N., and W. Balamuth. 1974. Hemin replaces serum as a growth requirement for Naegleria. Applied Microbiology, 28:64-65. Baverstock, P.R., Illana, S., Christy, P.E., Robinson, 8.3. and Johnson, A.M. 1989. srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista:Rhizopoda) Molecular Biology and Evolution, 6(3):243-257. Ben Amar, M.F., Pays, A., Tebabi, P., Dero, B., Seebeck, T., Steinert, M. and Pays, E. 1988. Structure and transcription of the actin gene of Trypanosoma brucei. Molecular and Cellular Biology, 8:2166-2176. Ben Amar, M.F., Jefferies, D. Pays, Bakalara N., Kendall, G. and Pays, E. 1991. The actin gene promoter of Trypanosoma brucei . Nucleic Acids Research, 19 : 5857-5862 . Bennetzen, J.L. and Hall, B.J. 1981. Codon selection in yeast. The Journal of Biological Chemistry, 257 (6) :3026- 3031. Blum, H., Hannappel, M., and Arnold, G.J. 1990. Nucleotide and deduced amino acid sequence of an actin cDNA clone from Plasmodium falciparum. Nucleic Acids Research, 18(23):7140. Clark, C.G., and Cross, G.A.M. 1988. Molecular Biology and Evolution, 5(5):512-518. Cresnar, B., Mages, W., Muller, K., Salbaum, M.J. and Schmitt, R. 1990. Structure and expression of a single actin gene in Volvox carteri. Current Genetics, 18:337- 346. Cupples,C.G., and Pearlman,R.E. 1986. Isolation and characterization of the actin gene from Tetrahymena thermophila. Proc. Natl. Acad. Sci. USA, 83:5160-5164. Dudler, R. 1990. The single-copy actin gene of Phytophtora megasperma encodes a protein considerably diverged from any other known actin. Plant Molecular 33 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 34 Biology, 14:415-422. Duma, R.J., Rosenblum, W. and McGhee, R.F. 1971. Primary amebic meningoencephalitis caused by Naegleria. Two new cases, response to amphotericin B, and a review. Ann. Intern. Med., 74:923-931. Durica, D.S., Garza, D. Restrepo, M.A. and Hryniewicz, M.M. 1988. DNA sequence analysis and structural relationships among cytoskeletal actin genes of the sea urchin Strongylocentrotus purpuratus. Journal of Molecular Evolution, 28:72-86. Edman, U., Meza, I. and Agabian, N. 1987. Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica. Proc. Natl. Acad. Sci. USA, 84:3024-3028. Fidel, 5., Doonan, J.H. and Morris, N.R. 1988. Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a gamma-actin. Gene, 70:283-293. Firtel, R. A., Timm, R. Kimmel, A.R. and McKeown, M. 1979. Unusual nucleotide sequences at the 5' end of actin genes in Dictyostelium discoideum. Proc. Natl. Acad. Sci. USA, 76: 6206-6210. Fitch,W.M. and Margoliash, E. 1979. Construction of phylogenetic trees. 1967. Science:279-284. Fletcher L.D., McDowell, J. H., Tidwell, R.R., Meagher, R. B. and Dykstra, C.C. 1994. Structure, expression and phylogenetic analysis of the gene encdong actin I in Pneumocystis carinii. Genetics, 137:743-750. Fowler, M., and Carter, R.F. 1965. Acute pyogenic meningoencephalitis probably due to Acanthamoeba spp.: a preliminary report. Br. Med. J. 3:740-742. Francisco, J.A. 1986. On the virtues and pitfalls of the molecular evolutionary clock. The Journal of Heredity, 77:226-235. Genetics Computer Group. 1994 . Program manual for the GCG package, version 8, University of Wisconsin. Gonzalez,I.L. and Schmickel, R.D. 1986. The Human 18S Ribosomal RNA gene: evolution and stability. American Journal of Human Genetics, 38:419-427. Gouy,M. and Gautier, C. 1982. Codon usage in bacteria: correlation with gene expressivity. Nucleic Acids 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 35 Research, 10:7055-7074. Grantham, R. Gautier, C. and Gouy, M. 1980. Codon frequencies in 119 individual genes confirm consistent choices of degenerate bases according to genome type. Nucleic Acids Research, 8(9):1893-1912. Greslin, A.F. Loukin, S.H., Oka, Y. amd Prescott, D. M. 1988. An analysis of the macronuclear actin genes of Oxtricha. DNA, 7 (8):529-536. Grosjean,H. and Fiers, W. 1982. Preferential codon usage in prokaryotic genes: The optimal codon:anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene, 18:199-209. Hamelin, H., Adam, L. Lemieux, G. and Pallotta, D. 1988. Expression of the three unlinked isocoding actin genes of Physarum polycephalum. DNA, 7(5):317-328. Harper, D. S. and Jahn, C.L. 1989. Differential use of termination codons in ciliated protozoa. Proc. Natl. Acad. Sci. USA, 86: 3252-3256. Hasegawa, H., Iida, Y., Yano, T., Takaiwa, F. and Iwabuchi M. 1985. Phylogenetic relationships among eukaryotic kingdoms inferred from ribosomal RNA sequences. Journal of Molecular Evolution, 22:32-38. Hightower, R. C. and Meagher, R. B. 1986. The molecular evolution of actin. Genetics, 114:315-332. Hirono, M., Endoh, H., Okada, N., Numata, O. and Watanabe, Y. 1987. Tetrahymena actin- Clonining and sequencing of the Tetrahymena actin gene and identification of its gene product. Journal of Molecular- Biology, 194:181-192. Hu, Wang-nan. 1992. Gene expression associated with virulence in the amoeba Naegleria fowleri. Ph.D. dissertation. Hu, W.N., Kopachik, W., and Band, R.N. 1992. Cloning and characterization of transcripts showing virulence-related gene expression in Naegleria fowleri. Infection and Immunity, 60:2418-2424. Hyde, J.E., Kelly, S.L., Holloway. S.P., Snewin, V.A. and Sims, P.F.G. 1989. A general approach to isolating Plasmodium falciparum genes using non-redundant oligonucleotides inferred from protein sequences of other organisms. Molecular and Biochemical Parasitology, 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 36 32:247-262. John, D.T. 1982. Primary amebic meningoencephalitis and the biology of Naegleria fowleri. Annual Review of Microbiology, 36:101-123. Kim, K., Gooze, L., Peterson, C., Gut, J. and Nelson, R.G. 1992. Isolation, sequence and molecular karyotype analysis of the actin gene of Cryptosporidium parvum. Molecular.and Biochemical Parasitology, 50: 105-114. Kimmel, A. R. and Firtel, R. A. 1983. Sequence organization in Dictyostelium: unique structure at the 5’-ends of protein coding genes. Nucleic Acids Research, 11(2): 541-552. Knecht, D.A., Cohen, S.M., Loomis, W.F. and Lodish, H. F. 1986. Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors. Molecular and.Cellular Biology, 6(11):3973-3983. Kowbel, D.J. and Smith, M.J. 1989. The genomic nucletide sequences of two differentially expressed actin-coding genes form the sea star Pisaster ochraceus. Gene, 77: 297-308. Krause, M., Wild, M., Rosenzweig, B. and Hirsh, D. 1989. Wild-type and mutant actin genes in Caenorhabditis elegans. Journal of Molecular Biology, 208: 381-392. Lane, D.J., Pace, B., Olsen,G.J., Stahl, D.A., Sogin, M.L. and Pace, N.R. 1985. Rapid determination of 163 ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA, 82:6955-6959. Loomis, W. F. and Smith, D. W. 1990. MOlecular phylogeny of Dictyostelium discoideum by protein sequence comparison. Proc. Natl. Acad. Sci. USA, 87:9093-9097. Ma, P., Visvesvara, G.S., Martinez, A.J., Theodore, F.H., Daggett, P.M. and Sawyer, T.K. 1990. Naegleria and Acanthamoeba infections:Review. Reviews of Infectious Diseases, 12(3):490-513. Maddison, W.F., and Maddison, D.R. 1992. MacClade: Analysis of phylogeny and character evolution. Version 3.0. Sinauer Associates, Sunderland, Massachusetts. Marciano-Cabral, F. 1988. Biology of Naegleria spp. Microbiological Reviews: 114-133. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 37 Martindale, D.W. 1989. Codon usage in Tetrahymena and other ciliates. Journal of Protozoology, 36:29-34. McKeown, M. and Firtel, R.A. 1982. Actin multigene family of Dictyostelium. Cold Spring Harbor Symp. Quant. 3101.,x211: 495-505. McKeown, M. and Firtel, R.A. 1981. Differential expression and 5'end mapping of actin genes in Dictyostelium. Cell,24:799-807. Mertins, P. and Gallwitz, D. 1987. A single intronless actin gene in the fission yeast Schizosaccharomyces pombe: nucleotide sequence and transcripts formed in homologous and heterologous yeast. Nucleic Acids Research, 15(18):?369-7379. Milligan, S.M. and Band, R.N. 1988. Restriction endonuclease analysis of mitochondrial DNA as an aid in the taxonomy of Naegleria and Vahlkampfia. Journal of Protozoology, 35:198-204. Nairn, C.J., Winesett, L. and Ferl, R.J. 1988. Nucleotide sequence of an actin gene from Arabidopsis thaliana. Gene, 65:247-257. Nellen, W., and Sures, I. 1980. Structure of a split yeast gene:complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, 77:2546-2550. Nellen, W., Datta, S., Reymond, C., Sivertsen, 8., Mann, Crowley, T., and Firtel, R.A. 1987. MOlecular biology in Dictyostelium: tools and applications. Methods in Cell Biology. Nellen, W. and Gallwitz, D. 1982. Actin genes and actin messenger RNA in Acanthamoeba castellanii. Nucleotide sequence of the split actin gene I. Journal of Molecular Biology, 159:1-18. Nerad, T.A., and Daggett P.M. 1979. Starch gel electrophoresis: and effective method for separation of pathogenic and nonpathogenic Naegleria strains. Journal of Protozoology, 26:613-615. Pearson, L. and.Meagher, R.B. 1990. Diverse soybean actin transcripts contain 'a large intron in the 5’ untranslated leader: structural similiarity to vertebrate muscle actin genes. Plant Molecular Biology, 14: 513-526. Pernin, P., Cariou, M.L. and Jacquier, A. 1985. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 38 Biochemical identification and phylogenetic relationships in free-living amoebas of thelgenus Naegleria. Journal of Protozoology, 32(4):592-603. Pollard, T.D., and Cooper, J.A. 1986. Actin and actin- binding proteins. A critical evaluation of mechanisms and functions. Annual review of Biochemistry, 55:987-1035. Reddy, 8., Ozgur, K., Lu, M., Chang, W., MOhan, S.R., Kumar, C. and Ruley, H.E. 1990. Structure of the human smooth muscle alpha-actin gene. Analysis of a cDNA and 5' upstream region. The Journal of Biological Chemistry, 265(3):1683-1687. Sambrook,J., Fritsch, E., and Maniatis, T. 1989. Molecular cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Saul, A., and Battistutta,D. 1988. Codon usage in Plasmodium falciparum. Molecular Biochemical Parasitology, 27:35-42. Schuster, F.L. 1979. Biochemistry and physiology of protozoa (volume 1): Small amebas and ameboflagellates. Academic press. New York. Sogin, M.L., Elwood, H.J. and Gunderson, J.H. 1986. Evolutionary diversity of eukaryotic small-subunit rRNA genes. Proc. Natl. Acad. Sci. USA, 83:1383-1387. Sogin, M.L., Ingold, A., Karlok, M., Nielson, H. and Engberg, J. 1986. Phylogenetic evidence for the acquisition of ribosomal RNA introns subsequent to the divergence of some of the major Tetrahymena groups. The EMBO Journal, 5(13):3625-3630. Stranathan, M., Hastings, C., Trinh, H. and Zimmerman, J.L. 1989. Molecular evolution of two actin genes from carrot. Plant Molecular Biology, 13:375-383. Starner, W.T. and Sullivan, D.T. 1989. Letter to the Editor. A shift in the third-codon-position nucleotide frequency in alcohol dehydrogenase genes in the genus Drosophila. Molecular Biology and Evolution, 6:546-552. Sussman, D.J., Lai, E.Y. and Fulton, C. 1984. Rapid Disappearance of translatable actin mRNA during cell differentiation in Naegleria. The Journal of Biological Chemistry, 259: 7355- 7360. Swofford, D.L. 1993. PAUP: phylogenetic analysis using parsimony, version 3.1. Illinois Natural History Survey, 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 39 Champaign. Tannich, E. and Horstmann, R.D. 1992. Letter to the Editor. Codon usage in pathogenic Entamoeba histolytica. Journal of Molecular Evolution, 34:272-273. Tsang, A.S., Mahbubani, H. and.Williams, J.G. 1982. Cell- type-specific actin mRNA populations in Dictyostelium discoideum. Cell, 31: 375-382. Unkles, S.E., Moon, R.P., Hawkins, A.R., Duncan, J.H. and Kinghorn, J.R. 1991. Actin in the oomycetous fungus Phytophthora infestans is the product of several genes. Gene, 100:105-112. Vandekerckhove, J. and Weber, K. 1978. Mammalian cytoplasmic actin are the products of at least two genes and.differ in primary structure in at least 25 identified positions from skeletal actins. Proc. Natl. Acad. Sci. USA, 75(3):1106-1110. Vandekerckhove, J. and Weber, K. 1979. The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. Differentiation, 14: 123-133. Vandekerckhove, J. and Weber, K. 1980. Vegetative Dictystelium cells containing 17 actin genes express a single major actin. Nature, 284:475-477. Vandekerckhove, J. and Weber, K. 1984. Chordate muscle actins differ distinctly from invertebrate muscle actins. Journal of Molecular Biology, 179:391-413. Wada, K., Wada, Y., Doi, H. Ishibashi, F., Gojobori, T. and Ikemura, T. 1991. Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Research, 19:1981-1986. Warrick, H.M. 1987. Codon frequency in Dictyostelium discoideum, In Methods in Cell biology, Vol. 28. Academic Press. N.Y. Wesseling, J.G., de Ree, J.M., Ponnudurai, T., Smits, M.A. and Schoenmakers, J.G.G. 1988. Nucleotide sequence and deduced amino acid sequence of a Plasmodium falciparum actin gene. Molecular and Biochemical Parasitology, 27:313-320. Wesseling, J.G., Smits, M.A. and Schoenmakers, J.G.G. 1988. Extremely diverged actin proteins in Plasmodium falciparum. Molecular and Biochemical Parasitology, 81. 40 30:143-154. Wildeman, A.G. 1988. A putative ancestral actin gene present in a thermophilic eukaryote: novel combination of intron positions. Nucleic Acids Research, 16(6):2553- 2564. Figure 1. Autoradiograph of a Dot Blot of 96 independent clones of N. fowleri. The blot was probed with a 32P end- labelled actin oligonucleotide, one clone, #47, was detected. 41 42 Figure 2. Autoradiograph of a Northern Blot of RNA isolated from amoebae cultured with different food sources and different strains of Naegleri fowleri. Total RNA (10ug) was electrophoretically separated on an agarose gel containing formaldehyde. Lane 1- LEE strain cultured Axenically (Ax), Lane 2- LEE strain cultured with bacteria as a food source (Bact), Lane 3- L.L. strain cultured Axenically (Ax), Lane 4- L.L. strain cultured with bacteria as food source (Bact), Lane 5- Hb-l strain cultured Axenically (Ax), Lane 6- Hb-l strain cultured with bacteria as a food source (Bact) and Lane 7- LEE strain cultured and obtained from the third passage through a mouse (Mp). The blot was probed with a 32P oligolabelled 250bp cDNA actin insert. 43 44 Figure 3. Autoradiograph of a Southern Blot of genomic clones from Naegleria fowleri. Top half of the blot, Lanes 1-12- genomic clones with inserts, size range (500bases to 3.0kb). Lane 13- Bluescript KS+ vector cut with BamH I, and Lane 14- Molecular size marker, lambda restricted with Hind III. Bottom half of blot, 15-25- genomic clones with inserts, size range (250 bases to 2.5kb). Lane 26- Bluescript KS+ vector cut with BamH I, and Lane 27- Molecular size marker, lambda restricted with Hind III. The blot was probed with a ”P end-labelled oligonucleotide specific to the 5' end of the actin gene. 45 46 Figure 4. Autoradiograph of a Southern Blot of restriction fragments from clones #12, #20, #19. Lane 1- clone #19 restricted with Hinc II. Lane 2- clone #19 restricted with Cla I. Lane 3- clone #12, restricted with Cla I and 591 II. Lane 4- clone #12 restricted with EcoR V. Lane 5- clone #20 restricted with 891 II and C1a I. Lane 6- clone #20 restricted with Cla I and Hinc II. Lane 7- cDNA insert 250bp that was used for screening the genomic library. Lane 8- molecular size marker, lambda restricted with EcoR I and Hind III. The blot was probed with an ”P end-labelled oligonucleotide specific to the 3' end of the actin gene. 47 48 Figure 5. Autoradiograph of a Minisouthern Blot of restriction fragments from clones #19, #15, #12 and #20. Lane 1- clone #19 restricted with Hinc II. Lane 2- clone #19 restricted with Cla I. Lane 3- clone #12 restricted with Cla I and Hinc II. Lane 4- clone #15 restricted with Cla I and Hinc II. Lane 5- clone #12 restricted with EcoR V. Lane 6- clone #20 restricted with Cla I and Hinc II. Lane 7- cDNA insert used for screening the genomic library. Lane 8- molecular size marker, lambda restricted with EcoR I and Hind III. The blot was probed with a ”P end-labelled oligonucleotide specific to the 5’ end of the actin gene. 49 50 Figure 6 GENOMIC ACTIN of Naegleri fowleri CAAGCCTCATTCTTGAAGTTGTCAATTTGAAAGGGAGAAATTGTTGGCATTTACAGTAAGACAGT TGCTTTCTTTGAGGATGATCAGACATCTCTCAGAAATGCACACCTTTCATCAAAGTGAATGACZZAA TTTCATTGGGAAGGCAACTTTCATTTATGGTTTGGGTCATCATCCATCACTATCTTGTTTCAA‘I‘IZK CATCAAAAATATCATTGGTTTGTTGAAGGTTGTTGAAGGTCCAGCAACACGTCACACCAAATCT‘I‘ TAAATTTTTTCWTTAACAGCATTCTT'I‘CACACAAACAAAAAACTCAACAACAACTTG CTCTCCAACAAGAACAACAAA - 1 51 M C D D V Q A L V V D N G S G M C ATG TGT GAC GAC GTT CAA GCA CTC GTA GTT GAT AAC GGA TCT GGT ATG TGT ggtatgtgt 102 K A G F A G D D A P R A V F P S I AAG GCT GGT TTC GCT GGT GAT GAT GCA CCA AGA GCT GTC TTC CCT TCC ATC aag gct ggt ttc gct 153 I G R P K Q K S I M V G M G N K D ATT GGT AGA CCA AAG CAA AAG TCC ATC ATG GTT GGT ATG GGT AAC AAG GAT - 204 AYVGDEAQSKRGILTLK GCC TAT GTT GGT GAT GAA GCT CAA TCC AAG AGA GGT ATT TTG ACT TTG AAG 255 YPIEHGIVTNWDDMEKI TAT CCA ATT GAA CAC GGT ATT GTC ACC AAT TGG GAT GAT ATG GAA AAG ATC atg gaa aag atc 306 WHHTFYNELRVAPEEHP TGG CAT CAC ACC TTC TAC AAT GAA TTG AGA GTT GCT CCA GAG GAA CAT CCA tgg cat cac acc ttc tac aat gaa ttg aga gtt gct cca gag gaa cat cca 357 V L L T E A P L N P K A N R E K M GTC TTG TTG ACT GAA GCT CCA TTG AAT CCA AAG GCT AAC AGA GAA AAG ATG gtc ttg ttg act gaa gct cca ttg aat cca aag gct aac aga gaa aag atg 408 TQIMFETFSVPAMYVAI ACT CAA ATC ATG TTT GAA ACC TTC TCT GTT CCA GCC ATG TAT GTT GCC ATT act caa atc atg ttt gaa acc ttc tct gtt cca gcc atg tat gtt gcc att 459 QAVLSLYASGRTTGIVL CAA GCT GTC TTG TCT TTG TAT GCT TCT GGT CGT ACC ACT GGT ATT GTT TTG caa gct gtc ttg tct ttg tat gct tct ggt cgt acc act ggt att gtt ttg 510 DSGDGVSHTVPIYEGYA 51 cont. Figure 6 GENOMIC ACTIN of Naegleria fowleri GAC TCT gac tct L TTG ttg P CCT cct Y TAC L TTG E GAG R AGA D GAC TTT S TCT TAT R AGA aga C TGT tgt G V GGT GTC ggt gtc R K AGA AAG aga aag E G GAA GGT 988 ggt S M TCC ATG tcc atg I G ATT GGA att gga Figure 6. .N..fow1eri. 5’ region and ending at +1071 GATC. actin nucleotide sequence determined from clone ACT 1. GGT GAT GGT GTC TCT CAC ACT GTT CCA ATT TAT GAA GGT ggt gat ggt gtc tct cac act gtt cca att tat gaa ggt H A I L R L D L A G R D L CAT GCT ATT TTG AGA TTG GAT TTG GCT GGT AGA GAT TTG cat gct att ttg aga ttg gat M K I L M E R G Y S F N T ATG AAG ATT CTC ATG GAA CGT GGT‘TAC TCA TTC AAT ACC E GAA I ATT V GTC R AGA D GAT I ATC K E K L C Y I AAG GAA AAG CTC TGT TAT ATT Ii ATT E GAA E GAA M ATG K AAG A A E S S S V GCT GCT GAA TCA TCC TCC GTT Q CAA E GAA N AAC V I T V G N E GTG ATT ACT GTT GGA.AAT GAA gtg att act gtt gga aat gaa L TTG P CCA D GAC G GGT M ATG atg E GAA gaa V GTT gtt P N F I G CCA AAC TTC ATT GGT cca aac ttc att ggt E GAA gaa CCA cca TTG ttg TTC ttc CAA 088 E GAA gaa T ACA aca F TTC ttc S I TCG ATT tcg att G GGA 993 S TCT tct K C AAG TGT aag tgt D I GAT ATT gat att CAT cat AAC aac TCT tct D GAT gat V GTC gtc K AAG G G GGT GGT ggt ggt T N ACC AAC T ACT act L TTG ttg G GGT ggt R AGA N AAC aac L TTG ttg TAT tat GTT gtt ACC acc I A ATT GCT M ATG E GAA L TTG M ATG A GAG ACC GCT TAT tat ACT ACT A GCT E GAA R AGA aga A GCT gct D GAT gat M ATG atg P CCT GCT gct 561 D CAT 612 A GCT 663 L TTG 714 K AAG 765 F TTC ttc 816 A GCT gct 867 I ATC atc 918 F TTT ttt 969 A GCT att gct K I AAG ATT aag att G S GGT TCC ggt too 939 K AAG aag I ATC atc aga atg V GTT gtt V GTG gtg L A TTG GCT ttg gct GCC A GCT gct S TCA tca aag P CCA cca L TTG ttg gaa CCA cca S TCC tcc ttg E GAA gaa T ACC acc 8C0 aac R K AGA AAG aga aag F 0 TTC CAA ttc caa atg Y TAC tac Q CAA caa gct S TCG tcg M ATG atg cct gct 1020 V W- GTC TGG gtc tgg 1071 W I TGG ATC tgg atc Nucleotide and deduced amino sequence of a genomic of Negative numbers indicate 5' upstream sequence. line- deduced amino acid sequence. First Second line- partial genomic actin nucleotide sequence determined from clone ACT 8, encompassing putative TATA-like sequence is underlined. 52 Third line- partial genomic The Figure 7. GENOMIC ACTIN of Naegleria fowleri 1122 T K E E Y E D A G P G I V H R K 8 ACC AAG GAA GAA TAT GAG GAT GCC GGT CCA GGT ATT GTC CAC AGA AAG AGC F * TTC TAA +65 ATTGACCTTGGATGCACATTATCAAATTCCATTGTAAIAAAACATAAAATCTATGTAAAATCATG +130 CATGAGTTGTGTCTTTGTAAAATTGATTTGTAGTCTCCTATTATGTCAATTTTGTGTTTGGTTAT GGTT FIGURE 7. The partial 3’ nucleotide sequence and deduced amino acid sequence of clone ACT 1. Clone ACT 1 encompassed the 3’f1anking region and coding region ending at +38 GATC. The stop codon TAA is designated with a (*). The polyadenylation signal AATAAAA is underlined and occurs 36 bases after the stop codon. 53 wwocnm mm. mmnmwsos< msmwwmwm 0m monw: muonmw: mmncmsomm. one on nsm nsnmm sow» cmnmwsoswocm nnmmm Om Hanan: 8mm A0.H.uo.mm. w.H.no.muv. wmoosmnnconwos om nso nnmm cmwso smonwmdm. oosvwmnm won»: pnonmw: mmacmsom om mm nmxm ammo msmwwnmd flung sawso woman” HNH UswpoomsmnwomeK MBHOHSmnw