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Characterization of nuclear basic proteins in sperm and erythrocytes of vertebrates Su, Hua Wei 2004

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Characterization of Nuclear Basic Proteins in Sperm and Erythrocytes of Vertebrates by HUAWEI S U B.Sc., East China University of Science & Technology, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE R E Q U I R E M E N T S FOR THE D E G R E E OF MASTER OF S C I E N C E In THE FACULTY OF G R A D U A T E STUDIES Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 2004 © H u a W e i Su, 2004 Library Authorization In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. HUAWEI SU 16/03/2004 Name of Author (please print) Date (dd/mm/yyyy) Title of Thesis: Characterization of Nuclear Basic Proteins in Sperm and Erythrocytes of Vertebrates Degree: Master of Science Department of Zoology The University of British Columbia Vancouver, BC Canada Year: 2004 A B S T R A C T I have examined the arginine-enriched nuclear basic proteins (NBPs) that condense chromatin in two kinds of transcriptionally inactive, terminally differentiated cells of non-mammalian vertebrates: sperm and erythrocytes. I have focused on the characterization of sperm nuclear basic proteins (SNBPs) in teleost fish and erythrocyte nuclear basic proteins (ENBPs) in reptiles and birds by electrophoresis, high pressure liquid chromatography and amino acid analysis to ask: 1) Is internal fertilization a constraint on the diversity of SNBPs seen in teleost fish and frogs? 2) Is bird linker histone H5 also present in alligator erythrocyte nuclei? I have found that internally fertilizing Xiphophorus helleri guentheri (swordtail), Xiphophorus maculatus (platyfish), Poecilia reticulata (guppy), Poecilia picta (guppy) and Cymatogaster aggregata (shiner perch) all contain protamines as their SNBPs. Amongst frogs, there is a sporadic reversion from protamines in the primitive, internally fertilizing frog Ascaphus truei, to sperm histones among several more advanced, externally fertilizing frogs (Kasinsky et al., 1999). This parallels a simplification of elongated introsperm to more rounded ectaquasperm in advanced neobatrachians that is regarded as a secondary reversion to external fertilization (Lee and Jamieson, 1992). However, characterization of SNBPs from the internally fertilizing neobatrachian frog Eleutherodactylus coqui shows the presence of four protamines. These results favor the hypothesis (Kasinsky, 1989) that fish and frogs with internal fertilization tend to select arginine-rich protamines as their SNBPs rather than sperm histones, whereas external fertilization is compatible with protamine-to-histone reversions in some advanced frogs. Is linker histone H5 unique to bird erythrocyte nuclei? H1°, another differentiation-specific linker histone in vertebrates, is not erythrocyte-specific. I have characterized ENBPs from Ictalurus punctatus (catfish), Rana catesbeiana (bullfrog), Chelonia mydas (sea turtle), Alligator mississippiensis (alligator), and Gallus gallus (chicken), as well as possible NBPs in the condensed nucleus of mouse normoblasts, the precursors of mature enucleated red blood cells. Alligator ENBPs appear to contain a linker histone H1°/H5 which is midway in the evolutionary trend from H1° to H5. This is consistent with the fact that alligators have a higher average blood pressure than sea turtles but much lower than in birds. i i TABLE OF CONTENTS Abstract ii List of Tables vi List of Figures vii Abbreviations x Acknowledgements xii 1 Introduction 1 1.1 DNA and nuclear basic proteins (NBPs) ....1 1.2 Sperm nuclear basic proteins (SNBPs) and erythrocyte nuclear basic proteins (ENBPs) 3 1.2.1 Classification of SNBPs 4 1.2.2 SNBP transitions during spermiogenesis 8 1.2.3 Evolutionary relationship of SNBPs to somatic histones 12 1.2.4 Classification of ENBPs in vertebrates 12 1.2.5 ENBP transitions during erythropoiesis 17 1.2.6 Evolution of linker histones 17 1.3 Questions to be examined in this thesis 20 1.3.1 Does internal fertilization constrain the range of SNBP diversity in teleost fish and frogs? 20 1.3.2 Is linker histone H5 present in alligator erythrocytes? 23 2 Materials and Methods 28 2.1 Living organisms 28 2.1.1 Internally fertilizing teleost fish examined for sperm nuclear basic proteins (SNBPs) 28 2.1.2 Internally fertilizing frog examined for SNBPs 28 2.1.3 Vertebrates examined for erythrocyte nuclear basic proteins (ENBPs)..29 2.2 Nuclear basic protein extraction 32 2.2.1 Extraction of SNBPs 32 2.2.2 Extraction of ENBPs 32 2.2.3 Extraction of liver nuclear basic proteins (NBPs) 33 2.3 Gel electrophoresis • 34 2.3.1 Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis 34 2.3.2 Acid urea polyacrylamide gel electrophoresis 36 2.3.3 Acid urea polyacrylamide gel electrophoresis for resolving peptides 37 2.3.4 Acid urea Triton polyacrylamide gel electrophoresis 37 2.4 Protein fractionation 38 2.4.1 Fractionation of SNBPs 38 2.4.2 Fractionation of ENBPs 38 2.5 Chymotrypsin digestion 39 2.6 Amino acid analysis 39 iii 3 Results 40 3.1 Characterization of SNBPs from internally fertilizing teleost fish 40 3.1.1 Comparison of SNBPs from Xiphophorus, Poecilia, and CymatogasterXo see if protamines or protamine-like proteins are present 40 3.1.2 SNBPs from testis of Xiphophorus helleri guentheri (swordtail) and Xiphophorus maculatus (platyfish) with different times of sexual maturity 41 3.1.3 Fractionation of SNBPs from testis of Cymatogaster aggregata (shiner perch) 42 3.1.4 Amino acid analysis of the protamine fraction from Cymatogaster aggregata (shiner perch) 42 3.1.5 SNBPs in teleost fish with male preganancy 48 3.2 Characterization of SNBPs from an internally fertilizing frog 48 3.2.1 SNBPs from Eleutherodactylus coqui 48 3.2.2 Fractionation of the SNBPs from Eleutherodactylus coqui 51 3.2.3 Amino acid analysis of the protamine-like protein fractions from Eleutherodactylus coqui 51 3.3 Characterization of ENBPs from vertebrates 54 3.3.1 Comparison of ENBPs from Gallus domesticus (chicken), Alligator mississippiensis (alligator), Rana catesbeiana (bullfrog), Ictalurus punctatus (channel catfish), and NBPs from Mus musculus (mouse) spleen normoblasts 54 3.3.2 Fractionation of ENBPs from Gallus domesticus (chicken), Alligator mississippiensis (alligator), Rana catesbeiana (bullfrog), and Ictalurus punctatus (channel catfish) 55 3.3.3 NBPs from erythrocytes of Chelonia mydas (sea turtle) compared to NBPs from spleen normoblasts of Mus musculus (mouse) 61 3.3.4 Fractionation of the ENBPs from Chelonia mydas (sea turtle) 61 3.3.5 Comparison of the H1 and/or H5 linker histone fractions of erythrocyte and liver NBPs from Gallus domesticus (chicken), Alligator mississippiensis (alligator), Chelonia mydas (sea turtle), Rana catesbeiana (bullfrog), and Ictalurus punctatus (channel catfish) 62 3.3.6 Chymotrypsin digestion pattern of linker histone fractions of erythrocyte and/or liver NBPs from Gallus domesticus (chicken), Alligator mississippiensis (alligator), Rana catesbeiana (bullfrog), and Ictalurus punctatus (channel catfish) 68 3.3.7 Amino acid analysis of the linker histone fractions from erythrocytes of Gallus domesticus (chicken), Alligator mississippiensis (alligator), Chelonia mydas (sea turtle), Rana catesbeiana (bullfrog), and Ictalurus punctatus (channel catfish) 72 4 Discussion 75 4.1 Internally fertilizing fish tend to have protamine-like SNBPs 75 4.2 The internally fertilizing frog Eleutherodactylus coqui has protamine-like SNBPs 77 iv 4.3 ENBPs from vertebrates 79 4.3.1 "Linker histone H1°/H5" in Alligator mississippiensis (alligator) ENBPs 80 4.3.2 Linker histone H1° in Chelonia mydas (sea turtle) ENBPs 82 4.3.3 Linker histone H1° in Rana catesbeiana (bullfrog) ENBPs 82 4.3.4 Linker histone H1° in Ictalurus punctatus (channel catfish) ENBPs 83 4.3.5 ENBPs in Mus musculus (mouse) normoblasts 84 4.4 The evolutionary trend of linker histones 84 4.5 Future work 87 4.6 General conclusion 87 5 References 89 LIST OF TABLES 1. Historical classifications of SNBPs 5 2. Historical classification of the linker histone variants in Xenopus laevis (frog) erythrocytes 16 3. Criteria used in this thesis to determine the SNBP type 26 4. Criteria to determine the linker thistone variants in vertebrates used in this thesis 27 5. Criteria to determine the linker thistone variants in vertebrates used in this thesis 35 6. Amino acid composition (mol%) of SNBPs from testis of Cymatogaster aggregata (shiner perch) after HPLC 47 7. Amino acid composition (mol%) of SNBPs from testis of Eleutherodactylus coquiafter HPLC 53 8. Amino acid composition (mol%) of linker histones from vertebrates after HPLC 74 vi LIST OF FIGURES 1. The way linker histone H1 is thought to bind to the nucleosome 2 2. Representative examples of SNBPs from the H/H1, PL, and P types 7 3. Developmental history of a single spermatocyst in Scylliorhinus caniculus (dogfish) 9 4. Interaction of cc-helical protamines with DNA 11 5. Evolutionary relationship of SNBPs to nucleosomal and linker histones 13 6. Schematic diagram of a nucleus extruded from a developing mammalian red blood cell (eryth rob last) 18 7. Schematic diagram showing the evolutionary trend of linker histones 19 8. SNBP diversity in the chordates 21 9. Comparison of systolic and diastolic levels of systemic blood pressure in vertebrates 25 10. Phylogeny of internally fertilizing teleost fish examined for SNBPs 30 11. Phylogeny of vertebrate orders examined for ENBPs 31 12. Acid-urea-PAGE of SNBPs in testis of internally fertilizing fish 43 13. Acid-urea-PAGE of SNBPs in testis of X. maculatus and X. helleri guentheri with different times of sexual maturity 44 14. Acid-urea-PAGE of SNBPs in testis of X. maculatus with different times of sexual maturity 45 15A. Reverse phase HPLC fractionation of C. aggregata (shiner perch) SNBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column 46 15B. Acid-urea-PAGE analysis of C. aggregata (shiner perch) SNBP fractions obtained from the HPLC shown in Figure 15A 46 16. Acid-urea-PAGE of SNBPs in testis of S. schlegeli 49 17A. Acid-urea-PAGE of SNBPs in internally fertilizing E. coqui 50 17B. Acid-urea PAGE of SNBPs from different frogs 50 vii 18A. Reverse phase HPLC fractionation of E. coqui SNBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column 52 18B. Acid-urea-PAGE analysis of E. coqui SNBP fractions obtained from the HPLC shown in Figure 18A 52 19. SDS-PAGE analysis of NBPs from erythrocytes and livers of chicken, alligator, frog, and catfish 56 20A. Reverse phase HPLC fractionation of G. domesticus (chicken) ENBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column 57 20B. SDS-PAGE analysis of G. domesticus (chicken) ENBP fractions obtained from the HPLC shown in Figure 20A 57 21 A. Reverse phase HPLC fractionation of A. mississippiensis (alligator) ENBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column 58 21B. SDS-PAGE analysis of A. mississippiensis (alligator) ENBP fractions obtained from the HPLC shown in Figure 21A 58 22A. Reverse phase HPLC fractionation of R. catesbeiana (bullfrog) ENBPs, using a (25x0.46 cm) 300-A Vydac C 1 8 column 59 22B. SDS-PAGE analysis of R. catesbeiana (bullfrog) ENBP fractions obtained from the HPLC shown in Figure 22A 59 23A. Reverse phase HPLC fractionation of /. punctatus (channel catfish) ENBPs, using a (25x0.46 cm) 300-A Vydac Ci 8column 60 23B. SDS-PAGE analysis of /. punctatus (channel catfish) ENBP fractions obtained from the HPLC shown in Figure 23A 60 24. SDS-PAGE analysis of NBPs from mouse spleen normoblasts, and chicken, alligator, sea turtle, frog, and catfish erythrocytes and livers 63 25A. Reverse phase HPLC fractionation of C. mydas (sea turtle) ENBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column 64 25B. SDS-PAGE analysis of C. mydas (sea turtle) ENBP fractions obtained from the HPLC shown in Figure 25A 64 26. Acid-urea-Triton-PAGE analysis of erythrocyte and liver NBPs from alligator and chicken, and their HPLC fractions (H1 and/or H5 linker histones) 65 27. Acid-urea-Triton-PAGE analysis of erythrocyte and liver NBPs from alligator viii and catfish, and their HPLC fractions (H1 and/or H5 linker histones) 66 28. Acid-urea-Triton-PAGE analysis of the erythrocyte and liver NBPs from bullfrog and chicken, and their HPLC fractions (H1 and/or H5 linker histones) 67 29. Acid-urea-Triton-PAGE analysis of ENBPs from chicken, alligator, and sea turtle, arid HPLC fractions (H1 and/or H5 linker histones) from sea turtle ENBPs in Figure 25A 69 30. Composite figure of acid-urea-Triton-PAGE showing lack of H5 linker histone in sea turtle ENBPs 70 31 A. Reverse phase HPLC fractionation of C. mydas (sea turtle) ENBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column 71 31B. Acid-urea-Triton-PAGE analysis of C. mydas (sea turtle) ENBP fractions obtained from the HPLC shown in Figure 31A 71 32. Acid-urea -PAGE of chymotrypsin digestion of NBP HPLC fractions from chicken, alligator, bullfrog and catfish 73 33. SNBP diversity in internally fertilizing bony fish and frogs 76 34. Distribution of SNBP types as related to sperm head morphology in anurans 78 35. A proposed hypothesis for the evolutionary trend of linker histones 86 ix ABBREVIATIONS ACN - acetonitrile APS - ammonium persulfate AU - acid-urea AUT - acid-urea-Triton bp - base pair BPB - bromophenol blue cDNA - complementary deoxyribonucleic acid Da - Dalton DNA - deoxyribonucleic acid EC - enzyme commission EDTA - ethylenediaminetetraacetic acid E. F. - external fertilization EGME - ethylene glycolmonoethylether ENBPs - erythrocyte nuclear basic proteins H - core histone H2A, H2B, H3, H4 - core histone H1 - linker histone H1° - linker histone of differentiated cell H1°/H5 - linker histone of alligator erythrocytes H5 - arginine-enriched linker histone of bird erythrocytes HCI - hydrochloric acid HPLC - high performance liquid chromatography I. F. - internal fertilization KCI - potassium chloride KP - keratinous protamine LB - Luria-Bertani MgCI2 - magnesium chloride NaCl - sodium chloride NBPs - nuclear basic proteins P - protamine PAGE - polyacrylamide gel electrophoresis PL - protamine-like or intermediate proteins RBC - red blood cell SDS - sodium dodecyl sulphate SNBPs - sperm nuclear basic proteins TEMED - N,N,N\N'-tetramethylethlenediamine TFA - trifluoroacetic acid TLCK - tosyllysine chloromethyl ketone OHZ - phenylhydrazine-treated xi ACKNOWLEDGEMENTS I would like to express my gratitude to the people who have inspired and helped me to produce this thesis: I am forever indebted to my thesis supervisor, Dr. Harold Kasinsky, for his enthusiasm, guidance, patience, dedication, encouragement, and understanding. I couldn't find the word to express my appreciation. Thank you so much! And I wish to thank Vicki, Leah, and Jeremy for their hospitality and friendship. I am grateful to Dr. Juan Ausio, for his inspiration and enthusiasm. I had a great time in his lab with Wade, John, Yue, Allison, Lindsay, Deanna, Susan, Pepita and Xiaoying. I would like to thank my committee members, Dr. Wayne Vogl, Dr. Rosie Redfield, Dr. Eric Taylor and Dr. William Milsom who kindly gave me comments and suggestions during the progress of my project. I also want to thank Amanda Southwood, Manuela Gardner, Howard Rundle, Ramona deGraff, Andrew Hendry, Aydan Peterson, Satoshi Watanabe, Dr. Robin Liley, Dr. David Jones, Dr. Keith Humphries, Dr. Tom Mommsen, Dr. James Hanken, and Dr. Jurgen Vielkind for providing me valuable experimental materials. Finally, my thanks go to my wife, Yi Zhou and my family. Without the support from them, I would never have been able to finish my thesis. xii 1. Introduction 1.1 DNA and nuclear basic proteins (NBPs) Chromatin, the complex of nuclear DNA and nuclear basic proteins (NBPs), including histones and nonhistone chromosomal proteins, is the basic structure of the eukaryotic chromosome in somatic cells. Histones can be structurally grouped into two main categories: core histones with the characteristic histone fold motif and linker histones with the unique globular winged helix domain. Histones make up the majority of nuclear basic proteins and play the key role in packing the 46 long DNA molecules in a human cell totaling about 2m in length (2 million |jm) into a tiny nucleus only about 10 um in diameter, with a volume of about 400 um3. The most fundamental structural unit of chromatin is the nucleosome core particle without the linker histone (Figure 1A). 146 bp DNA are wrapped around an octamer of the core histones, consisting of a tetramer of histones H3 and H4 ((H3/H4)2) and two dimers of H2A/H2B, to form the nucleosome core particle. The core histones are small basic proteins (11,000-16,000 daltons) with more than 20 mol% of the amino acid residues being lysine and arginine (Wolffe, 1998). Each of the core histones contains a histone fold domain with amino- and carboxyl-terminal tails which have a highly basic amino acid composition (Arents et al., 1991). Electrostatic interaction and hydrogen-bonding between the phosphodiester backbone of DNA and the basic amino acid residues present in the histone octamer of the core histones are the most important interactions for organizing DNA in the nucleosome. Linker histone H1 is also lysine-rich and a larger protein (>20,000 daltons) compared to the core histones (Wolffe, 1998). Linker histone H1 contains a globular 1 A) nucleosome DNA helix histone octamer B) linker histone H1 N globular domain * - C N C) H1 and nucleosome interaction Figure 1. The way linker histone H1 is thought to bind to the nucleosome. A: nucleosome; B: linker histone H1; C: H1 and nucleosome interaction in which the globular domain of linker histone H1 binds to nucleosome near the site where the DNA helix enters and leaves the histone octamer. N and C designate the N- and C- terminal of linker histone H1, respectively. 2 domain flanked by two less basic amino- and carboxyl-terminal domains (Figure 1B). This globular domain consists of a unique winged helix motif (Ramakrishnan et al., 1993). The highly tissue-specific proteins such as linker histone H5 from the nucleated erythrocytes of birds (Neelin et al., 1964) are also included in the linker histone H1 family. Nucleosomes are usually packed together by linker histone H1 through the interaction with linker DNA (Figure 1C) to form regular higher-order chromatin structure (van Holde, 1989). Core histones are among the most highly evolutionarily conserved proteins (Isenberg, 1978) and are present in somatic cells of all eukaryote except dinoflagellates, which package the major portion of their DNA with small basic proteins completely unlike histones (Vernet era/., 1990). However, linker histone H1 is less evolutionarily conserved than core histones (Isenberg, 1978; Cole, 1984), especially in the amino-and carboxyl-terminal domains. 1.2 Sperm nuclear basic proteins (SNBPs) and erythrocyte nuclear basic proteins (ENBPs) In this thesis, I will examine two categories of nuclear basic proteins (NBPs) that condense chromatin in two kinds of transcriptionally inactive, terminally differentiated cells: sperm and erythrocytes. Although the inactivation of chromatin in these two cell types proceeds by different routes, the final products of these processes, i.e., sperm and erythrocytes, have been inactivated by interaction with their respective SNBPs or ENBPs that are usually arginine enriched. I will pay special attention to the presence of arginine side chains in both SNBPs of sperm and ENBPs of erythrocytes during the condensation of chromatin in non-3 mammalian vertebrates. This is because positively charged arginine side chains can not only form ionic linkages with negatively charged phosphodiester bonds of DNA, as can lysine side chains, but can also form more hydrogen bonds with DNA than can lysine residues, thus leading to a more condensed state of chromatin. 1.2.1 C l a s s i f i c a t i o n o f S N B P s Sperm nuclear basic proteins (SNBPs) are found in the sperm of both vertebrates and invertebrates (Kasinsky, 1989). SNBPs contain large amounts of basic amino acid residues which are tightly associated with the DNA, resulting in extremely condensed chromatin in the sperm. In contrast to the evolutionary conservative nucleosomal histones in somatic cells and the somewhat variable H1 linker histones, SNBPs that compact DNA in the nucleus of a sperm head are highly diverse (Kasinsky etal., 1985; Poccia, 1976; Kasinsky, 1989; Ausio, 1999; Lewis etal., 2003). Historically, there have been several attempts to define SNBPs (Table 1). B l o c h : Based on cytochemical criteria, Bloch (1969, 1976) proposed five different classes of sperm nuclear basic proteins: Type 1. Protamine type: these are low molecular weight (3000-5000 Da), arginine-rich proteins. This type of proteins is also known as monoprotamines (Kossel, 1928). Examples are salmon, herring, and mackerel. Type 2. Stable protamine or keratinous protamine type: these proteins contain cystine as well as arginine. An example of this type is found in the bull (Krawetz et al., 1987). Type 3. Intermediate sperm basic protein type: also known as diprotamines or triprotamines as they contain the basic amino acids lysine and/or histidine, as well as arginine (Kossel, 1928). They are intermediate in their properties between histones and protamine and have a higher molecular weight (6500-50000 Da). Examples of this type 4 CO D_ CD Z CO CO c o co o co co JO O "CO o o w I 0) .Q CC c g "55 o CL E o o •g o CO o c 'E < o *-75.21 0 Q . Q_ CD -z. in "to 'E CD . C O o % o c o T3 0 CO CO -Q CO c _o *-*—• CO o co co J O O CD s i E g . co Q-(D O « c 3 ° tO Q_ C/D ^ T - 1 c\i 0 CD CL CL h - l -O CO CO CD C L CO CD CO T3 0 0 O « co . E Z ® . +—. 0 2 •= Q-6.2x3 CO 0 -* CO 05 ^ E - " £ _ Q.C0 Z -a cd ^r ' in 0 0 0 Cu Q . Q . h- h h o o CQ CO CO CD 0 5 0 £ 5 c <-•T co co a> •-_Q CO 0 £ C CO CO 0L9 I ^ S m f l c T3 c CO ^ o o E LO CM o 0 ~ 0 8 I S + s 0 £ .E + « CD — E 0 '>-> 0 CO « X co CO 0 ' CO cO C N E . E - c X 0 ^ " - a a o D c ^3* 0 CO1 c c o o o col c CO !5 (/) m CO 00 CD o " E = °-3.S2 t o ' 0 - Q E o S " ^ ^ 0 £ 0 J 5 ^ 5 T < 1 ) C C L " " " Q . C L C L -> S > ^ > . 5^ h- h- coT E o 9 ' i O-r, ^ C I75 « +- £ s i -I—* C CO o c ° 0 C C L CO . t r J l € CO CO 1— L>»0 cO 0"§ 2 > — - O CO O O l l £ o i CO CO E E o o o o 6 o o 00 0 CL X X 0 c CO + 0 c 'c 'cn CO "o 2^  o E o up LO co < CO CO 0 CO c CD 0 > c CO o X o o O O o LO I o o LO CO 0 CL '35 < CD CT> 0 0 O ) c c *= CD 0 ^ 0 45 O O O X O 1 - O O O O LO 6 o o CO 0 C L are found in bivalve mollusks, such as surf and razor clams (Ausi6,1986). Type 4. Somatic-like histone type: An example of this type is found in grass carp (Kadura, 1983). Type 5. No basic proteins detected in the nucleus of the mature sperm. An example of this type is found in crab (Bloch, 1969). Subirana: Since some species may contain sperm nuclear basic proteins falling within several of the above types, Subirana (1983) proposed a broader classification: sperm protamines and sperm histones. Sperm protamines are SNBPs with a lysine plus arginine composition of 45 to 80 mol% and a serine plus threonine content of 10 to 25 mol%. Sperm histones are SNBPs that can have four different types of nuclear composition: (1) no detectable change in the family of very lysine-rich H1 linker histones; (2) slight changes in the family of H1 histones; (3) additional sperm-specific basic proteins; (4) considerable changes in histones H1 and H2B. Ausio: Based on current information, Ausio (1986, 1999) proposed a new classification in which SNBPs are grouped into three major categories: histone H/H1 type, protamine-like PL type and protamine P type (Figure 2). H/H1 type consists of histones that are found in the nuclei of somatic cells, but are somewhat more arginine-rich with 20-30 mol% of lysine plus arginine content (Ausio, 1995). This group includes germinal histones and contains a trypsin-resistant globular domain. Their molecular weights are around 8,000 to 20,000 daltons. H/H1 type is equivalent to the somatic-like histone type in Bloch's (1969, 1976) classification. 6 globular domain 1) H/H1 N - 0 " - >c 2) PL N § - 111 Rim Jit. . R Run 3) PL ^ R B R R 4) P N —R rt R R R R—R—R—3^ Q 5) KP ^ —R—| R—R R—R—R ^-C I s N — ^ « - R — R — f t — R >- C Figure 2 . Representative examples of SNBPs from the H/H1, PL, and P types. H/H1 = histones; PL = protamine-like proteins; P = protamines; KP = keratinous protamines. PL type contains protamine-like proteins with and without the globular domain. P type contains protamines and keratinous protamines which can form disulfide bonds with each other. N and C designate the N- and C- terminal of linker histone H1, respectively. R represents the arginine residue in the SNBPs. 7 PL type is the most structurally heterogeneous group with molecular weight ranging from 6,500 to 50,000 daltons. PL type is closely related to somatic very lysine-rich histone H1 (Ausio, 1992) and includes transition proteins PL-I, Pl-ll, and PL-IV (Jutglar et al., 1991; Giancotti et al., 1992; Carlos et al., 1993) with a globular domain and transition protein PL-Ill (Rocchini era/., 1995) without a globular domain. It consists of basic proteins having an arginine plus lysine content that usually amounts to at least 35 to 50 mol% (Ausio, 1995). This type is equivalent to the intermediate sperm basic protein type in Bloch's (1969; 1976) classification. P type consists of relatively small (3,000 to 5,000 daltons), arginine-rich highly basic proteins called protamines and keratinous protamines which are also cystine-rich. Their arginine content is at least 30 mol%, their histidine plus lysine plus arginine content is 55 to 80 mol% and their serine plus threonine plus glycine content is 10 to 25 mol% (Hunt et al., 1996). P type is equivalent to the protamine type and the stable protamine (or keratinous protamine) type in Bloch's (1969; 1976) classification. In this thesis, I am using Ausio's (1999) classification in which SNBPs are grouped into three categories: H/H1 type, PL type and P type. 1.2.2 SNBP transitions during spermiogenesis During spermiogenesis, somatic histones in spermatocytes are almost always replaced by spermatid-specific histones. The transition proteins replace germinal histones during the initial stages of condensation of chromatin. They are finally replaced by protamines, which are almost the only SNBPs in the sperm nuclei of most mammals with some H/H1 still present and the only SNBPs in many fish (Steger et al., 1998). A diagram of the developmental history of a single spermatocyte in Scylliorhinus caniculus (dogfish) is shown in Figure 3 (Stanley, 1966), where histones are gradually replaced by 8 Figure 3. Developmental history of a single spermatocyst in Scylliorhinus caniculus (dogfish). The main point of this figure is to show the SNBP transitions during spermiogenesis in S. caniculus where H/H1 type (steps l-V) SNBPs are gradually replaced by PL type (step VI) SNBPs, and finally by P/KP type (step VII) SNBPs. As this occurs, the chromatin becomes more and more condensed as indicated by the increasing darkness of the sperm heads. Development proceeds from lower right (I) around the upper right (VII). H/H1 = histones; PL = protamine-like proteins; KP = keratinous protamines. (Figure and legend are modified from Stanley, 1966). 9 protamine-like proteins, and finally by keratinous protamines. As this occurs during spermiogenesis the chromatin becomes more and more condensed (Gusse and Chevaillier, 1978). The degree of histone replacement by protamines may vary among species (Wolfe, 1993). In human sperm, approximately 15% of the DNA is associated with histones, whereas the other 85% interacts with protamines (Oliva and Dixon, 1991). In some sperm of molluscs, around 80-90% of the DNA is complexed with protamines, while in the winter flounder, this percentage is much lower, at 25% (Wolfe, 1993). In the presence of protamines, the DNA found in the newly formed sperm is extremely condensed and transcriptionally inactive (Oliva and Dixon, 1991). However, not all species have some of their histones replaced by protamines during spermiogenesis. This is the case in some bony fish like grass carp (Kadura era/., 1983) and in sea urchins (Oliva and Dixon, 1991). The fact that the DNA in the mature sperm is much more condensed than the DNA found in the somatic cells reflects the difference in the amino acid composition and structure of histones and protamines. The histone folds of the slightly lysine-rich and arginine-rich histones constitute an octamer core around which negatively charged DNA is wrapped in the nucleosome structures that resembling a string of beads (Luger et al., 1997). Protamines are highly arginine-rich basic proteins, much smaller than histones, which can run parallel to DNA fibers in the sperm nucleus and bind to them in a partial helical form (Figure 4) (Subirana, 1983). Protamines can bind more efficiently to DNA than histones because arginine, the major component of protamines, can interact more strongly with the DNA backbone and potentially form more hydrogen bonds than lysine, the major component of histones (Helene and Lancelot, 1982; Ausio 10 11 era/., 1984). In additional, cysteines, found in mammalian protamines, can form cross-linking disulfide bonds to provide further condensation of the chromatin in mammalian sperm (Balhorn era/., 1992, 1999). Such structures can condense nuclear chromatin in mammalian sperm DNA, the most tightly compacted eukaryotic DNA, at least six fold greater than in metaphase chromosomes of somatic cells (Ward and Coffey, 1991). 1.2.3 Evolutionary relationship of SNBPs to somatic histones Subirana et al (1973), Subirana and Colom (1987) and Ausio (1986, 1995, and 1999) proposed that protamine evolved from the PL protein intermediate, which in turn arose from the primary H/H1 histone precursor. Linker histone H1 gave rise to the more arginine-rich PL-I protein. Post-translational cleavage of the PL-I protein (loss of its globular domain) resulted in the PL-protein, which eventually leads to protamine: H / H 1 ^ P L ^ P (Ausio, 1986, 1995, 1999) (Figure 5). Support for this view comes from the demonstration of a frameshift mutation in the tunicate Styela montereyensis that enhances the content of arginine side chains in the SNBP of the mature sperm (Lewis et al., 2003). 1.2.4 Classification of ENBPs in vertebrates Red blood cells (RBCs) of vertebrates are circulating cells containing hemoglobin. In all vertebrates but mammals, RBCs are nucleated. RBCs and sperm share some common characteristics: (1) They are both motile cells. Sperm move by means of a flagellum, while RBCs are pushed by the blood pressure of the animal and are deformable in the capillaries. (2) They are both terminally differentiated cells. (3) They are both transcriptionally inactive. 12 globular domain Figure 5. Evolutionary relationship of SNBPs to nucleosomal and linker histones. H = nucleosomal core histones; H1 = linker histone; PL = protamine-like proteins; P = protamines. N and C designate the N- and C- terminals, respectively. R = arginine residue. 13 (4) They both contain highly condensed chromatin (except for mammalian erythrocytes). (5) They both may contain arginine-enriched nuclear basic proteins. Proteins, that we call "erythrocyte nuclear basic proteins" (ENBPs) are found in the nuclei of erythrocytes from non-mammalian vertebrates. No ENBPs are detected in the mature enucleated mammalian RBC. ENBPs of vertebrates contain core histones as well as linker histones. The latter can be grouped into the following categories: (1) Linker histone H1: somatic-like linker histone H1 (H1A-E) (Wells and Brown, 1991), a lysine-rich basic protein, can be found in erythrocytes of the bullfrog Rana catesbeiana (Shimada etal., 1981) and the Japanese quail (Palyga and Neelin, 1998). (2) Linker histone H1°: differentiation-specific histone H1°, a more lysine-rich protein, was first found in mammalian tissues with little cell division (Panyim and Chalkley, 1969). H1° type can be found in erythrocytes of fish (Miki and Neelin, 1975, 1977), sea turtle (Rutledge et al., 1981), frogs Xenopus laevis (Rutledge et al., 1984) and Rana catesbeiana (Shimada et al., 1981), and birds (Moorman et al., 1986; Srebreva era/., 1983). Linker histone H1°is not unique to erythrocytes, but is specific to differentiated cells. For example, it has been shown to appear in the brain, retina and liver in mice (Gjerset era/., 1982). A typical mammalian linker histone H1° differs from a typical H1 in containing histidine (1.0 to 1.9 mol%) and methionine (0.4 to 0.8 mol%), more acidic amino acid residues (8.4 to 10.9 mol% vs. 5.1 mol%), a higher arginine content (2.3 to 4.1 mol% vs. 1.5 mol%) and less alanine (13.1 to 16.8 mol% vs. 22.8 mol%) (Panyim and Chalkley, 1969; Smith etal., 1984). (3) Linker histone H5: differentiation-specific and erythrocyte-specific linker histone 14 H5, an arginine-enriched linker histone, was first characterized in nucleated erythrocytes of chicken Gallus gallus (Neelin et al., 1964; Hnilica, 1964) and is only found in erythrocytes of birds (Khochbin and Wolffe, 1994), such as the Japanese quail {Coturnixjaponica) (Neelin era/., 1995), the goose (Brasch era/., 1974; Yaguchi era/., 1979) and the duck (Erea et al., 1978). Compared to the typical mammalian linker histone H1°, chicken linker histone H5 contains more arginine (11.2 mol% vs. 2.3 to 3.9 mol%) and serine (12.6 mol% vs. 8.4 to 9.9 mol%) (Smith etal., 1984). There are two more subtypes of linker histones: Hit, a male germ line subtybe (Panyim and Chalkley, 1969; Bucci era/., 1982) and B4 (H1M), which is the only linker histone found in Xenopus eggs (Smith et al., 1988). Linker histones Hit and B4 (H1M) are specific to particular cell types and are not found in erythrocytes. There is confusion regarding the linker histone subtype in Xenopus laevis erythrocytes: H1° or H5? (Table 2) The two linker histones in the erythrocytes of X. laevis discovered by Risley and Eckhardt (1981) and Mann et al. (1982) and called by them H1D and H1E were considered to be H1 s (satellite linker histone H1S, an H1°-like histone) by Neelin's group (Brown era/., 1981; Rutledge era/., 1984). These two linker histones were called H1°/H5, an H5-like or H1°-like protein (Moorman et al., 1984; Smith et al., 1984). Later, Neelin's group referred to two variants of H1 s as H5 linker histone (Rutledge et al., 1988; Shwed et al., 1992). However, Khochbin and Wolffe (1994) cited three lines of evidence that H1D and H1E belong to the H1° subtype. First, the promoter region of the Xenopus linker histone variant has an H1 box, which is characteristic of H1°. Secondly, the C-terminal of the protein sequence of the Xenopus linker histone variant contains only two arginine residues, whereas a typical linker histone H5 has a very arginine-rich C-terminal. Moreover, H5 is erythrocyte-specific, but 15 Table 2. Historical classification of the linker histone variants in Xenopus laevis (frog) erythrocytes. Name Reference H1D, H1E Risley and Eckhardt (1981) H1 ! H5 Brown etal. (1981) Rutledge etal. (1984) O/LJC Moorman etal. (1984) m / M b Smith etal. (1984) Rutledge etal. (1988) Shwed etal. (1992) Khochbin and Wolffe (1993) H1 0 Khochbin and Wolffe (1994) Brocard et al. (1997) 16 these two H1 variants were also found in other tissues (Khochbin and Wolffe, 1994). Now it is generally accepted that the H1 linker histone variant in Xenopus belongs to the H1° subtype (Khochbin and Wolffe, 1993 and 1994; Brocard etal., 1997). 1.2.5 ENBP transition during erythropoiesis During erythropoiesis, somatic-like linker histones are replaced by differentiation-specific H1° in Xenopus laevis (Appels and Wells, 1972; Rutledge et al., 1988; Khochbin and Wolffe, 1993) and H5 in the chicken Gallus gallus (Neelin, 1964) and the chromatin sequentially loses its transcriptional activity. In Xenopus, a three-fold increase in the relative content of H1° was demonstrated during erythroid maturation (Rutledge etal., 1984). In mammals, late orthochromatic erythroblasts extrude their nuclei to become immature erythrocytes (reticulocytes), which then leave the bone marrow and pass into the bloodstream (Alberts et al., 2002) (Figure 6). Chromatin condensation progresses until just before extrusion. 1.2.6 Evolution of linker histones Schulze (1995) and Peretti and Khochbin (1997) proposed that the ancestor of the differentiation-specific linker histones H1° and H5 and the ancestor of somatic-like linker histone H1 of vertebrates diverged from linker histone H1 of invertebrates. They found that the structures of vertebrate differentiation-specific H1° and H5 genes appear to be close to that of invertebrate somatic H1 genes (Figure 7). Based on the comparison of the structure of known linker histone H1° in vertebrates and the structure of avian linker histone H5, Brocard et al. (1997) hypothesized that the linker histone H5 gene may have been derived from an ancestral linker histone H1° gene that became erythrocyte-specific during the evolutionary appearance of birds (Figure 7). 17 erythroblast Figure 6. Schematic diagram of a nucleus extruded from a developing mammalian red blood cell (erythroblast). "The cell is shown extruding its nucleus to become an immature erythrocyte (reticulocyte), which then leaves the bone marrow and passes into the bloodstream. The reticulocyte will lose its mitochondria and ribosomes within a day or two to become a mature erythrocyte." (Figure and legend slightly modified from Alberts etal., 2002, p.1292). 18 H 5 Vertebrate HI Vertebrate H l u Vertebrate HI Invertebrate Figure 7. Schematic diagram showing the evolutionary trend of linker histones (Schul and Schulze, 1995; Peretti and Khochbin, 1997; Brocard etal. 1997). 19 1.3 Questions to be examined in this thesis 1.3.1 Does internal fertilization constrain the range of SNBP diversity in teleost fish and frogs? An analysis of SNBP distribution in animals (Kasinsky, 1985, 1989, 1995; Rosenberg et al., 1991) suggests that the mode of fertilization might serve as a constraint on SNBP diversity. For example, amongst the chordates, frogs and bony fish show a diversity of SNBP types (H/H1, PL, P), whereas internally fertilizing cartilaginous fish, urodeles and amniotes (reptiles, birds and mammals) have either protamines or keratinous protamines (P, KP) in their sperm nuclei (Figure 8). Internally fertilizing sperm tend to be elongated with a more condensed nucleus (Franzen, 1977; Baccetti, 1982; Wirth, 1984; Jamieson, 1991). Kasinsky (1985, 1989, 1995) has hypothesized that SNBPs in internally fertilizing animals tend to be more protamine-like than histone-like. This may be due to the fact that during internal fertilization, the sperm have to move through the more viscous reproductive tract of the female rather than sea water. This requires the chromatin in the sperm head to be more condensed in order to resist drag forces and thus increase the efficiency of movement. Protamines are smaller structural proteins than histones and rich in arginine side chains. Since arginine residues can bind more strongly to the phosphodiester backbone of DNA with more hydrogen bonds than lysine residues, as well as by electrostatic interaction (Helene and Lancelot, 1982; Ausio etal., 1984), the SNBPs of internally fertilizing (LF.) animals should be enriched in arginine over lysine residues with respect to SNBPs in externally fertilizing (E.F.) animals. 20 CD C L >^  C D C "E CC -*—» o CO u o c •4—• CC CD CD" C L CD C "E CO -i—• o C L CD C L > * - * — ' J D | s "is 2 cl C L , II o> _l CO co Q- 1 CD • CO TD \ O o CD g_2 > » Q) CD C C CC CO _ ^ - Z o .E >- X *S E 'co j | O CD "cn M ~ > c -o =5 o B oVl & CD .c "o Z CD ^ 00 CD O 0 C O o CN L L X C L Cataetyx laticeps is a deep sea viviparous benthic fish in the order Gadiformes, family Bythitidae. This is the only internally fertilizing teleost fish that has been examined so far (Saperas et al., 1993; Chiva et al., 1995). Its sperm contain somatic type histones and an additional arginine-enriched H1 -like basic protein (which is not a low molecular weight protamine). Does this contradict Kasinsky's hypothesis (1985, 1989, 1995)? To test this hypothesis further, we need to look at more examples of internally fertilizing teleost fish. In this thesis, I examine the SNBPs from Xiphophorus helleri guentheri (swordtail), Xiphophorus maculatus (platyfish), Poecilia reticulata (black-banded guppy), Poecilia picta (guppy) and Cymatogaster aggregata (shiner perch), all internal fertilizers, as well as Synganthus schlegeli (seaweed pipefish), with open brood pouches in their belly region. It has been found (Kasinsky et al., 1999) that testes from the internally fertilizing frog Ascaphus true! (family Ascaphidae), which appeared early in evolution, contain protamine-like SNBPs. This is in accordance with Kasinsky's hypothesis (1985, 1989, 1995) that internal fertilization selects for more arginine-rich SNBPs in the sperm chromatin. However, a trend can be seen towards P —• H reversions in externally fertilizing Silurana (Kobel et al., 1996), a subgenus of the frog genus Xenopus laevis (family Pipidae) (Mann et al., 1982), as well as in the advanced, externally fertilizing neobatrachian frogs Crinia signifera (family Myobatrachidae) and Rana catesbeiana (family Ranidae) (Kasinsky et al., 1985; Itoh et al., 1997). Such a trend parallels the simplification of elongated, introsperm in primitive Ascaphus true! to more rounded ectaquasperm in more advanced neobatrachians by Jamieson and coworkers (Jamieson etal, 1993; Lee and Jamieson, 1993; Jamieson, 1999; Scheltinga era/., 2001) and attributed by them as a secondary reversion to external fertilization. One way 22 to test this hypothesis (Kasinsky, 1985, 1989, 1995) is to see whether an advanced neobatrachian that has secondarily acquired internal fertilization might also have converged to arginine-rich protamines. In this thesis, using gel electrophoresis, HPLC and amino acid analysis, I examine the SNBPs from the neobatrachian frog, Eleutherodactylus coqui, (family Leptodactylidae) from Puerto Rico that has internal fertilization, in order to determine its SNBP type. This frog is the only other anuran with internal fertilization that is presently not endangered (Sever et al., 2003; M. H. Wake, personal communication). The criteria I use in this thesis to determine the SNBP type are described in Table 3. 1.3.2 Is linker histone H5 present in alligator erythrocytes? Linker histone H5 is the H1 subtype most strongly associated with chromatin (Koutzamani et al., 2002) in the erythrocyte of birds. Since birds have a high systemic blood pressure (Figure 9), this might require the presence of a more arginine-rich linker histone, H5, rather than H1°, to replace lysine-rich linker histone H1 in order to achieve greater condensation of the chromatin in erythrocytes. If this is the case, then the alligator might contain an H5 linker histone in its nucleated erythrocytes as it has a higher systemic blood pressure than sea turtle and frog (Figure 9), in which it is already known that there is an H1° linker histone (Rutledge et al., 1981, 1984). Since an analysis of the SNBPs indicates that alligator is more closely related to birds than is sea turtle (Hunt et al., 1996), we hypothesize that there should be a linker histone H5-like protein present in alligator ENBPs. This is a way to test the hypothesis of Brocard et al. (1997) that the linker histone H5 gene may have been derived from an ancestral linker histone H1° gene that became erythrocyte-specific during the evolutionary appearance of birds. 23 In this thesis, I again utilize gel electrophoresis, HPLC and amino acid analysis to purify and examine ENBPs from nucleated erythrocytes of catfish, bullfrog, sea turtle, alligator, and chicken to see if linker histone H5 is, in fact, unique to birds. In addition I also look for possible H5-like NBPs in mouse normoblasts, the precursors of mature enucleated RBCs with condensed nuclei (Figure 6). The criteria I use in this thesis to determine the linker histone type in vertebrates are amino acid analysis and electrophoretic mobility in SDS-PAGE and AUT-PAGE (Table 4). Typical linker histone H1° differs from a typical linker histone H1 in containing histidine (1-1.9 mol% versus 0) and methionine (0.4-0.8 mol% versus 0), greater acidic residue (8.4-10.9 mol% versus 5 mol%), greater arginine content (2.3-4.1 mol% versus 1.5 mol%), and less alanine (13.1 mol% versus 23 mol%) (Panyim and Chalkley, 1969; Smith et al., 1984). Chicken H5 is like linker histone H1°, but has more arginine (11.2 mol% versus 2.3-4.1 mol%) and more serine (12.6 mol% versus 8.4-9.9 mol%) (Smith et al., 1984). Among these amino acid contents, arginine content is the most important indicator. Linker histone H5 runs faster in both SDS and AUT gels than linker histone H1°, which runs faster than linker histone H1. 24 200 20 1 6 Q . "HT 1 12 Systemic blood pressure • 180 160 torr 140 120 100 80 60 40 20 Lungfish Amphibian Reptile (frog) (turtle) Reptile Mammal Bird (crocodile) (man) (chicken) Figure 9. Comparison of systolic and diastolic levels of systemic blood pressure in vertebrates. The values refer to nonanesthetized resting animals. The top of each box indicates the systolic level while the bottom indicates the diastolic level of blood pressure (Figure and legend modified from Withers, 1992, p. 1136, as adapted from Johansen etal., 1970). 25 Table 3. Criteria used in this thesis to determine the SNBP type. SNBP type Electrophoretic mobility in AU-PAGE and AUT-PAGE Amino acid composition H/H1 Slow Lys+Arg: 20-30 mol% PL In between Lys+Arg: 35-50 mol% His+Lys+Arg: 55%-80 mol% P Fast Arg > 30 mol% Ser+Thr+Gly: 10-25 mol% H/H1 = core histone/linker histones; PL = protamine-like proteins; P = protamines. Lys = lysine residue; Arg = arginine residue; His = histidine residue; Ser = serine residue; Thr = threonine residue; Gly = glycine residue. AU- PAGE = acid-urea polyacrylamide gel electrophoresis; AUT-PAGE = acid-urea-Triton polyacrylamide gel electrophoresis. 26 Table 4. Criteria to determine the linker histone variants in vertebrates used in this thesis. Amino acid composition Electrophoretic mobility in SDS-PAGE and AUT-PAGE H1 Arginine*: ~1.5mol% No histidine No methionine Acidic residue: ~5mol% Alanine: ~23mol% Slow Arginine* 2.3-4.1 mol% Histidine: 1-19 mol% H1° Methionine 0.4-0.8 mol% Acidic residue 8.4-10.9 mol% Serine 8.4-9.9 mol% In between H5 Similar to H1° but has more arginine* (~11.2 mol%) and more serine (-13 mol%) Fast * Arginine composition is the most important indicator. SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; AUT-PAGE = acid-urea-Triton polyacrylamide gel electrophoresis. 27 2. Materials and Methods 2.1 Living organisms 2.1.1 Internally fertilizing teleost fish examined for sperm nuclear basic proteins (SNBPs) Specimens of Cymatogaster aggregata (shiner perch) (Figure 10) were collected at Bamfield, British Columbia, by Ramona deGraaf in Prof. Eric Taylor's laboratory, Zoology, University of British Columbia. Specimens were kept in 90% ethanol and stored at 4°C until dissection. Specimens of Poecilia reticulata (black-banded guppy) (Dawes, 1991) and Poecilia picta (guppy) were collected from Prof. Robin Liley's laboratory, Zoology, University of British Columbia. Specimens of Xiphophorus helleri guentheri (swordtail) and Xiphophorus maculatus (platyfish) were a generous gift from Dr.Jurgen Vielkind at the British Columbia Cancer Centre in Vancouver, Specimens of Synganthus schlegeli (seaweed pipefish) were collected from Otsuchi-Bay, Japan by Dr. Satoshi Watanabe at the University of Tokyo. The gonadal tissue was dissected from these animals and stored at - 80°C until further processing. 2.1.2 Internally fertilizing frog examined for SNBPs Gonadal tissues of Eleutherodactylus coqui, order Anura, family Leptodactylidae (Pough et al., 1998, p.64) were supplied by Prof. James Hanken's laboratory, Harvard University. 2 8 2.1.3 Vertebrates examined for erythrocyte nuclear basic proteins (ENBPs) Figure 13 shows the vertebrates examined for ENBPs. Blood specimens from Alligator mississippiensis (alligator) were collected by Manuela Gardner in Prof. David R. Jones' laboratory, University of British Columbia. Blood specimens from Chelonia mydas (sea turtle) were collected by Dr. Amanda Southwood in Prof. David R. Jones' laboratory, University of British Columbia. Blood and liver specimens of Gallus domesticus (chicken) and Rana catesbeiana (bullfrog), as well as liver specimens of Alligator mississippiensis (alligator) were supplied by Prof. J . Ausio, University of Victoria. Spleen specimens of Mus musculus (mouse) were generous gifts from Prof. Keith R. Humphries at the British Columbia Cancer Agency. Blood and liver specimens of Ictalurus punctatus (channel catfish) were collected by Prof. Tom Mommsen at the University of Victoria. Blood specimens were collected into heparinized tubes, and then strained through 2 layers of cheesecloth. The original volume of the blood specimen was measured. The sample was then spun down for 10 minutes at 2000g at 4°C in a Beekman AJ - 20 rotor. The supernatant was discarded and the pellet was resuspended in 50% - 75% of its original volume of buffer, containing 0.15 M NaCl, 15 mM sodium citrate, 10 mM sodium phosphate pH 7.2, and 10 ug/ml protease inhibitor cocktail tablets ("Complete" from Roche Molecular Biochemicals). An equal volume of 80% glycerol was added to this suspension. The sample was frozen with dry ice immediately and then stored at -80°C until further processing. 29 Order Perciformes Cymatogaster aggregate (shiner perch) Ui -4-1 o. o •£ o o Order Scorpaeniforrnes Sebastes (rockfish) 0 -*-> (/) O 0 I -•4-1 tfi O 0 3 LU o a o £ c 'd Order Cy p ri n o d o nt ifo rtn es Poecilia reticulata (black-banded guppy) Poecilia picta (guppy) Xiphophorus heiferi guenther (swo rdtail) CD a o c to o Xiphophorus macuiatus (platyfish) Order Gadiformes Cataetyx faticeps (brotula) Figure 10. Phylogehy of internally fertilizing teleost fish examined for SNBPs. Phylogeny adapted from Nelson, 1994, frontispiece. 30 — cu =s -ST £ t f S 2 1 S a — w Jo E • » - B = S J i u. (o o o T : ^ O c3 •sa '& ra .g i f , ° S3 o o co ra d | 5 cu cu 5 & ines ines •§ " 3 = S CD 3 — QJ .s. •a turti ,1- ra ' l— CU ,o 9 cu tf> o « ra .eg ra ='5 si 1 » 3 3 £ " 5 a -e ra o '** tf) CO c ra 3 C J I ! a ra w CL CQ Z . LU Z. « •a ^ rz ^ E ° : co r~~ <D CD C/3 T -•a cc B c CO co 25 E £ o g > ± &s o a. >, co .c -a Q_ CO • T - C •i- CD 2> C? LL CL CU cu O ^ 2.2 Nuclear basic protein extraction 2.2.1 Extraction of SNBPs Two frozen testes from a single animal were suspended in 60 ul of 0.15 M NaCl, 10 mM Tris - HCI buffer (pH 7.5), containing 10 (jg/ml N - tosyllysine chloromethylketone (TLCK) (Sigma St. Louis, MO). The sample was then homogenized in this buffer in a Polytron homogenizer (Barnant, Barrington, IL) for 1 ~ 2 minutes at speed 4 - 5 . The homogenate was spun down for 10 minutes at 16,0000 at 4°C in an Eppendorf microfuge (Brinkmann Instruments Inc., Westbury, NY). The pellet was resuspended in the previous buffer (same volume) but containing, in addition, 0.5% (v/v) Triton X-100. It was then homogenized and centrifuged as before. This step was usually repeated twice. The supernatant was discarded and the final pellet was resuspended in 80 pi of 0.4 N HCI, homogenized in a Polytron and kept at 4°C with occasional stirring for 1 minute. The HCI extract was next spun at 16,000c/ in the Eppendorf microfuge for 10 minutes. The supernatant thus obtained was aliquoted into a 1.5 ml Eppendorf tube. The acid-soluble protein was then precipitated overnight with 6 volumes of cold acetone at - 20°C. The tube was centrifuged as above, the supernatant was discarded and the pellet was dried using a Speedvac concentrator. The dried pellet was labeled and stored at - 80°C until further use. Each experiment was repeated 5 times for different fish. 2.2.2 Extraction of ENBPs The frozen blood specimen (5 ml) from a single animal was thawed on ice, and then centrifuged at 16,000c; in the Eppendorf microfuge for 10 minutes. The supernatant was discarded and the pellet was resuspended in 1.5 ml of buffer containing 0.1 M KCI, 32 1 mM MgCI 2- 6H 2 0, 50 mM Tris-HCl (pH 7.5), 10 ug/ml protease inhibitor cocktail tablets (Roche) and 0.5% (v/v) Triton X-100, and incubated on ice for 10 minutes. The sample was then spun down for 15 minutes at 16,000c; at 4 ° C in an Eppendorf microfuge. This step was usually repeated twice. The pellet thus obtained was resuspended in the previous buffer (same volume) but without Triton X-100 and centrifuged as before for 5 minutes. The final pellet was resuspended in approximately 2 ml of 0.4 N HCI, homogenized in a Polytron and kept at 4 ° C with occasional stirring for 1 minute. The HCI extract was next spun at 16,000c; in the Eppendorf microfuge for 15 minutes. The supernatant thus obtained was aliquoted into 1.5 ml Eppendorf tubes and the acid-soluble protein was then precipitated overnight with 6 volumes of cold acetone at - 2 0 ° C . The tubes were centrifuged as above, the supernatant was discarded and the pellet was dried using a Speedvac concentrator. The dried pellet was labeled and stored at - 80 ° C until further processing. Each experiment was repeated 5 times for different animal. 2.2.3 Extraction of liver nuclear basic proteins (NBPs) A piece of liver tissue (around 0.5 ml in volume) from a single animal was minced with scissors in 2 ml of buffer containing 0.1 M KCI, 1 mM MgCI2- 6H 2 0, 50 mM Tris-HCl (pH 7.5), 10 ug/ml protease inhibitor cocktail tablets (Roche), 0.5% Triton X-100 and incubated on ice for 10 minutes. The sample was then homogenized in this buffer in a homogenizing tube for 1 - 2 minutes on ice. The homogenate was transferred to two 1.5 ml Eppendorf tubes and spun down for 10 minutes at 16,000c; at 4 ° C in an Eppendorf microfuge. This step was usually repeated twice. The pellet thus obtained was resuspended in the previous buffer (same volume) but without Triton X-100. After 33 being transferred to the homogenizing tube, the pellet was well homogenized in this buffer for 1 - 2 minutes on ice. The suspension thus obtained was aliquoted into 1.5 ml Eppendorf tubes and centrifuged as before for 10 minutes. The final pellet was resuspended in approximately 2 ml of 0.4 N HCI, homogenized as before and kept at 4°C with occasional stirring for 1 minute. The HCI extract was next spun at 16,000gr in the Eppendorf microfuge for 10 minutes. The supernatant thus obtained was aliquoted into 1.5 ml Eppendorf tubes and the acid-soluble protein was then precipitated overnight with 6 volumes of cold acetone at - 20°C. The tubes were centrifuged as above. The supernatant was discarded and the pellet was dried using a Speedvac concentrator. The dried pellet was labeled and stored at - 80°C until further use. Each experiment was repeated 5 times for different animal. 2.3 Gel electrophoresis The gel electrophoresis used in this thesis is summarized in Table 5. The electrophoresis apparatus is from Idea Scientific, Minneapolis, MN. All gels were stained for 30 minutes in 10% (v/v) acetic acid, 25% (v/v) isopropanol, and 0.1% (w/v) Coomassie blue, and then destained overnight in 10% isopropanol (v/v) and 10% (v/v) acetic acid. A picture was taken with Kodak® 667 film. 2.3.1 Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis This gel is used to separate histones but protamines generally can not be separated. Vertical 15% polyacrylamide separating gels for analysis of proteins were prepared by mixing 5 ml of 30% (w/v) acrylamide to 0.8 % (w/v) bis-acrylamide, 2.5 ml 34 Table 5. Gel electrophoresis used in this thesis. Gel type Final % of polyacrylamide (w/v) Direction Separation SDS-PAGE 15% 0 ^ 0 Separate histones AU-PAGE 15% 0 ^ 0 Separate histones from protamines AU-PAGE 20% © 0 Resolve peptides AUT-PAGE 15% 0 ^ 0 Separate H5 from other linker histones SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; AUT-PAGE = acid-urea-Triton-polyacrylamide gel electrophoresis; AU-PAGE = acid-urea-polyacrylamide gel electrophoresis. 35 1.5 M Tris (pH 8.82), 2.32 ml water, 100 ul 10% SDS, 4.5 ul TEMED, and 56.7 ul 10% APS (10 ml total in volume). This separating gel was poured into the electrophoretic apparatus and layered with water saturated butanol. When the separating gel was polymerized, butanol was poured off. The gel apparatus was rinsed with water and then dried with filter paper. Then a 6% polyacrylamide stacking gel prepared by mixing 400 ul of 30% (w/v) acrylamide to 0.8% (w/v) bis-acrylamide, 500 ul 0.5 M Tris (pH 6.8), 1.06 ml water, 20 ul 10% SDS, 2 ul TEMED, and 20 ul 10% APS (2 ml total volume) was poured on top of the separating gel. Immediately the comb was applied and the gel was allowed to polymerize for about 30 minutes. The running buffer was a mixture of 0.05 M Tris, 0.38 M glycine and 0.1% (w/v) SDS. The protein sample dissolved in distilled water was mixed with 1 volume of 2 x sample buffer: 2.5 ml 0.5 M Tris pH 6.8, 4 ml 10% (w/v) SDS, 2 ml 100% (v/v) glycerol, 1 ml B-mercaptoethanol, 0.5 ml BPB and then loaded. The gel was run at 100 volts from (-) to (+) until BPB was approximately 1 cm from the bottom of the gel (about 1 hour). 2.3.2 Acid urea (AU) polyacrylamide gel electrophoresis This gel is used to separate histones and protamines. Vertical 15% polyacrylamide, 2.5 M urea, 5% acetic acid gels for analysis of proteins were prepared by mixing 4 ml of 30% (w/v) acrylamide to 0.8 % (w/v) bis-acrylamide, 2 ml of 10 M urea, 1 ml of 43% (v/v) acetic acid, 7 mg of thiourea, and 45 ul of 30% (v/v) hydrogen peroxide. The running buffer was 5% (v/v) acetic acid. The protein sample was dissolved in distilled water and mixed with 1 volume of 2 x sample buffer: 8 M urea, 10% (v/v) acetic acid, and 0.1% (w/v) pyronin Y and loaded. The gel was run at 120 volts from (+) to (-) until the pink pyronin dye was about 0.5 cm from the bottom of the gel (about 1 hour). 36 2.3.3 Acid urea (AU) polyacrylamide gel electrophoresis for resolving peptides This gel contains a higher percentage of polyacrylamide (20%) and is used to resolve peptides. Vertical 20% polyacrylamide gels for resolving peptides were prepared by mixing 3.34 ml of 60% (w/v) acrylamide to 2% (w/v) bis-acrylamide, 2.5 ml of 10 M urea, 1.25 ml of 43% (v/v) acetic acid, 8.8 mg of thiourea, 2.91 ml water and 56.3 pi of 30% (v/v) hydrogen peroxide (10 ml total volume). The size of this gel was 7.3 cm x 10.2 cm x 0.075 cm. The electrophoresis apparatus was from Idea Scientific and set up in a 4°C cold room. The running buffer used was 5% (v/v) acetic acid. The protein sample was dissolved in distilled water and mixed with 1 volume of 2 x sample buffer: 8 M urea, 10% (v/v) acetic acid, 0.1% (w/v) pyronin Y and then loaded. The gel was run at 100 volts from (+) to (-) for 10 minutes, then at 250 volts until the pink pyronin dye was about 0.5 cm from the bottom of the gel (about 2.5 hour). 2.3.4 Acid urea Triton (AUT) polyacrylamide gel electrophoresis This gel contains the detergent Triton X-100 which can fill up the hydrophobic globular domain of the core histone and therefore retard its movement. As a result, linker histones are moving faster than core histones, and we can therefore get a better separation of the linker histones from the core histones. Vertical 15% polyacrylamide, 3.8 M urea, 4.8% acetic acid and 6 mM Triton gels for analysis of proteins were prepared by mixing 2.5 ml of 60% (w/v) acrylamide to 0.4 % (w/v) bis- acrylamide, 2.28 g urea, 480 ul of 100% (v/v) acetic acid, 7 mg of thiourea, 24 pi of freshly made 45 mM NH 4OH, 154 pi of 25% Triton X-100, 4.52 ml water, and 45 pi of 30% (v/v) hydrogen peroxide (10 ml total volume). The size of this gel was 7.3 cm x 10.2 cm x 0.075 cm. The electrophoresis apparatus was from Idea Scientific. The 37 running buffer used was 5% (v/v) acetic acid. The protein sample dissolved in distilled water was mixed with 1 volume of 2 x sample buffer: 8 M urea, 10% (v/v) acetic acid, and 0.1% (w/v) pyronin Y and loaded. The gel was run at 100 volts from (+) to (-) until the slow moving dye (second dye) was 2/3 through the gel (about 6 hours). The gel was then stained for 30 minutes in 25% (v/v) isopropanol, 10% (v/v) acetic acid, and 0.2% (w/v) Coomassie blue. The gel was then destained overnight in 10% (v/v) isopropanol and 10% (v/v) acetic acid. A picture was taken with Kodak® 667 film. 2.4 Protein fractionation by reverse phase high performance liquid chromatography (HPLC) 2.4.1 Fractionation of SNBPs Reverse phase HPLC was performed to fractionate the protamine and/or protamine-like protein components from SNBPs. The column used was a C 1 8 (25x0.46 cm) 300-A Vydac column. Buffer A was 0.1% trifluoroacetic acid (TFA) and Buffer B was 100% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile was recorded at a wavelength of 230 nm using a sensitivity of 0.2 absorbance units (AU). The elution gradient was as follows: 0% B for 5 min, 0% B - 60% B for 60 min, 60% B -80% B for 5 min, 80% B for 5 min, and 80% B - 0% B for 10 min. After fractionation, aliquots of each eluted peak were dried using a Speedvac concentrator and stored at 4 ° C . 2.4.2 Fractionation of ENBPs Reverse phase HPLC was performed to fractionate the H1 and/or H5 linker histone components from ENBPs, using a (25x0.46 cm) 300-A Vydac C 1 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and 15% ethylene glycolmonomethylether 38 (EGME), and Buffer B was 0.1% trifluoroacetic acid (TFA), 15% ethylene glycolmonomethylether (EGME), and 70% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile was recorded at a wavelength of 210 nm using a sensitivity of 0.2 AU. The elution gradient was as follows: 20% B for 15 min, 20% B -50% B for 50 min, 50% B -100% B for 15 min, 100% B for 5min, 100% B - 20% B for 10 min, and 20% - 0% B for 1 min. After fractionation, aliquots of each eluted peak were dried using a Speedvac concentrator and stored at 4TJ. 2.5 Chymotrypsin digestion Lyophilized HPLC fractions of H1 and H5 linker histones were dissolved in 2.5 ul 50 mM Tris-HCl (pH 7.5) and mixed with the same volume of chymotrypsin (EC 3.4.23.1) with the same buffer at an enzyme-to-substrate ratio E/S = 1:500 (w/w). The concentration of the substrate was 1 pg/pl. The mixture was incubated at room temperature for 100 minutes and the reaction was stopped by adding 5.0 ul of 2 x urea sample buffer (8 M urea, 10% (v/v) acetic acid, and 0.1% (w/v) pyronin Y) and then loaded directly onto the acid-urea-polyacrylamide gel electrophoresis for resolving peptides. 2.6 Amino acid analysis The lyophilized HPLC protein fractions (around 5 pg protein per sample) were sent to the Advanced Protein Technology Centre at the Sick Children Hospital in Toronto for amino acid analysis. 39 3. Results 3.1 Characterization of SNBPs from internally fertilizing teleost fish As described in Table 3, the criteria used in this thesis to determine the SNBP type are: 1) Electrophoretic mobility: Protamines move more rapidly in AU and AUT gels than protamine-like proteins, which move faster than histones H/H1. 2) Amino acid analysis: Protamines consist of arginine-rich (Arg > 30 mol%) highly basic proteins (His + Lys + Arg = 55-80 mol%, Ser + Thr -i-Gly = 10-25 mol%). Protamine-like proteins consist of 35-50 mol% arginine + lysine. Histones H/H1 have a low (20-30 mol%) lysine + arginine content. 3.1.1 Comparison of SNBPs from Xiphophorus, Poecilia, and Cymatogaster to see if protamines or protamine-like proteins are present The SNBPs from testis of X. maculatus (platyfish), X. helleri (swordtail), P. reticulata (black-banded guppy), P. picta (guppy) and C. aggregata (shiner perch), all internal fertilizers (Figure 10), were extracted by 0.4 N HCI, and then subjected to acid-urea-PAGE. Figure 12 shows the electrophoretic analysis of these SNBPs. Lanes 1 and 2 are histone and protamine markers from Gallus domesticus (chicken) and Oncorhynchus keta (chum salmo), respectively. Lanes 3-6 are SNBPs from internally fertilizing fish showing both histone and a protamine- like protein that is moving more slowly than the typical chum salmon protamine. SNBPs from different species of guppy show variability. The band of protamine-like protein in P. picta SNBPs (lane 4) is moving faster than that in P. reticulata SNBPs (lane 3). SNBPs in Lane 7 are from shiner perch, which contains a protamine moving faster than those of the other internally fertilizing 40 fish, but slightly more slowly than chum salmon protamine. Therefore, according to electrophoretic mobility, protamine-like proteins are present in each of these internally fertilizing teleost fish. 3.1.2 SNBPs from testis of Xiphophorus helleri guentheri (swordtail) and Xiphophorus maculatus (platyfish) with different times of sexual maturity. The acid extracted SNBPs from testis of X. helleri and X. maculatus were used to examine the variability of SNBPs between species and within species but with different times of sexual maturity (Figure 13). Both histones and protamine-like proteins are present in swordtail and platyfish SNBPs (lanes 4 - 1 2 ) (except in one swordtail testis (lane 3) which probably was aspermatogenic). However, the NBPs from brain only contain histones (lanes 2 and 13). The SNBPs from swordtail with late maturity (maturation time: 10-11 months, J . Vielkind personal communication) (lanes 6 and 7) contain more histones than those with early maturity (maturation time: 6-7 months, J. Vielkind personal communication) (lanes 4 and 5), as well as three additional faint protamine-like protein bands (indicated by arrows) which are not seen in lanes 4 and 5. Because I used the protease inhibitor cocktail tablets (Roche), it is not likely that these faint bands are artifacts. The SNBPs from platyfish seem to show a similar effect when comparing a late maturing platyfish (maturation time: 10 months, sd/sr strain, J . Vielkind personal communication) (lane 11) with a platyfish with normal maturity (maturation time: 5-7 months, sr'/sr' strain, J . Vielkind personal communication) (lane 12). However, in another gel with different platyfish (Figure 14), exactly the opposite result is obtained. SNBPs from platyfish with normal maturity (lane 5) are observed to contain more histone, and less of the major protamine, as well as three minor protamine-like protein bands, as compared to SNBPs from platyfish with late maturity (lane 3), which show 41 only the major protamine band. This suggests that the three faint bands may be intermediate proteins, perhaps the precursors of the main protamine. 3.1.3 Fractionation of SNBPs from testis of Cymatogaster aggregata (shiner perch) The acid extracted SNBPs of C. aggregata were fractionated by reverse phase HPLC. Figure 15A shows the elution profile of the SNBPs from C. aggregata. Figure 15B shows the electrophoretic analysis of the SNBP fractions from Figure 15A. Fractions 1 and 2 are the protamine fractions, while fractions 3-12 are histone fractions. 3.1.4 Amino acid analysis of the protamine fraction from Cymatogaster aggregata (shiner perch) Reverse phase HPLC fraction #1 (Figure 15A) from C. aggregata was dried in a Speedvac to remove TCA and ACN. This fraction was then analyzed for amino acid composition. Table 6 shows the amino acid composition of this fraction from C. aggregata. The sample contains 66.7 mol% arginine, which is very close to 67.2 mol% arginine from chum salmon protamine. And it contains 16.9 mol% content of Ser + Thr + Gly. According to the amino acid analysis criteria in Table 3, it clearly indicates that C. aggregata contains primarily protamine in its SNBPs. Because I used a testis preparation to obtain the SNBPs from shiner perch, the histones seen in Figure 12 lane 7 probably have come from contamination of somatic cells in the testis prepation. 42 Figure 12. Acid-urea-PAGE of SNBPs in testis of internally fertilizing fish. 1, Chicken (G. domesticus) histones; 2, chum salmon (O. keta) protamine; 3, P. reticulata (black-banded guppy) SNBPs, 4, P. picta (guppy) SNBPs; 5, X. helleri (swordtail) SNBPs; 6, X. maculatus (platyfish) SNBPs; 7, C. aggregata (shiner perch) SNBPs. Electrophoresis is from top (+) to bottom (-). 43 X. helleri X. maculatus EM. LM. LM. N.M. Figure13. Acid-urea-PAGE of SNBPs in testes of X. maculatus (platyfish) and X. helleri guentheri (swordtail) with different times of sexual maturity. 1, Chicken (G. domesticus) histones; 2, X. helleri brain NBPs; 3-5; X. helleri (early maturity of 6-7 months; three different fish) SNBPs; 6-7, X. helleri (late maturity of 10-11 months) SNBPs; 8-9, X. maculatus (no indication about sexual maturity) SNBPs; 10-11, X. maculatus (late maturity of 10 months; sd/sr strain; two different fish) SNBPs; 12, X. maculatus (normal maturity of 5-7 months; sr'/sr' strain) SNBPs; 13, X. maculatus brain NBPs; 14, chum salmon (O. keta) protamine. Protamine-like proteins are shown by arrows. E. M. = early maturity; L. M. = late maturity; N. M. = normal maturity. Electrophoresis is from top (+) to bottom (-). 44 L M . N.M. 1 2 3 4 5 6 Figure 14. Acid-urea-PAGE of SNBPs in testis of X. maculatus with different times of sexual maturity. 1, X. maculatus (no indication about sexual maturity) SNBPs; 2, X. maculatus (no indication about sexual maturity) brain NBPs; 3, X. maculatus (late maturity of 10 months; sd/sr strain) SNBPs; 4, X. maculatus (late maturity of 10 months; sd/sr strain) brain NBPs; 5, X. maculatus (normal maturity of 5-7 months; sr'/sr' strain) SNBPs; 6, X. maculatus (normal maturity of 5-7 months; sr'/sr' strain) brain NBPs. Three faint protamine-like protein bands in lane 5 are indicated by arrows. N. M. = normal maturity; L. M. = late maturity. Electrophoresis is from top (+) to bottom (-). 4 5 15A 0.18 0.09 0.00 protamine 1 histone 7 2 80 <t> 40 o 45 Time (min) 90 15B protamine histone S 1 2 3 4 5 6 7 8 9 10 11 12 S —I l l L _ J • — J L _ J >—I I—! \—' (—i l ) \ » PLII !Mf H»4 C 3 PLIII • PLIV protamine— w Figure 15A. Reverse phase HPLC fractionation of C. aggregata (shiner perch) SNBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and Buffer B was 100% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile was recorded at a wavelength of 230 nm (solid line) using a sensitivity of 0.2 absorbance units (AU). The elution gradient (dotted line) was as follows: 0% B for 5 min, 0% B - 60% B for 60 min, 60% B - 80% B for 5 min, 80% B for 5 min, and 80% B -0% B for 10 min. Figure 15B. Acid-urea-PAGE analysis of C. aggregata (shiner perch) SNBP fractions obtained from the HPLC shown in Figure 15A. Samples from each peak were run alongside M. californianus (mussel) protein markers in S lanes which contain three protamine-like (PL) proteins: PLII, PLIII and PLIV. Electrophoresis is from top (+) to bottom (-). 46 Table 6. Amino acid composition (mol%) of SNBPs from testis of Cymatogaster aggregata (shiner perch) after HPLC. C. aggregata 1 #13 (protamine) O. keta (chum salmon)2 Protamine Lys 0.1 — His 0.6 — Arg 66.7 67.2 Asx 0.2 — Glx 0.4 — Thr 2.9 — Ser 4.5 9.9 Pro 3.3 9.1 Gly 9.5 6.5 Ala 3.2 1.3 1/2 Cys — Val 0.3 4.7 Met 0.3 — He 0.1 1.3 Tyr 0.3 — Phe 7.5 — Trp — Lys/Arg 0.002 — 1 Internal fertilization 2 External fertilization in Oncorhynchus keta control (Ando etal., 1973) 3 Fraction number from HPLC (Figure 15A) and AU-PAGE (Figure 15B, lane 1) 47 3.1.5 SNBPs in teleost fish with male preganency The SNBPs from Syngnathus schlegeli (seaweed pipefish) with open brood pouches in the male belly region were extracted from testis with 0.4 N HCI and then subjected to acid urea PAGE. Figure 16 shows the electrophoretic analysis of these SNBPs. Lanes 1 and 2 are S. schlegeli (seaweed pipefish) SNBPs. These lanes contain two faint bands (indicated by arrows) in the region between histone markers (lane 4) and chum salmon protamine (lane 3). The low level of protein in lanes 1 and 2 may be due to the low sperm count in the testis. Because fertilization occurs in the pouch, a low sperm count is sufficient to fertilize the egg (Watanabe etal., 2000). 3.2 Characterization of SNBPs from an internally fertilizing frog 3.2.1 SNBPs from Eleutherodactylus coqui The SNBPs from testis of E. coqui were extracted by 0.4 N HCI, and then subjected to acid urea PAGE. Figure 17A shows the electrophoretic analysis of these SNBPs. Lanes 1-3 are salmon protamine markers and lanes 12-14 are mussel protamine-like protein markers of increasing protein concentration. Lanes 4-11 are SNBPs from E. coqui with increasingly higher protein content (lanes 4-7 and 9-11). E. coqui clearly contains four protamine-like proteins (A, B, C, and D, Figure 17A) with the fastest band (A) moving more rapidly than the protamine-like SNBPs of internally fertilizing A. truei (Figure 17B). Compared to other frogs (Figure 17B), the protamine-like protein B from E. coqui (lane 1) appears to have the same mobility on the acid urea PAGE as the protamine-like proteins b1/b2 from A. truei (Kasinsky etal., 1999) (lane 2). The fastest moving band in lane 1, which is the protamine-like protein A from E. coqui, runs slightly faster than the major protamine-like band P1 from B. japonicus (Takamune 48 1 2 3 4 Figure 16. Acid-urea-PAGE of SNBPs in testis of Syngnathus schlegeli (seaweed pipefish; two different fish). 1 and 2, S. schlegeli SNBPs; 3, O. keta (chum salmon) protamine; 4, G. domesticus (chicken) histones. Two faint protamine-like protein bands in lane 2 are indicated by arrows. Electrophoresis is from top (+) to bottom (-). 49 17A O. keta E. coqui M. californianus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 - - - n- - ~ • ~ ~ ~ p Wp» ^Km ^Kw- mmm -ni„j *jggi -PLII D • c »- « # - P L I I I mm - PLIV protamine- *** 17B i/H1 • P H 5 1-H2B/H2A/H3 H4 PUP Figure 17A. Acid-urea PAGE of SNBPs in internally fertilizing (I. F.) E. coqui. 1-3, 0. keta (chum salmon) protamine; 4-11, E . coqui SNBPs; 12-14; M. californianus (mussel) protamine-like (PL) protein markers (PLII, PLIII and PLIV). A, B, C and D are four protamine-like protein components in E. coqui SNBPs. — I n d i c a t e s SNBPs from a single frog with increasing amount of sample. Figure 17B. Acid-urea PAGE of SNBPs from different frogs. 1. E . coqui (I. F.), 2. A. truei (I. F.), 3. B. japonicus (E.F.), 4. X. laevis (E.F.), 5, R. catesbeiana (E.F., external fertilization), in comparison to CE: chicken (G. domesticus, I. F.) erythrocyte histones; MC: SNBPs from muscle M. californianus (E.F.) and SL: salmon (E.F.) protamine used as histone and protamine markers. A, B, C, and D indicate major SNBPs of E. coqui; a, b1/b2, and c indicate major SNBPs of A. truei; P1 indicates major SNBPs of B. japonicus; SP6 indicates major SNBPs of X. laevis. Electrophoresis is from top (+) to bottom (-). 50 etal., 1991) in lane4. 3.2.2 Fractionation of the SNBPs from Eleutherodactylus coqui The acid extracted SNBPs from testis of E. coqui were fractionated by reverse phase HPLC. Figure 18A shows the elution profile of the SNBPs from E. coqui. And Figure 18B shows the electrophoretic analysis of these SNBP fractions. Lanes 1-5 contain four purified protamine-like protein fractions (A, B, C and D) from E. coqui SNBPs. Lanes 3 and 4 show a single band A, and lane 5 a single band B. 3.2.3 Amino acid analysis of the protamine-like protein fractions from Eleutherodactylus coqui Reverse phase HPLC fractions #1 - #7 (Figure 18A) from E. coqui SNBPs were dried in a Speedvac to remove TCA and ACN. Fractions were then analyzed for amino acid composition, as shown in Table 7. Although I did not succeed in separating E. coqui proteins C and D from proteins A and B, the amino acid analysis of the latter two SNBPs indicate that E. coqui SNBPs include protamine-like proteins. Proteins A and B have an arginine content of 35-38 mol% and a Lys + His + Arg content of 44-47 mol%, which is less than that of a typical protamine (at least 55 mol% of basic amino acid residues). However, the Arg content and Lys + His + Arg content of E. coqui proteins A and B are very similar to the composition of protamine-like proteins a, b1, b2, and c in the internally fertilizing A. truei (Kasinsky era/., 1999). They are also similar to protein SP6 of externally fertilizing frog X. laevis (Yokota et al., 1991), but have fewer arginine residues than that in protamine P1 (42.3 mol%, 57.3 mol%) of externally fertilizing B. japonicus (Takamune et al., 1991). 51 18A histone Time (min) 18B protamine histone S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 S Figure 18A. Reverse phase HPLC fractionation of E. coqui SNBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and Buffer B was 100% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile was recorded at a wavelength of 230 nm (solid line) using a sensitivity of 0.2 absorbance units (AU). The elution gradient (dotted line) was as follows: 0% B for 5 min, 0% B -60% B for 60 min, 60% B - 80% B for 5 min, 80% B for 5 min, and 80% B - 0% B for 10 min. Figure 18B. Acid-urea-PAGE analysis of E. coqui SNBP fractions obtained from the HPLC shown in Figure 18A. Samples from each peak were run alongside the starting sample (s) of E. coqui SNBPs, which contain four protamine and protamine-like protein fractions: A, B, C, and D. P/PL designates the range of electrophoretic mobility of protamine and protamine-like proteins. Electrophoresis is from top (+) to bottom (-). 52 5 i r o-_i -CL _ X CD "cO "3 Cr o O 05 j O s CD s-CD Ul o" co GO CD 2 *+— I CO -CL CQ Z co -o O E,. c o CO o_ CL E o o . TJ O CC o c -"E < 0 -Q cc I-r o | m I r o S oa i -4 C N I L O I o 3 O ' I C M - * CM I CN _ l _ a < C 7 \ 1 ^ 5 r o ?> a o) >i! 10 H I H I H n. o < CD C _CC ccf 00 CD cn LU (3 < CL Z> < C CO CO CO 00 „ CD ca O • - CL co =p CC -t * E c 2 9 *" '•cS § N -Q Zi c CM CD c _CC CQ 1^  CD co .9? CD i j_ L L ^ CO C0 CO oo •>-00 CC CC CC 0 0 CO S .E 0 (0 2 r - CL § • - E t c _ r 0 O CC CC "O 0 1 < § o c o E ^ 0 $ 5 c _cc cc . t - C Q CO CQ Is-0 i -Jj' 2 CQ COLL Is- L L CD ^ 05 <£ 0 CD ' <" 0 c c 0 E 2 -5 0 Zi CO Zi CO 0 0 CO 0 "CC . c c 0 ~ 0 CL -J=; CO co CL 2 E C L E co 0 3 CL-C 0 cq ^ Q - . C "cc co o_ c E g o (8 (fl X CQ LU ® cc <o ^ I U " - J r— CL C L - f c O O O CQ >< O C M co •>* m co co 3.3 Characterization of ENBPs from vertebrates As described in Table 4, the criteria to determine the linker histone variants in vertebrates used in this thesis are electrophoretic mobility in SDS and AUT gels and amino acid composition. The mobility of linker histones in SDS-PAGE and AUT-PAGE is: H5 > H1°> H1. With respect to amino acid composition of linker histones, arginine content is the most important indicator. Typical linker histone H1° differs from a linker histone H1 in having a greater arginine content (2.3-4.1 mol% versus 1.5 mol%), containing histidine (1-1.9 mol% versus no histidine) and methionine (0.4-0.8 mol% versus no methionine), more acidic residue (8.4-10.9 mol% versus 5 mol%), and less alanine (13.1 mol% versus 23 mol%) (Panyim and Chalkley, 1969; Smith era/., 1984). Chicken H5 has a similar composition to linker histone H1°, but has more arginine (11.2 mol% versus 2.3-4.1 mol%) and more serine (12.6 mol% versus 8.4-9.9 mol%) (Smith era/., 1984). 3.3.1 Comparison of ENBPs from Gallus domesticus (chicken), Alligator mississippiensis (alligator), Rana catesbeiana (bullfrog), Ictalurus punctatus (channel catfish), and NBPs from Mus musculus (mouse) spleen normoblasts The NBPs from erythrocytes of G. domesticus (chicken), A. mississippiensis (alligator), R. catesbeiana (bullfrog), and /. punctatus (channel catfish) were extracted by 0.4 N HCI, and then subjected to SDS-PAGE. The NBPs extracted from the liver of these species by 0.4 N HCI were used as controls. Figure 19 shows the electrophoretic analysis of these NBPs. The ENBPs and liver NBPs from the catfish, bullfrog, alligator, and chicken all contain core histones and H1 linker histones. Chicken ENBPs show a dark and sharp H5 linker histone band, as seen in lane 8. The faint band at the H5 position in chicken liver NBPs (lane 7) may come from blood contamination. 54 Interestingly, alligator ENBPs (lane 6) also show a band at the H5 linker histone position, which indicates that alligator ENBPs might have an H5 linker histone. However, it is a minor component compared to H1 linker histones. Alligator ENBPs also show many faint bands in the linker histone region. These may come from proteolytic degradation. Neither the ENBPs from the channel catfish (lane 2) nor the ENBPs from the bullfrog (lane 4) show any H5 linker histone. 3.3.2 Fractionation of ENBPs from Gallus domesticus (chicken), Alligator mississippiensis (alligator), Rana catesbeiana (bullfrog), and Ictalurus punctatus (channel catfish) The acid extracted ENBPs from G. domesticus (chicken), A. mississippiensis (alligator), R. catesbeiana (bullfrog), and I. punctatus (channel catfish) were fractionated by reverse phase HPLC. Figures 20-23 show the elution profiles and subsequent electrophoretic analysis of these ENBPs. While the HPLC profile of chicken ENBPs (Figure 20A) shows a large H5 linker histone peak and a small H1 linker histone peak, the HPLC profile of alligator ENBPs (Figure 21 A) shows the reverse: a small putative H5 linker histone peak and a prominent H1 linker histone peak. These results again suggest that this putative H5 linker histone is a minor component in alligator ENBPs but a major one in chicken ENBPs. Fraction #3 from the HPLC (Figure 22A) is probably a degradation product of H1 linker histones, as seen by the more rapidly migrating band (indicated by an arrow) in lane 3, Figure 22B. 55 8 CD C o "55, 0) l C D C o in o o Figure 19. SDS-PAGE analysis of NBPs from erythrocytes and livers of chicken, alligator, frog, and catfish. 1, /. punctatus (catfish) liver NBPs; 2, catfish ENBPs; 3, R. catesbeiana (bullfrog) liver NBPs; 4, bullfrog ENBPs; 5, A. mississippiensis (alligator) liver NBPs; 6, alligator ENBPs; 7, G. domesticus (chicken) liver NBPs; 8, chicken ENBPs (histones are designated). Electrophoresis is from top (-) to bottom (+). 56 20A 4.00 ^2.00 0.00 baJL 0 core histone 78910 linker histone 2 f 50 Time (min) 6 J J 1100 50 CD 20 100 20B linker histone core histone S 1 2 3 4 5 6 7 8 9 10 S linker histone! core histone 1 Figure 20A. Reverse phase HPLC fractionation of G. domesticus (chicken) ENBPs, using a (25x0.46 cm) 300-A Vydac C 1 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and 15% ethylene glycolmonomethylether (EGME), and Buffer B was 0.1% trifluoroacetic acid (TFA), 15% ethylene glycolmonomethylether (EGME), and 70% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile (solid line) was recorded at a wavelength of 210 nm using a sensitivity of 0.2 AU. The elution gradient (dotted line) was as follows: 20% B for 15 min, 20% B - 50% B for 50 min, 50% B -100% B for 15 min, 100% B for 5 min, 100% B - 20% B for 10 min, and 20% - 0% B for 1 min. Figure 20B. SDS-PAGE analysis of G. domesticus (chicken) ENBP fractions obtained from the HPLC shown in Figure 20A. Samples from each peak were run alongside chicken ENBP markers (S). Fractions #1 and #6 contain little amount of proteins (small peaks in Figure 20A), which results in very faint bands in lanes 1 and 6 in Figure 20B. Electrophoresis is from top (-) to bottom (+). 57 21A 0.35 S0.18 core histone linker histone 7G. 2 0.00 21B 50 Time (min) l inker h i s t o n e c o r e h i s t o n e S _ J _ 2 _ 3_ 4 5 6 7 8 S linker histone f f f -core histone Figure 21 A. Reverse phase HPLC fractionation of A mississippiensis (alligator) ENBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and 15% ethylene glycolmonomethylether (EGME), and Buffer B was 0.1% trifluoroacetic acid (TFA), 15% ethylene glycolmonomethylether (EGME), and 70% acetonitrile (ACN). The flow rate was 1 mL/min and the elution profile (solid line) was recorded at a wavelength of 210 nm using a sensitivity of 0.2 AU. The elution gradient (dotted line) was as follows: 20% B for 15 min, 20% B - 50% B for 50 min, 50% B -100% B for 15 min, 100% B for 5 min, 100% B - 20% B for 10 min, and 20% - 0% B for 1 min. Figure 21B. SDS-PAGE analysis of A. mississippiensis (alligator) ENBP fractions obtained from the HPLC shown in Figure 21 A. Samples from each peak were run alongside alligator ENBP markers (S). Electrophoresis is from top (-) to bottom (+). 58 22A 1.20 S 0.60 1 linker histone u B6 .7 core histone 1&4 10 J 1 0.00 22B 50 Time (min) linker histone core histone S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 S W* W W W <0K W U-^ *MM<MP* W * ^ - " " S « f * W W L_J 100 linker histone| core histone I Figure 22A. Reverse phase HPLC fractionation of R. catesbeiana (bullfrog) ENBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and 15% ethylene glycolmonomethylether (EGME), and Buffer B was 0.1% trifluoroacetic acid (TFA), 15% ethylene glycolmonomethylether (EGME), and 70% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile (solid line) was recorded at a wavelength of 210 nm using a sensitivity of 0.2 AU. The elution gradient (dotted line) was as follows: 20% B for 15 min, 20% B - 50% B for 50 min, 50% B -100% B for 15 min, 100% B for 5 min, 100% B - 20% B for 10 min, and 20% - 0% B for 1 min. Figure 22B. SDS-PAGE analysis of R. catesbeiana (bullfrog) ENBP fractions obtained from the HPLC shown in Figure 22A. Samples from each peak were run alongside bullfrog ENBP markers (S). The band (indicated by an arrow) in lane 3 is probably ae enriched degradation product of H1 linker histone. Electrophoresis is from top (-) to bottom (+). 59 23A 1.00 core histone ^ 0.50 j linker histone 8g1<ii 4 JH ! '.5' 3 6 | | $ S 7 I It 'i ! 'A, : fa 0.00 50 Time (min) 100 23B linker histone core histone S 1 2 3 4 5 6 7 8 9 10 11 12 S linker histone j «••# core histone Figure 23A. Reverse phase HPLC fractionation of /. punctatus (channel catfish) ENBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and 15% ethylene glycolmonomethylether (EGME), and Buffer B was 0.1% trifluoroacetic acid (TFA), 15% ethylene glycolmonomethylether (EGME), and 70% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile (solid line) was recorded at a wavelength of 210 nm using a sensitivity of 0.2 AU. The elution gradient (dotted line) was as follows: 20% B for 15 min, 20% B - 50% B for 50 min, 50% B -100% B for 15 min, 100% B for 5 min, 100% B - 20% B for 10 min, and 20% - 0% B for 1 min. Figure 23B. SDS-PAGE analysis of /. punctatus (channel catfish) ENBP fractions obtained from the HPLC shown in Figure 23A. Samples from each peak were run alongside catfish ENBP markers (S). Due to an accident, lane 2 in Figure 23B was not loaded. Electrophoresis is from top (-) to bottom (+). 60 3.3.3 NBPs from erythrocytes of Chelonia mydas (sea turtle) compared to NBPs from spleen normoblasts of Mus musculus (mouse) Two preparations of mouse spleen normoblasts were examined. In the first preparation, a control spleen contained a mixture of some normoblasts, along with lymphocytes and myeloid cells of various descriptions. In the second preparation, normoblasts were isolated from a phenylhydrazine - treated mouse spleen (0HZ spleen). In this case, the spleen was over 99% enriched for normoblasts. The NBPs from erythrocytes of C. mydas (sea turtle) and these two kinds of spleen normoblasts from M. musculus (mouse) were extracted by 0.4 N HCI, and then subjected to SDS-PAGE in order to compare them with ENBPs from chicken, alligator, bullfrog, and catfish. Figure 24 shows that normoblast NBPs from both normal mouse spleen and mouse spleen 0HZ (lanes 8 and 9) contain two dark bands in the H1 linker histone region but no band in the H5 linker histone region. Normoblast NBPs from these two types of mouse spleen show no difference in their migration patterns on the gel. Sea turtle ENBPs (lane 5) contain a dark band of H1 linker histones and a faint band migrating slightly more slowly than the H5 linker histone band in chicken ENBPs (lane 11) and the putative H5 linker histone band in alligator ENBPs (lane 7). 3.3.4 Fractionation of the ENBPs from Chelonia mydas (sea turtle) The acid extracted ENBPs from C. mydas (sea turtle) were fractionated by reverse phase HPLC. Figure 25 shows the elution profile and subsequent electrophoretic analysis on an SDS gel. The band in the core histone region in lane 5 probably comes from degradation of the linker histone. 61 3.3.5 Comparison of the H1 and/or H5 linker histone fractions of erythrocyte and liver NBPs from Gallus domesticus (chicken), Alligator mississippiensis (alligator), Chelonia mydas (sea turtle), Rana catesbeiana (bullfrog), and Ictalurus punctatus (channel catfish) The H1 and/or H5 linker histone fractions obtained by the HPLC were lyophilized to remove EGME, TFA and ACN, and then subjected to acid-urea-Triton-PAGE to confirm that there is an H5 linker histone in alligator erythrocyte nucleus. Figure 26 compares the H1 and the putative H5 linker histone fractions from alligator ENBPs with those from chicken ENBPs. Again, alligator ENBPs (lane 1) show a light band (the fastest band) at the H5 linker histone position compared to the dark H5 linker histone band in lane 6 from chicken ENBPs. Lanes 2 and 7 show enrichment of the H5 linker histone by HPLC fractionation. The H5 linker histone in chicken liver (lane 10) may be due to the presence of blood in the liver. Figures 27 and 28 show the H1 and/or H5 linker histone fractions of erythrocyte and liver NBPs from chicken, alligator, bullfrog, and catfish. These gel profiles also suggest that alligator ENBPs include a putative H5 linker histone which is not a component in alligator liver NBPs. Neither NBPs from bullfrog or catfish erythrocytes and liver contain an H5 linker histone. However, ENBPs from both bullfrog and catfish contain H1 linker histones (lanes 9-12 in Figure 27 and lanes 8-11 in Figure 28) that are different from the H1 linker histone components in chicken (lane 3 in Figure 28) and alligator (lane 3 in Figure 27) ENBPs. 62 1 2 3 4 5 6 7 8 9 10 11 linker histone core histone Figure 24. SDS-PAGE analysis of NBPs from mouse spleen normoblasts, and chicken, alligator, sea turtle, frog, and catfish erythrocytes and livers. 1, /. punctatus (catfish) liver NBPs; 2, catfish ENBPs; 3, R. catesbeiana (bullfrog) liver NBPs; 4, bullfrog ENBPs; 5, C. mydas (sea turtle) ENBPs; 6, A. mississippiensis (alligator) liver NBPs; 7, alligator ENBPs; 8, M. musculus (mouse) normal spleen normoblast NBPs; 9, mouse spleen 0HZ normoblast NBPs; 10, G. domesticus (chicken) liver NBPs; 11, chicken ENBPs (histones are designated). Electrophoresis is from top (-) to bottom (+). 63 25A core histone 0.70 i 0.35 0.00 • 25 B 50 Time (min) linker histone 100 core histone linker histone core histone Figure 25A. Reverse phase HPLC fractionation of C. mydas (sea turtle) ENBPs, using a (25x0.46 cm) 300-A Vydac C i 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and 15% ethylene glycolmonomethylether (EGME), and Buffer B was 0.1% trifluoroacetic acid (TFA), 15% ethylene glycolmonomethylether (EGME), and 70% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile (solid line) was recorded at a wavelength of 210 nm using a sensitivity of 0.2 AU. The elution gradient (dotted line) was as follows: 20% B for 15 min, 20% B - 50% B for 50 min, 50% B -100% B for 15 min, 100% B for 5 min, 100% B - 20% B for 10 min, and 20% - 0% B for 1 min. Figure 25B. SDS-PAGE analysis of C. mydas (sea turtle) ENBP fractions obtained from the HPLC shown in Figure 25A. Samples from each peak were run alongside sea turtle ENBP markers (S). Fractions #1 and #2 contain little amount of proteins (small peaks in Figure 25A), which results in very faint bands in lanes 1 and 2. Electrophoresis is from top (-) to bottom (+). 64 a l l i g a t o r c h i c k e n 1 2 3 4 5 6 7 8 9 10 11 Figure 26. A c i d - u r e a - T r i t o n - P A G E analysis of erythrocyte and liver NBPs from alligator and chicken, and their HPLC fractions (H1 and/or H5 linker histones). 1, alligator ENBP markers; 2, alligator ENBP HPLC fraction (putative H5, Figure 21B, lane 1); 3, alligator ENBP HPLC fraction (H1, Figure 21B, lane 2); 4, alligator liver NBP markers; 5, alligator liver NBP HPLC fraction (H1); 6, chicken ENBP markers; 7, chicken ENBP HPLC fraction (H5, Figure 20B, lane 2); 8, chicken ENBP HPLC fraction (H1, Figure 20B, lane 4); 9, chicken liver NBP markers; 10, chicken liver NBP HPLC fraction (H5); 11, chicken liver NBP HPLC fraction (H1). Electrophoresis is from top (+) to bottom (-). 65 alligator catfish Figure 27. Acid-urea-Triton-PAGE analysis of erythrocyte and liver NBPs from alligator and catfish, and their HPLC fractions (H1 and/or H5 linker histones). 1 and 5, alligator ENBP markers; 2, alligator ENBP HPLC fraction (putative H5, Figure 21B, lane 1); 3 and 4, alligator ENBP HPLC fraction (H1, Figure 21B, lane 2); 6, alligator liver NBP markers; 7, alligator liver NBP HPLC fraction (H1); 8, catfish ENBP markers; 9-13, catfish ENBP HPLC fractions (H1, Figure 23B, lanes 1, 3, 4, 6, and 7); 14, catfish liver NBP markers; 15-17, catfish liver NBP HPLC fractions (H1). Electrophoresis is from top (+) to bottom (-). 66 chicken bullfrog 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 28. Acid-urea-Triton-PAGE analysis of the erythrocyte and liver NBPs from bullfrog and chicken, and their HPLC fractions (H1 and/or H5 linker histones). 1, chicken ENBP markers; 2, chicken ENBP HPLC fraction (H5, Figure 20B, lane 2); 3, chicken ENBP HPLC fraction (H1, Figure 20B, lane 4); 4, chicken liver ENBP markers; 5, chicken liver NBP HPLC fraction (H5); 6, chicken liver NBP HPLC fraction (H1); 7, bullfrog ENBP markers, 8-11, bullfrog ENBP HPLC fractions (H1, Figure 22B, lane, 1,4, 7, and 9); 12, bullfrog liver NBP markers; 13-16, bullfrog liver NBP HPLC fractions (H1). Electrophoresis is from top (+) to bottom (-). 67 Figure 29 shows the acid-urea-Triton-PAGE analysis of ENBPs from the chicken, alligator and sea turtle. Sea turtle ENBPs, before HPLC, also contain proteins running in the H5 linker histone region of the AUT gel (the fastest bands in lanes 7 - 9 , indicated by ?). However, these bands are not present after HPLC. The HPLC fraction from the sea turtle linker histone region in Figure 31A , which is identical to Figure 25A, only shows the H1 linker histones (lanes 10 - 12) in Figure 29, but no H5 linker histone. The composite Figure 30 shows that ENBPs from both chicken and alligator contain proteins in the H5 linker histone region of the gel before and after HPLC. However, sea turtle ENBPs include proteins in the H5 linker histone region of gel only before HPLC. They are not present after HPLC. Therefore, another acid-urea-Triton-PAGE (Figure 31B), using sea turtle ENBP fractions from the linker and core histone regions of the chromatogram (Figure 31 A) was run to check if there is an H5 linker histone fraction in sea turtle. Figure 31B indicates that the protein running in the H5 linker histone region of gel (marked by ?) in lane S before HPLC is actually a component of the core histone region of the chromatogram after HPLC (the fastest band in lane 12, indicated by an arrow). The fastest band in lane 5 (Figure 31B) is probably a degradation artifact of linker histones. 3.3.6 Chymotrypsin digestion pattern of linker histone fractions of erythrocyte and/or liver NBPs from Gallus domesticus (chicken), Alligator mississippiensis (alligator), Rana catesbeiana (bullfrog), and Ictalurus punctatus (channel catfish) The lyophilized H1 and/or H5 linker histone fractions used in section 3.3.5 were incubated with chymotrypsin at room temperature for 100 minutes, and then subjected to 20% acid-urea-PAGE to esolve peptides. Figure 32 shows the chymotrypsin digestion pattern of these H1 and H5 linker histone fractions. Only the H5 linker histone 68 before HPLC after HPLC chicken alligator sea turtle 1 2 3 4 5 6 7 8 9 10 11 12 Figure 29. Acid-urea-Triton-PAGE analysis of ENBPs from chicken, alligator and sea turtle, and HPLC fractions (H1 and/or H5 linker histones) from sea turtle ENBPs in Figure 25A. 1-3, chicken ENBP markers; 4-6, alligator ENBP markers; 7-9, sea turtle ENBP markers; 10-12, sea turtle ENBPs from the HPLC fractions of the linker histone in Figure 25A. ? indicates proteins (from sea turtle) running in linker histone region of gel before HPLC but not present after HPLC. Electrophoresis is from top (+) to bottom (-). 69 Figure 30. Composite figure of acid-urea-Triton-PAGE showing lack of H5 linker histone in sea turtle ENBPs after HPLC. A: (taken from Figure 26) 1, alligator ENBP markers; 2, alligator ENBP after HPLC (H5 region of gel); 3, alligator ENBPss after HPLC (H1 region of gel); 6, chicken ENBP markers; 7, chicken ENBP after HPLC (H5 region of gel); 8, chicken ENBP after HPLC (H1 region of gel); B: (taken from Figure 29) 9, sea turtle ENBP markers; 10-12, sea turtle ENBPs after HPLC (H1 region of gel); ? indicates proteins from sea turtle running in linker histone region of gel before HPLC but not present after HPLC. B.H. = before HPLC, A.H. = after HPLC. Electrophoresis is from top (+) to bottom (-). 70 31A 0.70 i 0.35 0.00 core histone 13 12 linker histone 14 i 7*> 1 2 S 10c •10\l 50 Time (min) 100 20 too 31B linker histone core histone C S 2 3 4 6 6 7 8 9 10a 10b 10c 11 12 14 S C core histone linker histone Figure 31A (identical to Figure 25A). Reverse phase HPLC fractionation of C. mydas (sea turtle) ENBPs , using a (25x0.46 cm) 300-A Vydac C i 8 column. Buffer A was 0.1% trifluoroacetic acid (TFA) and 15% ethylene glycolmonomethylether (EGME), and Buffer B was 0.1% trifluoroacetic acid (TFA), 15% ethylene glycolmonomethylether (EGME), and 70% acetonitrile (ACN). The flow rate was 1ml/min and the elution profile (solid line) was recorded at a wavelength of 210 nm using a sensitivity of 0.2 A U . The elution gradient (dotted line) was as follows: 20% B for 15 min, 20% B - 50% B for 50 min, 50% B - 100% B for 15 min, 100% B for 5 min, 100% B - 20% B for 10 min, and 20% - 0% B for 1 min. Figure 31B. Acid-urea-Triton-PAGE analysis of C. mydas (sea turtle) ENBP fractions obtained from the H P L C shown in Figure 31 A. Samples from each peak were run alongside sea turtle E N B P markers (S) and chicken E N B P markers (C). The fastest band in lane S (indicated by ? in this figure and also in Figures 29 and 30) migrates in the fraction (indicated by an arrow) from core histone region (lane 12). Electrophoresis is from top (+) to bottom (-). 71 fraction from alligator ENBPs (lane 11) shows a similar digestion pattern to that of H5 linker histone fraction from chicken ENBPs (lane 9). All other H1 linker histone fractions show different patterns from the H5 linker histone fraction from chicken and alligator H5 linker histone fractions. 3.3.7 Amino acid analysis of the linker histone fractions from erythrocytes of Gallus domesticus (chicken), Alligator mississippiensis (alligator), Chelonia mydas (sea turtle), Rana catesbeiana (bullfrog), and Ictalurus punctatus (channel catfish) The linker histone fractions, #2 and #4 (Figure 20A) from G. domesticus (chicken), #1 and #2 (Figure 21 A) from A. mississippiensis (alligator), #3 and #7 (Figure 25A) from C. mydas (sea turtle), #1, #4, #7 and #9 (Figure 22A) from R. catesbeiana (bullfrog), and #1, #3, #4 and #6 (Figure 23A) from /. punctatus (channel catfish), by reverse phase HPLC were lyophilized to remove EGME, TCA and ACN, and then analyzed for amino acid composition. Table 8 shows the amino acid composition of these fractions. As we mentioned previously in section 3.3, the high mol% of arginine residues is the most important indicator for H5 linker histone. The putative H5 linker histone from alligator contains 6.1 mol% of arginine. Although this is lower than the typical H5 linker histone from chicken, which contains 12.4 mol% of arginine, it is much higher than the typical H1 linker histone from chicken which contains 3.6 mol% of arginine. The two fractions from sea turtle ENBPs after HPLC, #3 and #7, which contain 3.9 mol% and 2.1mol% of arginine respectively, belong to the H1° and H1 linker histone categories. All the other animals have ENBPs which contain less than 4 mol% of arginine except fraction #4 from bullfrog ENBPs, which contains 4.6 mol% of arginine. 72 X I 2 o rs 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 14 1 5 1 6 17 Figure 32. Acid-urea-PAGE of chymotrypsin digestion of NBP HPLC fractions from chicken, alligator, bullfrog and catfish. 1 and 17, M. californianus (mussel) protamine-like (PL) protein markers (PLII,III,IV); 2 and 16, undigested bullfrog ENBP HPLC fraction (Control); 3, catfish liver NBP HPLC fraction (H1), 4-7, catfish ENBP HPLC fractions (H1); 8, chicken ENBP HPLC fraction (H1); 9, chicken ENBP HPLC fraction (putative H5); 10, alligator ENBP HPLC fraction (H1); 11, alligator ENBP HPLC fraction (H5); 12-15, bullfrog ENBP HPLC fractions (H1); Arrow heads in lanes 9 and 11 indicate that chicken linker histone H5 and putative alligator linker histone H5 have similar chymotrypsin digestion pattern. Electrophoresis is from top (+) to bottom (-). 73 o _ l CL X CD •f—« M — CO to £ to i _ . 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In the following sections I will discuss the results as they relate to these two questions. 4.1 Internally fertilizing teleost fish tend to select protamine-like SNBPs On the basis of the seven teleost fish that have been examined thus far, the hypothesis (Kasinsky, 1985, 1989, 1995) that fish with internal fertilization have more protamine-like SNBPs, still holds. Six of these internally fertilizing fish (C. aggregata, P. reticulata, P. picta, X. h. guentheri, X. maculatus, Sebastes sp. and C. laticeps; see Figure 10) have protamines or protamine-like proteins as their SNBPs (Figure 33). The seventh Cataetyx laticeps (Saperas et al., 1993; Chiva et al., 1995) is an exception. It has somatic histones and an additional arginine-enriched H1 like protein, but no protamine. This may be due to the fact that this deep sea, viviparous benthic fish shows rapid development (progenesis) with short larval stages. It is possible that spermatogenesis occurs so rapidly, there might not be time to transcribe the protamine gene. As can also be seen in Figure 33, internal fertilization (Kasinsky, 1985, 1989, 1995; Chiva et al., 1995) appears to act as a constraint on SNBP diversity in cartilaginous fish 75 (P, KP), as well as in amniotes (reptiles (P), birds (P) and mammals (P, KP)). This is marked contrast to externally fertilizing agnathans (H/H1) and bony fish (H/H1, PL, P) where a more complete range of SNBP types has been observed. 4.2 The internally fertilizing frog Eleutherodactylus coqui has protamine-like SNBPs The sporadic reversion from protamines in the internally fertilizing frog Ascaphus truei, a primitive neobatrachian, to histones among more advanced, externally fertilizing frogs (Kasinsky et al., 1999) seems to contradict the general evolutionary trend of SNBPs from histone to protamine-like and then to protamine types (Ausio, 1995; Chiva era/., 1995). However, it parallels the simplification of elongated, internally fertilizing sperm in primitive A. truei (Jamieson et al., 1993; Jamieson, 1999; Scheltinga et al., 2001), to more rounded, externally fertilizing sperm in more advanced neobatrachians and attributed by Lee and Jamieson (1993) as a secondary reversion to external fertilization. On this basis, Kasinsky et al. (1999) proposed a model to explain SNBP evolution in anuran phylogeny in which the sporadic reversion from protamine or protamine-like proteins to histones has occurred concomitantly with the simplification from introsperm to less complex ectaquasperm (Figure 34). Kasinsky et al. (1999) predicted that protamine-like proteins would be present in the advanced neobatrachian Eleutherodactylus coqui, which is also an internal fertilizer (Figure 33) with an elongated sperm head (Singla, Ausio and Kasinsky, unpublished result). The data show that E. coqui SNBPs do indeed consist of four protamine-like proteins (Figure 17A, B, 18B) whose amino acid composition resemble those of A. truei (Table 7). This finding also 77 tn c M 'sz nt « o <D SZ • Leiopeimatidae L. hochstettcri(PL) c n) Z u re i 13 O c • Ascaphidae A. jwe^PL) Pipidae X. laevis(PL) X.(S.)tropicalis{H/M) • Pelobatidae S. couchii (PL) Bufonidae 6. japonicus (P.) Hylidae H. reg//fe (PL) .Leptodactylidae £ ™ 9 < « ( P L ) • Myobatrachidae C. signifera (H/H1) • Ranidae R. catesbeiana (H/H1) N/A N/A N/A N/A Figure 34. Distribution of SNBP types as related to sperm head morphology in anurans. H/H1 = core histone/linker histone type; PL = protamine-like type; P = protamine type; KP = keratinous protamine type. I.F. = internal fertilization; E.F. = external fertilization. N/A = sperm head morphology not available. Phylogeny adapted from Pough era/., 1997, p58, and modified as from Scheltinga et al., 2001. Sperm head morphology from Lee and Jamieson, 1993. 78 supports the model (Kasinsky era/., 1985, 1989, 1991) and suggests that this reversion is a result of different modes of fertilization: internal fertilization selects for more arginine-rich protamine-like proteins, whereas external fertilization is compatible with protamines or histones (Kasinsky, 1985, 1989, 1995). 4.3 ENBPs from vertebrates Since the discovery of linker histone H5 (histone V or f2c) in mature chicken erythrocytes (Neelin et al., 1964; Hnlica, 1964) and then in erythrocytes of several other avian species (Neelin, 1968). Research has been done to see if this erythrocyte-specific protein is unique to birds (Panyim era/., 1975; Miki and Neelin, 1975; Adams and Neelin, 1976; Grimmond and Holmes, 1980; Zlatanova, 1981; Rutledge et al., 1981; Shimada era/., 1981; Appels and Wells, 1972; Rutledge era/., 1988; Khochbin and Wolffe, 1993; Palyga and Neelin, 1998). Although there is some confusion in the literature, it is generally accepted that linker histone H5 is erythrocyte-specific and has so far only been found in avian nucleated erythrocytes (Khochbin and Wolffe, 1994). The other differentiation-specific linker histone H1°, however, is not erythrocyte-specific, and has been found in the terminally differentiated tissue in all vertebrates examined (Khochbin and Wolffe, 1994). One of the aims of my thesis is to determine if there is an H5 linker histone presents in A. mississippiensis (alligator) erythrocytes since alligator is closely related to birds with respect to SNBPs (Hunt et al., 1996). I also have examined NBPs in erythrocytes from G. domesticus (chicken), C. mydas (sea turtle), R. catesbeiana (bullfrog), and /. punctatus (catfish) (Figure 24). Livers from each of these species 79 (except sea turtle) were used as a tissue control to test the erythrocyte-specificity of linker histone H5. 4.3.1 "Linker histone H1°/H5" in Alligator mississippiensis (alligator) ENBPs Based on the pattern of HPLC, chymotrypsin digestion, SDS-PAGE, and AUT-PAGE, as well as amino acid analysis, alligator ENBPs appear to include an H5 linker histone. SDS-PAGE (Figures 19, 24) shows that alligator ENBPs contain a band at exactly the same position as the H5 linker histone band of chicken ENBPs. Although alligator liver NBPs also have a very faint band at the same position, it is very likely that this comes from the contamination of blood. The intensity of the H5 linker histone band in alligator ENBPs indicates that it is a minor component compared to H1 linker histone, whereas H5 linker histone is the major ENBP in the chicken RBC nucleus. The HPLC profile (Figure 21 A) confirms this conclusion, as we can see the same pattern. However, alligator ENBPs have a large linker histone H1 elution peak and a small linker histone H5 peak, while there is a small linker histone H1 peak and a prominent H5 linker histone peak in chicken ENBPs (Figure 23). In mature chicken erythrocytes, the ratio of linker histone H5 to H1 is around 2.5:1 (Appels and Wells, 1972). The relative amount of linker histone H5 to H1 in alligator and chicken ENBPs was measured by weighing their peak areas in the HPLC profiles. The ratio of linker histone H5 to H1 in alligator erythrocytes is 1:4.5, which is significantly lower than that (3.4:1) in chicken erythrocytes. This putative linker histone H5 is further confirmed by AUT-PAGE (Figure 29) and chymotrypsin digestion (Figure 35) because of the similar patterns that appear on each gel. 80 The data from amino acid analysis (#1 in Table 4) after HPLC (Figure 24A) call into question the authenticity of the alligator linker histone as an H5 histone. This alligator histone contains histidine (1.2 mol%) and methionine (0.4 mol%), so it is not a histone H1 (Section 1.2.4). It looks more like a linker histone H1°, because its content of histidine, methionine, and acidic residues (Asx+Glx: 9.4 mol%) falls into the typical linker histone H1° range (histidine: 1.0 to 1.9 mol%; methionine: 0.4 to 0.8 mol%; acidic residue (Asx+Glx): 8.4 to 10.9 mol%). But it is still not a typical linker histone H1°, since its arginine content (6.1 mol%) is much higher than that of a typical linker histone H1° (2.3 to 4.1 mol%) and its lysine to arginine ratio (3.2) is siganificantly lower than that of a typical linker histone H1° (10.0). These two parameters for a typical linker histone H5 are 11.2 mol% and 1.9, respectively. So, this alligator histone is actually in between linker histone H1° and H5. Consequently, we call this protein alligator "linker histone H1°/H5". Alligator "linker histone H1°/H5" has the same mobility as that of linker histone H5, which runs faster than does linker histone H1° on AUT-PAGE (Smith et al., 1984). The most important characteristic of alligator linker histone H1°/H5 is that it contains about 6.1 mol% arginine, which falls in between a typical H1° and H5. The trace amount of this protein found in alligator liver NBPs may have come from blood contamination. The alligator ENBPs also contain two H1 linker histones which could be separated by electrophoresis (Figure 19) but could not be separated by HPLC (Figure 21 A, B). These two H1 proteins (#2 in Table 8) contain no histidine and methionine, and low acidic residue (6.2 mol%), which is a marker for the typical linker histone H1 (Panyim and Chalkley, 1969; Smith et al., 1984). However, these two alligator H1 81 protein have more arginine (3.1 mol%) than that of a typical linker histone H1 (1.5 mol%). 4.3.2 Linker histone H1° in Chelonia mydas (sea turtle) ENBPs Earlier research (Rutledge et al., 1981) has shown that sea turtle erythrocyte contains a linker histone H1°. My results confirmed that this is the case as we can see a band running slightly slower than the chicken linker histone H5 on SDS-PAGE (Figure 24). The amino acid analysis data of this histone (#3 in Table 8) after HPLC (Figure 25A), shows small differences from the data of Rutledge et al. (1981), which is probably due to different species being examined (C. mydas in this thesis and P. scriptans elegans by Rutledge et al., 1981): for example, lower lysine (16.7 mol% vs. 28.0 mol%) but higher arginine (3.9 mol% vs. 1.7 mol%) and histidine (1.6 mol% vs. 0.3 mol%) content. However, it still falls into the linker histone H1° category (Section 1.2.4). Fraction #7, which contains no histidine, trace amount of methionine (0.2 mol%), and a high content of alanine (20.5 mol%), is clearly an H1 linker histone. 4.3.3 Linker histone H1° in Rana catesbeiana (bullfrog) ENBPs Linker histone H1° has been found in erythrocytes of the frog Xenopus laevis (Rutledge et al., 1984) and Rana catesbeiana (Shimada et al., 1981). The amino acid analysis data (Table 8) after HPLC (Figure 22A) suggest that the frog Rana catesbeiana examined in this thesis contains two linker H1° histones (fractions #1 and #9) and two linker H1 histones (fractions #4 and #7). Except for a much lower lysine content (16.6 and 14.6 mol%) than a typical bovine liver H1°a (Smith and Johns, 1980), the other amino acid compositions of these two Rana H1° linker histones are very similar to 82 bovine liver H1°a (Smith and Johns, 1980). The other two fractions clearly belong to the linker histone H1 category because they contain no methionine, have few acidic residues, a low arginine content (1.6 and 1.9 mol%, respectively), and a high alanine content (22.6 and 23.8 mol%, respectively). These two Rana H1 proteins contain trace amounts of histidine (0.7 and 0.6 mol%, respectively), which is close to that in the linker histone H1A (0.4 mol%) found by Shimada et al. (1981), while a typical linker histone H1 has no histidine at all (Panyim and Chalkley, 1969; Smith etal., 1984). 4.3.4 Linker histone H1° in Ictalurus punctatus (channel catfish) ENBPs It has been reported (Miki and Neelin, 1975, 1977) that erythrocytes of fishes (rainbowtrout and carp) contain linker histone H5, which was later grouped into the linker histone H1° category by Khochbin and Wolffe (1994). The amino acid analysis data (Table 8) after HPLC (Figure 23A) indicates that erythrocytes of /. punctatus (channel catfish) studied in this thesis includes both H1° (#1 in Table 8) and H1 (#3, #4, and #6 in Table 4) linker histones. Fraction #1 contains 1.6 mol% histidine, 0.1 mol% methionine, 9.6 mol% acidic residue, and 4.0 mol% arginine, which are characteristic of a typical linker H1° histone. However, its alanine content (22.3 mol%) is much higher than that in bovine liver H1°a (14.2 mol%) (Smith and Johns, 1980). Except for the trace amount of histidine (0.2 mol%), the other three fractions (#3, #4, and #6) contain a low amount of acidic residues and arginine, high alanine content, and no methionine, which suggests that they are H1 linker histones. 8 3 4.3.5 ENBPs in Mus musculus (mouse) normoblasts It is obvious that mature enucleated erythrocytes of mammals contain no ENBPs. But, is there a linker histone H5 present in the normoblast, which is the latest developmental stage during erythropoiesis where the nucleus gets very condensed just before it is extruded (Figure 6)? Adams and Neelin (1976) also reported the absence of cell-specific linker histone H5 in erythroid cells from rabbit marrow. However, Zlatanova (1981) reported a linker histone H5 in preparations from mouse spleen based on the electrophoretic pattern. In this thesis, I examined normal and enriched normoblast preparations from mouse spleen. SDS-PAGE (Figure 24) shows that there is no linker histone H5 present, in agreement with the findings of Adams and Neelin (1976). 4.4 The evolutionary trend of linker histones In both development (Khochbin and Wolffe, 1994) and evolution (Figure 7), it appears that nature proceeds from the general (somatic linker histone H1) to the specific (differentiation-specific linker histones H1° and H5). We have proposed that if this evolutionary trend for linker histones (Figure 7) (Schulze, 1995; Peretti and Khochbin, 1997; Brocard et al., 1997) is correct, we might find a linker histone H5-like protein in alligator erythrocytes. My data confirm this hypothesis. We do see a protein with 6.2 mol% arginine content, which is lower than that of a typical linker histone H5 but higher than that of a typical linker histone H1°. We call this protein "linker histone H1°/H5". This protein is a minor component in alligator ENBPs compared to linker histone H1 present in the alligator erythrocyte. From the data in this thesis, we can 84 modify the evolutionary trend of linker histones (Figure 7) as shown in Figure 35. Here the linker histone H1 of vertebrates and linker histones H1°, H1°/H5, and H5 have diverged from linker histone H1 of invertebrates. Linker histones H1° of fish, frog, and sea turtle have given rise to linker histones H1°/H5 of alligator erythrocytes and to H5 of bird erythrocytes, with increasing arginine content. Nature eventually removed linker histones altogether from mature enucleated red blood cells of mammals (Figure 6). Increased blood pressure might be an explanation for the presence of linker histone H1°/H5 in alligator erythrocytes. Higher blood pressure in alligator versus turtle could require the presence of a more arginine-rich H5-like linker histone in order to achieve greater condensation of chromatin in the nucleus of this motile red blood cell. Nature eventually solves the problem posed by even higher blood presure either by having a linker histone H5 in its ENBPs, as occurs in bird erythrocytes, or by eliminating the nucleus entirely, as is the case in the mature erythrocytes of mammals. Amongst the vertebrates, a bird (chicken) has the highest average blood pressure (Figure 9), so it has H5 as the major linker histone in its ENBPs. The average blood pressure in a reptile (crocodile), which is phylogenetically closer to bird in its SNBP profile (Hunt et al., 1996) than it is either to turtle or to mammal (man), is about one quarter that of chicken (Figure 9). Alligator has a minor component of H1°/H5 linker histone in its ENBPs. Turtle, frog and lungfish have relatively lower blood pressures than that of crocodile (Figure 9), and sea turtle, bullfrog and catfish contain linker histone H1° with a somewhat lower arginine content in their erythrocytes (Table 8). There is only one H1° in catfish, but there are two H1°s in bullfrog. Perhaps catfish shows the initial appearance of the linker histone H1° in vertebrate evolution. 85 No histone in mammal (mouse) RBC H5 Bird (chicken) H1°/H5 Reptile (alligator) H1 Fish (catfish) H1° Amphibian (frog), Reptile (sea turtle) H1 Invertebrate Figure 35. A proposed hypothesis for the evolutionary trend of linker histones. 86 4.5 Future work As we proposed in Figure 35 that alligator erythrocytes contain "linker histone H1°/H5", it makes sense to design PCR primers based on the conserved sequences of published bird H5 genes and the Xenopus H1° gene in order to clone the potential alligator H1°/H5 gene. Although I have failed to clone this alligator gene using genomic DNA in my first few attempts, it is still very necessary to isolate this gene in the future (using cDNA template might have a better chance) in order to distinguish between the following three possibilities: 1) it is an H5-like gene, 2) it is an H1°-like gene, 3) it is an intermediate H1°/H5 gene. Once this gene becomes available, we can determine exactly the protein sequence and the DNA sequence of flanking regions. This will allow us to find a category for alligator erythrocyte linker histones in order to confirm the evolutionary trend in vertebrates that I have proposed in my thesis at the molecular level. However, because the vertebrate linker histone genes are descendants of invertebrate "orphan" histone H1 genes (Schulze and Schulze, 1995), there is confusion about the exact type of vertebrate linker histone even when the gene is available as in Xenopus (Rutledge etal., 1988; Shwed etal., 1992). We might have a better chance to determine the linker histone type for alligator since it is closer phylogenetically to birds than is Xenopus. 4.6 General conclusion Arginine side chains tend to be more abundant in SNBPs of internally fertilizing non-mammalian vertebrates such as teleost fish and anurans, as well as in linker histones of ENBPs in alligator and birds, in comparison with NBPs in other differentiated 87 cell types. These cell types, sperm and erythrocytes, generally require a more condensed genome than other highly differentiated cells as they are transcriptionally inactive. The importance of arginine side chains in condensing chromatin is also apparent in SNBPs of invertebrates (Ausio, 1999), algae and plants (Reynolds and Wolfe, 1978, 1984; Rizzo et al., 1985; Kasinsky, 1989) and protozoans (Dacks and Kasinsky, 2000). Thus, arginine residues have played an important role in the evolution of condensed chromatin in at least two kinds of eukaryotic nuclei. 88 5. 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