UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The characterization and localization of inducible protein IP-25 Cserjesi, Peter 1980

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1980_A6_7 C84.pdf [ 7.06MB ]
Metadata
JSON: 831-1.0095240.json
JSON-LD: 831-1.0095240-ld.json
RDF/XML (Pretty): 831-1.0095240-rdf.xml
RDF/JSON: 831-1.0095240-rdf.json
Turtle: 831-1.0095240-turtle.txt
N-Triples: 831-1.0095240-rdf-ntriples.txt
Original Record: 831-1.0095240-source.json
Full Text
831-1.0095240-fulltext.txt
Citation
831-1.0095240.ris

Full Text

THE CHARACTERIZATION AND LOCALIZATION OF INDUCIBLE PROTEIN IP-25 by PETER CSERJESI B.Sc, The University of British Columbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR^THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY hie accept this thesis as conforming to the required standard THE UNIVERSITY DF BRITISH COLUMBIA May 1980 (c^ Peter Cserjesi, 1980 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t ten pe rm i ss i on . Department o f The Un i ve rs i t'y^Oyf B r i t ifeff Col umb i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ABSTRACT An in-depth study was undertaken to biochemically characterize and localize within the chromatin the inducible protein IP-25. This protein i s found in Friend-virus-transformed erythroleukemia c e l l s which have been induced to differentiate into orthochramic erythroblasts. It uas found that IP-25 i s one of a very limited number of proteins which appear de novo after the induction of Friend cells with dimethyl sulfoxide as an inducer. An apparent molecular weight of approximately 2D,DD0 daltons was obtained for IP-25 by SDS polyacrylamide gel electrophoresis. Acid-urea gel electrophoresis indicated that IP-25 is a basic protein which migrates in a manner similar to histones H1D or H5 in this gel system. Migration behaviour in urea gradient - TX-10TJ gels also indicated that this protein was similar to histone H 5 . Amino acid analysis revealed a similarity in amino acid composition between IP-25 and both histones H1 and H5. However the homology between IP-25 and these histones was not enough to enable a classification into either group. Peptide mapping of IP-25 and the H1b histone of Friend c e l l s indicated that fragments of similar molecular weights are generated when digestion i s carried out using chymotrypsin or papain. Chymotrypsin did not generate enough Fragments, while digestion with papain produced too many non-specific cleavages to make a definitive comparison between these two proteins. Localization of IP-25 within the chromatin repeat unit was accom-plished by micrococcal nuclease digestion of intact Friend c e l l nuclei. The protein composition of the nuclease generated fragments was analyzed by polyacrylamide gel electrophoresis. One dimensional analysis of nucleosomal proteins indicated that IP-25 maintained a constant quanti-t a t i v e relationship with H1 histories i n different sized chromatin frag^-ments. This suggests that l o c a l i z a t i o n of IP-25 p a r a l l e l s the i n t e r -nucleosDmal location of histone H1. In order to obtain more direct evidence for the l o c a l i z a t i o n of IP-25, two-dimensional electrophoresis mas performed. These experiments v e r i f i e d the one-dimensional electro-phoretic evidence. The presence of IP-25 i n dimethyl sulfoxide induced Friend c e l l s did not d r a s t i c a l l y a l t e r the nuclease generated repeat lengths. The function of IP-25 during Friend c e l l erythropoiesis could not be determined by the data obtained i n t h i s thesis. However, i t appears that the chromosomal accumulation of IP-25 and erythropoiesis i n these c e l l cultures are linked events. iv TABLE OF CONTENTS PAGE ABSTRACT •... • « * U TABLE OF CONTENTS . i u LIST OF FIGURES •> v i i LIST OF TABLES v i i i LIST OF ABBREVIATIONS * INTRODUCTION I Chromatin Structure • 1 A) The Nucleosome Core 2 B) Histone H1 ^ C) Histone Genes 6 D) Non-Histone Chromosomal Proteins 7 . II Erythropoiesis 8 A) Normal Erythropoiesis 8 B) Erythropoiesis in Friend Cells 10 III Thesis Objectives 13 MATERIALS AND METHODS I Materials 1^  II Li s t of Common Buffers 15 III Cell Line and Culture Conditions 16 IV Isolation of Histones from Whole Cells 16 V Electrophoretic Techniques 17 A) SDS Polyacrylamide Gel Electrophoresis 17 B) Acid-Urea - SDS Two-Dimensional Gel Electrophoresis 18 C) Urea Gradient Polyacrylamide Gel Electro-phoresis 19 V D) Isoelectric Focusing - SDS Two-Dimen-sional Electrophoresis 20 VI Amino Acid Analysis ••• 21 A) Sample Preparation •••• 21 B) Sample Hydrolysis 22 C) Sample Analysis 23 VII Peptide Mapping 23 VIII DansylatiDn • 25 IX One Dimensional Analysis of Nucleasomal Histones and DMA 26 A) Nucleosome Isolation 26 B) Histone Analysis 27 C) DNA Analysis ...27 X Two-Dimensional Aanalysis of Nucleosomal Proteins ........ 29 RESULTS I Protein Patterns of Uninduced and DMSO Induced Friend Cells .30 II Characterization of the IP-25 Molecule 35 A) Molecular Weight Determination of IP-25 by SDS Electrophoresis 35 B) Acid-Urea - SDS Two-Dimensional Electro-phoresis of IP-25 36 C) TX-100 with Urea Gradient Gel Electro-phoresis of Friend Cell Histones 39 D) Amino Acid Analysis hi E) Peptide Maps of H1 Histones and IP-25 57 F) Amino Terminal Group Determination 61 v i III Distribution of IP-25 on Friend Cell Chromatin 6k A) Determination of IMucleasomal Fraction Purity 64 B) IMucleosomal Protein Analysis 67 C) Tuia-Dimensional Examination of IMucleosomal Proteins 73 D) Examination of IMucleosome Repeat Lengths From Uninduced and DMSO Induced Friend Cells 76 IV Relationship Between IP-25 and Differentiation 79 A) IP-25 Accumulation During Differentiation 79 B) Effect of Various Inducers and Man-inducing Agents on IP-25 Accumulation Bk C) Effect of DMSO Induction on DMSO Resistant Cells 87 DISCUSSION I Characterization of IP-25 93 II Localization of IP-25 Attachment of Friend Cell Chromatin : 96 III The Relationship of IP-25 and Differentiation 99 LITERATURE CITED in* v i i LIST DF FIGURES Figure T i t l e Page 1 Two dimensional gel electrophoresis of uninduced and DMSO induced Friend c e l l proteins 32 2 Fluoragraphs of two dimensionally separated proteins from Friend c e l l cytoplasm and nuclei 34 3 Molecular weight determination of IP-25 by SDS polyacrylamide gel electrophoresis 38 k Acid-urea - SDS two dimensional gel electrophoresis of DMSO induced Friend c e l l histones 41 5 Urea gradient gel electrophoresis of histones kk 6 Identification of protein bands from urea gradient gel by SDS electrophoresis 46 7 Amino acid analysis elution profiles 49 8 Degradation of serine during acid hydrolysis 55 9 Peptide maps of the H1 histones and IP-25 59 10 Fractionation of Friend c e l l histones by Bio-Gel .60 chromatography 63 11 Sucrose gradient fractionation of nucleosomes 66 12 One dimensional analysis of nucleosomal proteins 69 13 Spectrophotometry scans of nucleosomal proteins separated on SDS polyacrylamide gels 7 2 14 Two dimensional electrophoresis of nucleosomal proteins 75 15 Comparison of DIMA lengths of micrococcal nuclease digested chromatin 78 16 Analysis of micrococcal nuclease digest products 81 17 Accumulation of IP-25 during induction with DMSO 33 a ratio of total Hi's 83 18 Effect of db cAMP an Friend c e l l chromosomal proteins ..... 89 19 SDS electrophoresis of chromosomal proteins from DMSO resistant Friend cells 91 v i i i LIST OF TABLES Table T i t l e Page 1 Amino acid analysis of H1a, H1b, and IP-25 52 2 Haemoglobin and IP-25 induction 66 3 Parial amino acid composition of lysine rich histones 101 ACKNOWLEDGEMENTS I wish to express my gratitude to Dr. R.O. Reeves who put up with and also supported me during the preparation of this thesis. I also wish to thank the graduate students and faculty members of the Genetics/Cell Biology Programme for their assistance and friendship. I would also like to thank my parents for their understanding and encouragement in my academic endeavours. And special thanks to Sherry for tolerating my strange hours and habits and helping to prepare and type this thesis. LIST OF ABBREVIATIONS AcH - acetic acid APS - ammonium persulfate cpm - counts per minute dansyl chloride - 1-dimethylaminonaphthalene-5-sulfochloride 6 2 db cAMP - IM -dibutyryladenosine 3':5'-cyclic monophasphoric acid NHP - non-histone chromosomal proteins PMSF - phenylmethylsulfonyl fluoride SDS - sodium, dodecyl sulfate TEA - triethanolamine TEMED - N,i\l,N' ,N'-tetramethylethylenediamine TX-100 - oc t y l phenoxy polyethoxyethanal 1 INTRODUCTION The organization and expression of the eukaryotic genome i s prob-ably the most studied problem i s molecular biology today. While these problems have been, or are rapidly being solved in v i r a l and prokaryotic organisms, there has been only limited success in eukaryotic c e l l s , especially in multicellular organisms. The reasons for the lack of success are two-fold: a) the eukaryotic genome i s several orders of magnitude larger than prokaryotic genomes, and b) the packaging of the chromatin i s far more complex and i s under continual change during the c e l l cycle and throughout differentiation. With the application of neu techniques and the rigorous use of established ones, i t mould appear that some progress i s being made in elucidating these problems. The structure of eukaryotic genomes consists of tuD components: a) the genetic material, deoxyribonucleic acid (DNA), and b) proteins associated with the DNA. As techniques in molecular genetics become more powerful, the known complexity of genome increases. This i s best illustrated by the finding of interrupted gene sequences which were neither found nor suspected u n t i l the advent of DNA cloning and sequencing methods. While much information i s being obtained on DNA sequence and structure, the research emphasis has been placed on chromosomal proteins. They are believed to be the components that control chromatin structure and function. I. Chromatin Structure It has become apparent that chromosomal proteins play an important role in regulating chromatin structure. The model for chromatin struc-ture proposed by Kornberg (1974) appears to be the organization adopted 2 by eukaryotes with feu, i f any, known exceptions. The unit structure in this model i s the nucleosome. It i s composed of an octomer of histone molecules, two each of histones H2A, H2B, H3 and H*+. Around this 'core' of histones i s wrapped a DNA segment of 1W] base pairs (Noll, 1977). The care particles are connected to each other by DNA which can vary in length from 5 to about 90 base pairs (Hornbery, 1977). It i s this linker region which gives the repeat unit of chromatin i t s length heterogeneity and i s the site of the f i f t h major histone H1 (Varshavsky, 1976). These five histones appear to remain quantitatively constant except in ce l l s where transcription i s terminated (e.g., sperm-atids, erythrocytes). A) The Nucleosome Core The dimensions of the nucleosome core has been determined using X-ray diffraction and electron microscopic measurements of crystallized nucleosome core preparations (Bush et a l . , 1977). The core appears to be a wedge-shaped, bilayered structure. The dimensions of 110 x 110 x 57 A1 includes the DNA which i s wound about the care for 1 3 A turns in a f l a t superhelix configuration with a pitch of about 28 8. The X-ray studies correlated well with neutron scattering studies (Randan et a l . , 1977) suggesting that this general morphology of the nucleosome i s correct. Throughout the eukaryotic c e l l kingdom, the histone involvement in nucleosome architecture appears to be the same or very similar. This involvement probably entails interaction of different histone species amongst each other and with DNA. These multiple interactions, and their presumably important role in maintaining c e l l v i a b i l i t y , are believed 3 to have lead to their extremely conservative evolution, especially those of H3 and Hk. Only recently has an Hk been found with a radically d i f f -erent amino acid sequence, and this was found in the macronucleus of Tetrahymena (Glover and Gorovsky, 1979). The macronucleus of this c i l i a t e functions as a gene amplification vessicle involved solely in transcription. It i s possible that transcription i s not regulated in macronuclei of Tetrahymena in a manner analogous to the lone nuclei of multi-cellular organisms. Such a difference in gene function may allow for a divergence in amino acid sequence to occur in histone H^. Due to homogeneity of structure and organization of the core histones within the genome of cells and between c e l l types of multi-cellular organisms, there i s l i t t l e possibility that histones are the exclusive regulators of gene expression. IMucleosomes are believed to play an important role as a general suppressor of transcription (Kleinsmith, 1975). The mechanism by which this i s accomplished i s not known, but simple steric hindrance of RI\IA polymerase could be a method. This does not imply that transcription-ally active chromatin i s free of core histones, for nucleosomes are present in active chromatin (Reeves, 1977; Ldeintraub and Groudine, 1976). These two inimical observations are acceptable i f the nucleosome can assume different configurational states in active and non-active chroma-t i n . For example, loosening of nucleosome structure could come about by changing the charge on histone molecules through post-synthetic modifi-cations. Modifications that are known to occur include phosphaylation, methylation and acetylation. Of the three, acetylation of histone appears to be associated with gene activity. This finding has been supported by histone analysis of nuclease enriched transcriptionally 4 active chromatin (Davie and Candida, 1978; Levy-Ldilsan et a l . , 1979; Reeves and Cserjesi, 1979) and by analysis of de novo synthesized mRl\!A and proteins in cells uhose histones were induced to hyper-acetylate (Reeves and Cserjesi, 198D). An extensive review on nucleosomes by Finch et a l . (1977) covers most features not covered here. B) Histone H1 The most lysine rich histone, H1, i s also the most variable of the histones. Variability i s found not only between species and tissues (Smerdon and Isenberg, 1976), but H1 subfractions are found within pure c e l l lines. Variations may be small, but can be as great as between the H1's and histone H5 in the nucleated erythroid cells of birds (Yaguchi et a l . , 1977). H5 is usually not classified as an H1, but an homology in amino acid sequence of close to 50 percent may warrant such a classification. However great variations in histone H1 are, they are limited to only certain areas of the molecule. There appear to be four domains: 1) the l\l-terminal 2D residues are nan-basic with a high proportion of proline and alanine; 2) residues 2D to 4D are very basic; 3) residues 4D to 123 have an apalar composition similar to globular proteins; and 4) a basic carboxyl end. The third domain contains the area where the molecule i s extensively folded (Chapman et a l . , 1976J Bradbury et a l . , 1975) and where the primary structure i s mast conserved. The roles of H1, like the other histones, have not been determined. The most widely held theory i s that i t i s responsible for stabilizing and compacting the chromatin strands. Nuclease studies have shown that H1 has an internucleosamal position (Varshavsky et a l . , 1976) and only harsh treatments have been able to attach i t to the core particle (Newman and 5 Moon, 1979). It i s in this exposed position that i t exerts a protection of the nucleosome linker region. If chromatin i s digested with micro-coccal nuclease, the H1 i s often associated with a DNA fragment about 35 base pairs long (Bakayev et a l . , 1977). When H1 i s removed prior to digestion, the rate of digestion increases up to eight fold (Moll and Hornberg, 1977). If nuclease activity can be related to accessibility of chromatin to transcription, i t would appear that H1 was involved in gene regulation, i f only at a gross lev e l . One way that transcription could be affected by H1 i s through i t s apparent stabilizing influence on nucleosomes. It has been suggested that nucleosomes may slide along the chromatin fiber to leave stretches of DMA exposed and accessible to transcription. If this were the case, the studies on nucleosome sliding using various ionic and temperature conditions indicate that H1 has the a b i l i t y to stabilize the core par-t i c l e (Spadatora et a l . , 1979). While non-histone chromosomal proteins (NHP) cannot be ruled out as contributing to this s t a b i l i t y , i t appears that H1 i s a major, or the major, stabilizing component. This conclusion i s further supported by the observation that disproportional elevated amounts of H1 are found in condensed, and probably transcriptionally inactive areas of the polytene chromosomes of D. melanoqaster (Janrich et a l . , 1977). Indirect lines of evidence also suggest a role for different subfractians of H1 in reducing or rendering genomes transcriptionally inert. During the differentiation of erythroid c e l l s which maintain their nuclei, the tissue specific histone H5 appears and replaces H1 during maturation of the c e l l from reticulocyte to erythrocyte (Dick and Johns,1969). During the maturation process, transcription ceases. 6 In somatic tissues, an H1 variant has been associated with slow or non-replicating tissue (Panyim and Chalkley, 1969; Varicchio, 1977). These observations indicate that different H1 fractions may have d i f f -erent involvements in transcriptional activity. More recent evidence for this has been obtained through nuclease digestion studies (Garka and Laurence, 1979). These authors observed that different H1 subfractions in rat l i v e r conferred different degrees of protection from micrococcal nuclease digestion. The formation of chromatin secondary structure has also been attributed ta the H1 molecule. These may include formation of 200 to 250 8 native chromosome fibres from 110 fibers (Renz et a l . , 1977) and the more complex compacting of chromatin into heterochromatin (Blumen-feld et a l . , 1977). Most studies to date have compared H1 depleted chromatin with native or reconstituted chromatin. As Cole et a l . (1977) have painted out, such studies must be interpreted with caution due to possible rearrangements and denaturatians that can be encountered during extraction procedures. C) Histone Genes The histane genes have been isolated and characterized from a number of species. For higher eukaryotic organisms, the five histones appear to be organized in tandem repeats. The number of reiterations varies from about 10 to 20 in mice (Jacob, 1976) to several hundred in sea urchin (Kedes and B i r n s t i e l , 1971). The general structure of a gene set i s similar. Each gene i s separated from the next by a linker region of various length. The spacer DIMA regions between genes have diverged in sequence, except for sequences immediately around the coding region which have been extensively conserved. 7 Differences i n the organization of histone gene 'blocks' have be-come apparent as more organisms are investigated. In sea urchin P.  m i l i a r i s the genes are found at one locus and are translated from the same strand (Gross et a l . , 1976). This i s not the case for the urchin L. pictus where the genes are found at two l o c i (Kedes, 1979) nor for the f l y , D_. melanaqaster which transcribes the histone genes H*+ and H2B from one strand and the rest from the complementary one (Lif t o n et a l . , 1977). The implications of having genes for each histone type clustered on the same stretch of the genome has not been determined. Possibly the genes are transcribed as polycistronic mRIMA but t h i s has not been established. However, such a mode of transcription i s consistent with the r esults of IMewrock et a l . (1977) which showed that synthesis and repression of histone subtypes H1, H2A and H2B are coordinated. while high molecular weight histone mRNA has been reported (Kunkel et a l . , 1978) the majority of evidence suggests independent transcription of each histone gene (Hackett et a l . , 1978; M e l l i et a l . , 1977). This of course, does not imply that a common regulator i s not present. D) IMan-Histone Chromosomal Proteins The IMHP are a heterogeneous group of proteins. This makes them a more l i k e l y candidate as the molecules that control t r a n s c r i p t i o n . Since IMHP have been extensively reviewed (Stein and Stein, 1976; Stein et a l . , 1977), only a very b r i e f summary w i l l be presented here. The IMHP include a l l proteins bound to chromatin which are not c l a s s i f i e d as histones. Because of th i s broad c l a s s i f i c a t i o n , there i s homogeneity between c e l l types i n IMHP content due to the inclusion of such house-keeping molecules 8 as RiMA polymerase, DMA repair enzymes, hormones and steroid receptor complexes, as well as proteins that may not be directly involved in chromatin structure or function but are associated to chromatin due to non-specific binding. This is not to suggest that a l l homogeneous IMHP can be excluded from any involvement in chromatin structure or function. To the contrary, there have recently been a large number of articles implicating specific NHP's in affecting both chromatin structure and function (Elgin et a l . , 1977; Hunt and Dayhoff, 1977; Javaherian and Sadeghi, 1979; Gates and Bekhor, 1979). The heterogeneous IMHP's show variation based upon a large number of factors. There are differences based on specificity for species (Elgin and Bonner, 1976) as well as tissues (Shelton and Neelin, 1971) and DfJA sequences (Gates and Bekhor, 1979). Changes in IMHP composition of chromatin are also observed during development (Johnson and Hnilica, 1971), differentiation (Vidali et a l . , 1973), stimulation of c e l l proliferation (Bush et a l . , 1977), and in response to other phenomena which affect gene expression. While many of these changes have been categorized, mainly through their gel electropharetic patterns, very l i t t l e i s known about the specific effects that each IMHP plays or how they interact with the other chromatin components. II Erythropoiesis A) Normal Erythropoeisis Erythropoiesis i s one line of differentiation that hemopoietic cells can take to become terminally differentiated. The stem c e l l s , which are located in the spleen and bone marrow, can differentiate into myeloblast, megakaryoblast, or when induced with the hormone 9 erythropoitin, become the precursor for the rest of the undifferentiated erythroblasts. Within the marrow the erythropoitin induced cells undergo the following maturation steps: Stem c e l l — proerythroblasts — basophilic erythroblasts polychromatophilic erythroblasts orthochromic erythro-blasts reticulocytes. As reticulocytes they enter the blood stream and become mature red blood c e l l s upon losing their nuclei. Each stage possesses i t s own characteristic morphology and biochemical changes which have been amply documented in texts (McDonald et a l . f 1978; Rifkind et a l . , 1976). As mentioned above, the main stimulus for erythrocyte formation is the hormone erythropoietin, but the commencement of multipotent stem ce l l s (CFU-S) towards erythropoeisis appears to be an effect of the cells micro^environment (Trentin, 1970). The in vitro characterization of different stages of erythroid development is based on proliferation capacity and responsiveness to erythropoitin. The most immature committed c e l l has the highest proliferation potential and the lowest erythro-poeitin sensitivity. As the committed c e l l matures i t s proliferative a b i l i t y declines and erythropoietin sensitivity increases. The stem c e l l goes through two stages, burst forming units-erythroid (BFU-S) and colony farming unit-erythroid (CFU-S) before differentiating into a proerythroblast. Erythropoietic c e l l culturing methods have been greatly improved in the past few years. It is now possible to isolate individual clones of erythropoietic c e l l s using methyl cellulose in the culture media (Tepperman et a l . , 197^). The main problem with such, a system i s the f i r s t few steps in the induction, and the regulation of the differentiation 1D pathway cannot be controlled irn vitro. Also, i t i s not possible to determine what stage the isolated cells are at un t i l they have d i f f e r -entiated, making collection of large numbers of cells from any specific stage d i f f i c u l t . The murine erythraleukemia c e l l s isolated by Charlotte Friend (1957) appears to have salved some of the in vitro drawbacks mentioned. B) Erythropoiesis in Friend Cells In 1957 Charlotte Friend reported recovering a virus from the spleen of Swiss mice incubated with disrupted Ehrlich ascites tumor c e l l f i l t r a t e which induced spleenomegaly and hepatomegaly upon incubation into susceptible mice. These pathological abnormalities are associated with immature hemopoietic cells i n f i l t r a t i n g these organs as well as peripheral blood. Terminally, the normal cells of these tissues are almost completely replaced with immature c e l l s . The erythropoietic nature af Friend disease has been demonstrated histopathalogically by electron microscopy (Ikawa and Sugano, 1967), the presence of erythrocyte specific surface antigens (Tabuse et a l . , 1977), and the in vitro stimula-tion of red c e l l production along with death of young nucleated erythroid cells (Tambourin et a l . , 1973). The relationship between FL ce l l s and normal erythroid precursor cells i s not yet clear, but the evidence indicates the possibility of several stages of erythropoietin responsive precursor cells as the target for Friend virus infection. Two lines of evidence support this hypothesis. F i r s t , elimination of multipotent stem ce l l s does not affect Friend virus induced proliferation of erythroid cells in the spleen of mice, while committed erythroid c e l l compartments which have been depressed give no response to Friend virus (Fredrickson et a l . , 1975). In the same line of evidence, tun l o c i responsible far hereditary anemias confer resistance to Friend virus infection. These l o c i appear to affect the avail a b i l i t y of erythropoietin responsive precursor c e l l s (Bennett et al.,. 1968; Steeves et a l . , 1968). The second line of evidence i s from the ino-culation of lethally irradiated mice uith normal spleen c e l l s . These give rise to erythroid, megakaryocyte and granulocytic colonies in the spleen. If Friend virus i s injected four days after spleen grafting, hyper-basophilic colonies (FL cells) also form but solely uithin the differentiated erythroid colonies (Tambourin and uendling, 1975). From these and other experiments (Host et a l . , 1979), i t appears that Friend virus induction takes place around the erythropoietic colony forming unit stage of erythropoiesis. The biology of Friend virus complex has been extensively studied. There are tuo v i r a l a c t i v i t i e s contained in Friend virus preparations, one of uhich i s responsible for the onset of spleenomegaly, hepato-megaly and erythroleukemia. The virus responsible for these actions i s termed Spleen Focus-Forming Virus (SFFV); It appears to be defective for replication and requires a second helper virus uhich may be one of a number of murine leukemia virus strains (MuLV). The SSFV genome is a recombinant molecule carrying a portion of the MuLV genome as uell as other murine viruses (Troxler et a l . , 1977). Genetic factors govern the susceptibility by the host to both MuLV helper virus and SFFV. The genes involved appear to be independently segregating ( L i l l y and Pincus, 1973). The effect of Friend virus complex on committed erythroid c e l l s i s tuo-fold; i t renders them capable of proliferation and differentiation independent of erythropoietin. Like moat malignant c e l l s , FL c e l l s are easily maintained in culture using standard tissue culture media supplemented with f e t a l calf serum. The ce l l s have been maintained in our laboratory for well over 200 passages. Most c e l l lines display low levels of spontaneous differentiation ranging from 1% - 20% as determined by benzidine reaction for haemoglobin. This range of differentiation i s not solely dependent on c e l l line, but also appears to be affected by culture conditions. These levels of induction can be increased dramatically by adding one of a number of chemical inducers (Marks and Rifkind, 1978). As was pointed out by Marks and Rifkind (1978), the act i v i t i e s of an inducer i s dependent on FL c e l l line and culture con-ditions so while some inducers such as butyric acid are l i s t e d as weak inducers in their table (Table 1 (Marks and Rifkind, 1978)), our lab has found i t to be one of the strongest using c e l l line Frc 18 clone 745. Morphological changes which occur after addition of an inducer to FL c e l l s , indicate that they differentiate from a BFU-E c e l l to the level of orthochromatic erythroblasts. These changes include nuclei condensation, as judged by nuclei to cytoplasm ratio (Friend et a l . , 1974), and decrease in c e l l size. In general, the ce l l s do not lose their nuclei as would differentiated normal erythrocytes. Along with these morphological changes are a number of molecular changes which resemble normal red blood c e l l differentiation. Some of these are: accumulation of globin messenger RNA (Nudel et a l . , 1977; Ross et a l . , 1974), o c - and j6-globin synthesis (Reeves and Cserjesi, 1979), uptake of iron into heme (Friend et a l . , 1974), increased levels of carbonic anhydrase (Habat et a l . , 1975), and accumulation of spectrin (Tabuse et a l . , 1977). while many changes do occur after FL c e l l induction uhich resembles normal erythropoiesis, i t i s important to bear in mind that there are many differences as u e l l . Some differences are: the ce l l s usually do not differentiate past orthochromatic erythroblasts, they lack 2,3 diphosphoglyceric acid (Kabat er a l . , 1975), do not increase catalase activity (Conscience et a l . , 1977), and uninduced ce l l s constitutively contain low levels of globin mRIMA (Conkie et a l . , 1974). It has also recently been shoun in our lab that even undifferentiated FL c e l l s contain lou levels of globin (Reeves and Cserjesi, 1979). Uhile this uas observed in only one c e l l l i n e , Frc 18, i t does bring into question the relevancy of using FL ce l l s as a model for normal erythropoiesis. I l l Thesis Objectives During Friend c e l l differentiation, a major chromatin bound protein appears. It has been reported that this protein, called IP-25, i s tightly bound to chromatin and i s not related to any knoun histone (Keppel et a l . , 1977). It uas further reported that IP-25 i s located uithin the internucleosomal region of the chromatin, and may be involved in reducing the susceptibility of chromatin to micrococcal nuclease d i -gestion (Keppel et a l . , 1979). The main objective of this thesis i s to confirm and extend the previous uork by examining the biochemical and physiological properties of this protein. The investigation in this thesis consists of three parts: 1) To characterize IP-25 biochemically to obtain a better understanding of i t s nature, uith the hope that i t u i l l lead to i t s cl a s s i f i c a t i o n . 2) To confirm i t s location uithin the chromatin structure. 3) To obtain a better understanding of the relationship of IP-25 uith Friend c e l l differentiation. MATERIALS AND METHDDS I Materials Materials for electrophoresis were obtained as follows: acrylamide and bisacrylamide were from Eastman Kodak Co., arnpholines and agarose from Bio-Rad Laboratories. Tissue culture materials were obtained from Grand Island Biological Co. with the exceptions of sodium p e n i c i l l i n G and dihydrostretomycin which were purchased from Sigma Chemical Co. The enzymes, micrococcal nuclease, pancreatic DNase, pancreatic RNase, papain, and chymotrypsin were from Sigma Chemical Co. Staphylo- coccus aureus US protease was from Miles Research Products and pronase was from CalbiDchem - Behring Corp. Isotopes and s c i n t i l l a t i o n fluors were from New England Nuclear. A l l common chemicals were of reagent grade and obtained commercially. 15 II List, of Common Buffers 1 0.8% NaCl - 0.02%' KC1 - 0.12% Na^PO^ - 0.02% KH^PQ^ (pH 7.2) 2 75 mM NaCl - 24 mM Na citrate (pH B.O) 3 25 mM KC1 - 10 mM Tris (pH 8.0) 4 10 mM Tris (pH 7.4) .5 1 M sucrose - 10 mM Tris (pH 7.4) 6 0.02% TX-100 - 10 mM MgClg - 50 mM Tris (pH 7.2) 7 10 mM NaH^PO^ - 20% 2-mercaptoethanol - 0.01% Bromophenol Blue -4% SDS -• 15% glycerol (pH 7.0) 8 0.9 N AcH - 10% glycerol (v/v) - 0.01% Pyronin Y METHODS III C e l l Line and.Culture Conditions Friend erythroleukemic c e l l s , Line 745A (Friend et a l . , 1974) were donated by Dr. R.O. Reeves. Cells were stored in a frozen state in liquid nitrogen with culture media plus 10% DMSO. When needed, the cell s were quick-thawed and grown in 90% Dulbecco's Modified Eagles medium supplemented with 1D% fet a l calf serum, 100 ug per ml dihydro-streptomycin, 1D0 ug per ml p e n i c i l l i n G, and 4 units per ml mycostatin. A l l experiments were performed on ce l l s between subculture passages 138 and 155. Cell papulations were expanded by culturing in 2 l i t r e r o l l e r bottles. Cells were collected by spinning a"t GOO g, resuspending twice in phosphate buffered saline (0.8% NaCl - 0.02% KC1 - 0.12% Na2HP0( 0.02% KH2P0^ pH 7.2) and storing at -80° C u n t i l required. Various chemical inducers were added to the tissue culture medium only after their pH had been adjusted to pH 7.2 with NaOH or HC1. DMSO induced c e l l s were obtained by adding 1.8% or 2.0% DMSO to exponentially dividing c e l l s . The ce l l s were then incubated in r o l l e r bottles with three volumes of fresh medium containing the appropriate concentration D f DMSO. If the medium became acidic, extra medium was added. IV Isolation of Histones from Whole Cells Histones were prepared two ways: 1) using a modified method of Marushige and Bonner (196S) and 2) using a TX-100 buffer system method. A l l procedures were performed at 0-4° C with PMSF added to buffers just prior to use. The f i r s t method consisted of resuspending a frozen c e l l pellet in 0.075 M NaCl - 0.024 M NB citrate (pH 8.0) and gently disrupting the ce l l membrane in a Potter - Elvehjem homogenizer. The nuclei were spun down at 3000 x g for 10 minutes, resuspended, and homogenized in 25 mM KC1 - 10 mM Tris (pH 8.0) - 1 mM MgCl2 using a Potter - Elvehjem homogenizer. The homogenate uas then spun at 3000 x g for 10 minutes and the pelleted nuclei taken up in 10 mM Tris (pH 7.4). The nuclei were disrupted in a ground glass homogenizer and spun through a sucrose cushion (1 M sucrose - 10 mM Tris pH 7.4) at 17000 x g for 20 minutes. The resulting chromatin pellet uas extracted uith 0.4 N HgSO^  for 15 minutes and the precipitate removed by centrifugation at 12000 x g for 10 minutes. The supernatant uas then dialyzed overnight against 0.1 M AcH and lyophilized. The second method consisted of isolating the nuclei uith 0.0256 TX-100 - 10 mM MgCl2 - 50 mM Tris (pH 7.2) in a Potter - Elvehjem homogenizer. The isolated nuclei were spun down at 1200 x g for 1 minute, washed uith 10 mM Tris buffer (pH 7.4) and respun. Nuclei were disrupted uith a ground glass homogenizer using 10 mM Tris (pH 7.4) as buffer. The homogenate uas layered over sucrose (Buffer 5) and spun at 17000 x g for 20 minutes. The resulting chromatin uas extracted as described above. \J Electrophoretic Techniques A 505 Polyacrylamide Gel Electrophoresis A l l SOS gels uere prepared by the method of Laemmli (1971). The separating gels were composed of varying concentrations of dHgO or chloroform recrystallized acrylamide (acrylamide : bisacrylamide = 100 : 1) 0.1% SOS - 0.375 M Tris (pH 8.8) - 0.05% TEMED (v/v) - 0.1% APS. The stacking gel contained k% acrylamide (acrylamide : bisacrylamide = 20 : 1) 0.1% SDS - 0.125 M Tris (pH 6.8) - 0.01% TEMED (v/v) - 0.2% APS. The gel dimensions, unless stated otherwise, were 13 x 13 x 0.15 cm for the separating gel with a 2 - 4 cm long stacking gel. The running buffer consisted of 0.38 M glycine - 0.05 M Tris (pH 8.6) - 0*1% SDS. Protein samples were prepared for SDS electrophoresis by complexing with SDS at, or near, 100° C for several minutes in a buffer containing 10 mM NaH2P0^ (pH 7.0) - 4% SDS - 20% 2-mercaptoethanol - 0.01% Bromophenol Blue 15% glycerol. 8 Acid-Urea - SDS Twa-Dimensional Gel Electrophoresis Acid-urea gel electrophoresis was by a modified procedure of Panyim and Chalkley (1969). The gel consisted of 15% acrylamide, (acrylamide: bisacrylamide = 150:1) - 5.4% glacial acetic acid (v/v) - 6.25% urea -0.5% TEMED (v/v) - 0.13% APS. Gels were cast in 0.6 cm internal diameter tubes of varying lengths. Pre-electrophoresis was performed overnight at 2 mM per tube using 0.9 W AcH (adjusted to pH 3.25 uith IMaOH) as the electrode solution. Samples were dissolved in 0.9 IM AcH (pH 2.25) -10% glycerol - 0.01% Pyronin Y and run using fresh 0.9 IM AcH (pH 2.25) electrode solution. The tube gels were frozen uith crushed dry-ice and cut longitudi-nally in hale. One half uas stained uith Coomassie Brilliant Blue R-250 while the other half uas equilibrated uith SDS buffer (Buffer 7) for 20 minutes and stored at -35° C in fresh buffer. The stained gel uas lined up uith the frozen and both trimmed to the appropriate length. The frozen gel uas thawed and equilibrated a further 20 minutes in SDS complexing solution. Tube gels were placed on top of a standard SDS slab gel by adhesion uith melted agarose (1% agarose in Buffer 7). The second dimension uas electropharesed overnight at 25 mA. C Urea Gradient Polyacrylamide Gel Electrophoresis An exponential urea gradient of 2.5 M to 7.5 M uas produced in an acid-TX-100 gel using the procedure of Zweidler (1978). The grad-ient uas generated by using 20 ml of solution A (12% acrylamide (acryl-amide :bisacrylamide = 150:1) - 7.5 M urea - 5% AcH - 6 mM TX-100 - 0.5% TEMED - 1.5% glycerol - 0.1% APS) in the front chamber of a gradient maker (Chrismac Plastic Fabrications Model LGM-1). Gels uere cast as a horizontal gradient by placing spacers on a l l four edges of the plates, leaving a small opening in the top l e f t corner to f a c i l i t a t e addition of the gel solution. The front chamber uas sealed and solution A allowed to flou u n t i l the reduced pressure in the chamber halted the flow (approx-imately 5 ml). The back chamber uas opened and the rest of the gel uas cast. Gels uere overlayed uith 5% AcH - 6 mM TX-100 and pre-electro-phoresed overnight using 5% AcH as the electrode solution. The gels uere then overlayed uith 0.5 M cysteamine-HCl and pre-electrophoresed for a further 30 minutes uith fresh electrode solution. The electrode solution uas again replaced and the histone sample uas layered along the length of the gel in a buffer containing 5% 2-mercaptoethanol - 5% AcH -10% glycerol. Electrophoresis uas carried out at 5 mA at room tempera-ture overnight. Gels uere stained uith Coomassie B r i l l i a n t Blue R-250, vertical strips cut out, and the strips subjected to second dimension SDS electrophoresis as described above. 20 D Isoelectric Focusing - SDS Tup-Dimensional Electrophoresis The protein patterns of uninduced and DMSO induced Friend whale c e l l , cytoplasm and nuclei were compared by high resolution electro-phoresis. The electrophoresis procedure of D'Farrell (1975) was followed with one exception, instead of bringing the sample urea con-centration up to 9 M, the sample was saturated with urea at roam temp-35 erature. Uninduced c e l l s were labelled with ( S) methionine (IMEN, spec. act. > 400 Ci/mmole) i n methionine-free culture medium. Induced c e l l s were grown in the presence of 2% DMSO for a total of 4 days, prior to and during isotopic labelling. Isotopic labelling of cell s varied in time, (from 0.5 to 12 hours) and in concentration (2 to 100 uCi per ml) in different experiments. The specific activity of the c e l l protein samples was determined as acid insoluble counts per ug pro-tein. Precipitation of proteins was carried out by the addition of 10 u l of bovine serum albumin (1 mg per ml). The protein precipitate was co l -lected on a glass f i l t e r and the insoluble radioactivity counted in 10 ml of liquid s c i n t i l l a t i o n fluor (Aquasol 2) using an Isocap 300 s c i n t i l l a -tion counter. The amount of activity electrophoresed per electrofocusing tube varied but a 200,000 cpm load was the minimum. Second dimension gels were 14.5% standard SDS gels. These gels were dried under vacuum and autoradiography directly using Kodak X-Omat R X*-ray film, or fluoro-graphed using the method D f Bonner and Laskey (1974). Fluorography was carried out using pre-flashed film with an of 0.18 (Laskey and Mi l l s , 1975). Autoradiographic exposures were varied in length to allow both the major and minor spots to be resolved on a given gel. \/I Amino Acid Analysis A Sample Preparation The chromatin from Friend cells induced uith 1.8% DMSO for 5 days uas extracted by the modified method of Marushige and Bonner (1966). Histones uere extracted from the chromatin uith either 0.4 IM H^SB^ or 5% HCIO^. After dialysis and lyophilization, the H2S0^ extracted samples uere complexed uith SDS (Buffer 7) uhile the HCIO^ sample uas dissolved in 0.9 IM AcH. The histones uere separated on SDS gels or acid-urea gels. The SDS gels uere 13 x 13 x 0.3 cm and consisted of 18% acrylamide. The Bromophenol Blue from uas run past the end of the gel for several hours to obtain better separation of the histones. The acid-urea gels uere of the same dimension as the SDS gels. The perchloric acid extracted samples uere electrophoresed on acid-urea gels as described above. After electrophoresis, the gels uere stained uith 0.25% Buffalo Black in 7% AcH - 35% MeOH solution and destained in the same solution minus the stain. The various histone fractions uere cut out and uashed ex-tensively in dH^ O in preparation for hydrolysis. Due to the small quantities obtainable, a l l amino acid analysis uas carried out on samples separated and hydrolyzed in polyacrylamide gels. IMeu modifications to the method of Levy-Wilson uere developed to obtain a f l a t base-line and to determine tryptophane content. It uas found that the bulk of acrylamide could be removed by freezing the hydrolysate mixture at -70° C. Houever, significant amounts remained after this treatment. It has been previously shoun that residual acrylamide causes base-line fluctuations as well as peak distortion during analysis (Huang, 1977). An additional cooling step at -70° C appears t D precipitate most of the remaining acrylamide. This precipitation i s carried out on the supernatant uhich is removed after the f i r s t freezing. By cooling the HC1 mixture to just above i t s freezing point, acrylamide recrystallizes. These crystals must be re-moved by centrifugation uhile s t i l l cold to prevent redissolving. Another modification to the procedure of Huang (1977) uas the use of thioglycolic acid as a means of preserving tryptophane during acid hydrolysis (Gehrke and Tabeda, 1973). This procedure uas tested by hydrolyzing human haemoglobin (Sigma Chemical Co.) uhich contain a tryptophane to arginine ratio of 1 : 2. The analysis of the haemoglobin hydrolysate gave a ratio of 1 : 2.13. The difference between the actual and experimental results may be due to some degradation of tryptophane during hydrolysis and/or impurities uhich uere evident in small quantities uhen the haemoglobin sample uas electrophoresed in an SDS gel. The major hindrance of this method uas found to be the formation of a colour reactive compound uhich produced a large peak at the position of the f i r s t feu amino acid peaks. This masks peaks that are in the nannomolar range. B Sample Hydrolysis The uashed gel slices uere ground to a powder in their hydrolysis tubes and lyophilized overnight. The hydrolyzing solution consisted of 6 IM HC1 - 0.05% 2-mercaptoethanol with one hydrolysis series containing 5% thioglycolic acid. Hydrolysis took place in IM^  flushed and evacuated tubes for 21, 42, 54, and 72 hours at 110° C. Upon completion of hydro-l y s i s , the tubes were allowed to coal to room temperature and immersed in a methanol - dry-ice bath. The supernatant was removed after the tubes had thawed, and the acrylamide precipitate discarded. The super-natant was again cooled and the precipitate that formed was spun out at 1600 x g while the tubes were s t i l l cold. The supernatant was removed and blow dried under at 50° C. Samples were taken up in 1.96% Na citrate 2H20 - 1.65% concentrated HC1 - 0.5% thiodiglycol - 0.01% caprylic acid (ph2.2) and f i l t e r e d through a 0.45 urn Millipore f i l t e r . C Sample Analysis A l l samples were run on a Beckman Amino Analyzer Model 118C. The column was 31.5 x 0.6 cm packed with Beckman Type AA-20 resin, and run using prepared Beckman Buffers. The running parameters were the same as in Beckman Application Notes 119C - AN - 00 (1975) with the following exceptions; 1) the column temperature was 52° C, 2) buffer changes consisted of Buffer A, 50 minutes; Buffer B, 16 minutes; Buffer C, 79 minutes; NaOH, 50 minutes; Buffer A, 35 minutes. The peaks were integrated by photocopying the printout and weighing the individual cut out peaks on an analytical balance. VII Peptide Mapping Cells growing exponentially in one l i t e r of standard medium or in the presence of medium containing 1.8% DMSO for 20 hours, were con-centrated by centrifugation at 700 x g and resuspended in 100 ml media lacking lysine. Cells were cultured for Ik hour prior to labelling. The induced sample contained 1.8% DMSO in the labelling medium. ^H-lysine was added to the media at a concentration of 10 uCi per ml (spec. act. = 60 Ci/mmole) and the ce l l s were cultured for 6 hours. 24 During, the incubation, the media was frequently adjusted with NaOH. After labeling, the c e l l s were collected by standard methods and frozen at -80° G. Histone purification consisted of isolating the nuclei in Buffer 6 using a Potter - Elvehjem homogenizer, grinding the isolated nuclei in Buffer k with a ground glass homogenizer and spinning the chromatin through a sucrose cushion (Buffer 5) at 12000 x g. Histones were extracted with 0.4 N H^ SO^ , dialysed overnight against dH^ O and lyoph-i l i z e d . Samples were complexed with SDS (Buffer 7) and the histones separated on a 22% SDS slab gels (14 x 14 x-0.3 cm). Protein bands were visualized by staining with Coomassie B r i l l i a n t Blue R-250. The histone bands were cut out and equilibrated in SDS buffer (0.125 M Tris-HCL (pH 6.8) - 0.1% SDS - 1 mM EDTA). Peptide mapping was by the method of Cleveland et al.(1977). Bel pieces containing histone were placed in 1 cm wide slots on a second 24% SDS slab gel which had, in addition, a 4% stacking gel containing 1 mM EDTA. The proteases used for histone cleavages (Staphylococcus aureus 178, chymotrypsin, and papain) were dissolved in a buffer containing 0.125 M Tris-HCL (pH 6.8) - 0.5% SDS - 10% glycerol - 0.001% Bromophenol Blue and then layered on top of the gel pieces in the slots. Protease concentrations and electrophoresis conditions are discussed in Results. Gels were flourographed by the method of Laskey and Mills (1975) as previously described. l/III Dansylation Chromatin uas isolated by the TX-100 method and the histones extracted uith either 0.4 N HgSO^  or 5% HCIO^. Samples uere dialysed and lyophilized prior to dansylatian, or in the case of the H^Sti^ extracted histones, a sample was fractionated by column chromato-graphy prior to dansylatian. Chromatography uas by the method described by Holt and Brandt (1977). Bio-gel P-60 (400 mesh) uas hydrated overnight in 0.02 N HCl - 0.02% NaN3 and the fines removed. The slurry uas poured uith the aid of an extension tube, and the gel alloued to settle for 2 days using the hydrating solution as buffer. The column uas made the desired height (90 x 1 cm) by removing gel from the top. Samples uere applied to the column in a buffer containing 8 M urea - 1% 2-mercaptoethanol -0.02 IM HCl and chromatographed at a flou rate of 5 ml per hour using a gravity fed buffer system. Elutant uas collected in 1 ml fractions and their 00 recorded using a Gilford 250 spectrophotometer at a uave-length of 220 nm. Appropriate fractions uere pooled and dialyzed over-night against dH^ O at 4° C and lyophilized. Samples were electro-phoresed on an SDS gel to determine the purity of each fraction. Dansylation of the histone samples folloued the procedure of Gray (1972). The sample uas dissolved at a concentration of approxi-mately 1 mg per ml in 1% SDS and heated in boiling uater for 5 minutes. One volume of N-ethylmorpholine uas added to the cooled mixture folloued by a 3/4 volume of dansyl chloride (25 mg per ml dissolved in dimethyl formamide). The reaction uas alloued to proceed for a minimum of 5 hours at 37° C. Proteins were precipitated uith 7 volumes of cold ( - 2 0 ° C) acetone. The precipitate uas dissolved in 0.9 N AcH and run using the acid-urea - SDS tun dimensional electrophoresis system pre-viously described. Gels uere vieued under UU illumination to determine uhich protein species had became dansylated. I X One Dimensional Analysis of IMucleosomal Histones and DMA A Nucleosome Isolation Cells used for nucleosome analysis uere never stored frozen (at -80° C) for more than a feu days prior to use. Frozen cells were resuspended in Buffer 2 uith a Pasteur pipet, and gently homo-genized in a Potter-Elvenjem hand homogenizer. The suspension uas centrifuged at 1000 x g for 2 minutes and the supernatant discarded. The nuclear pellet uas resuspended in nuclease digest buffer (50 mM -Tris-HCl (pH 7.4) - 25 mM KC1 - 2 mM MgClg - 2 mM CaCl 2 - 2 mM PMSF) to give a nuclei concentration of about 50 ODgSO u n i * s D e r m^ ar>d pre-incubated in a hot uater bath at 37° C for 5 minutes. Micrococcal nuclease (also dissolved in digest buffer at 17000 units (DD ) 2bU per ml) uas added to the nuclei at a final concentration of 200 ( D D 2 6 Q ) units per ml. Digestion uas allowed to take place for the .desired length of time uith intermediate mixing using a Pasteur pipet. The digestion uas terminated by adding EDTA (pH 7.4) to a final concentration of 10 mM and chilling on ice. Nuclei uere pelletted by a 2 minute spin at 600 x g, resuspended in 700 ul 10 mM EDTA (pH 7.4) and ground in a glass homogenizer to lyse the nuclei. The nuclear debris uas spun for 5 minutes at 1200 x g and the nucleosome containing supernatant layered on a sucrose gradient. Sucrose gradients of 5 to 30% uiere generated in Beckman poly-allomer tubes (1" x 3.5") using a two chamber gradient maker (Chrismac Plastic Fabrications Model LGM-1). The front chamber contained 17 ml of 3056 sucrose - 2 mM EDTA (pH 7.4) while the back chamber contained the same volume of 5% sucrose - 2 mM EDTA. The tubes were loaded into a Beck-man SbJ—27 centrifuge head and allowed to cool at 4° C for several hours prior to loading of sample. Up to 40 OD^grj units of nucleosome solution have been applied per tube. Gradients were centrifuged for 22 hours at 120,000 x g (26000 rpm) at 4 D C. The gradients were fractionated from the bottom in 20 drop aliquots. The sucrose was pumped (Buchler Polystatic Pump) through a photometer (LKB 8300 Uvicord II) measuring at 254 and the optical ..density on a Fisher Recordall Series 5000 chart recorder. Appropriate fractions, were pooled and dialysed against 10 mM Tris (pH 7.4) - 2 mM EDTA overnight using a 6000 - 8000 molecular weight cut-off dialysis bag (Spectropore 1) and lyophilized. Nucleosomes were redissolved in 200 ul of dH^ O arid 100 u l removed for DNA analysis. B Histone Analysis The nucleosomal histones were analysed by SDS electrophoresis. The samples were treated with 100 u l of SDS complexing solution (Buffer 7) and run an 22% SDS gels. After Caamassie B r i l l i a n t Blue R-250 staining, the gels were sliced and scanned at 550 nm with a Gilford 250 Spectro-photometer. C PIMA Analysis DNA was purified from the nucleosome fractions by digestion with Pronase at 20 ug per ml far 2 ta 3 hours at 37° C in a buffer containing 0.15 M NaCl - 0.25% SDS - 25 mM EDTA - 20% glycerin - 25 mM Tris-HCl (pH 8.0). The DNA uas extracted tuice uith tua volumes of chloroform -octanol (24:1 v/v) and precipitated uith 5 volumes ethanol overnight. The ethanol uas removed and the DNA dried under nitrogen prior to being taken up in 50 mM Tris (pH 8.4) - 50 mM Boric acid - 0.17 mM EDTA for application to agarose gels, and 10 mM TEA-HC1 - 2 mM EDTA - 20% glycerol (pH 7.6) for polyacrylamide gels. DNA uas analysed by tuo different electrophoretic systems : a) lou ionic strength polyacrylamide electrophoresis (l/arshavsky et a l . J1976) and b) agarose gel electrophoresis (Sharp et a l 1973). The polyacrylamide system used a vertical slab gel (16 x 13 x 0.15 cm) f i l l e d uith 7% acrylamide (acrylamide:bisacrylamide = 30:1) - 10 mM TEA-HC1 - 2 mM Na^EDTA - 0.1% APS - 0.05% TEMED (pH 7.6). Samples uere run uithout pre-electrophoresis using 10 mM TEA-HC1 - 2 mM Na^EDTA (pH 7.6) as the electrode buffer. DNA bands uere visualized under Ul/ light after soaking the gels in electrode buffer containing 2 ug per ml ethidium bromide. Agarose electrophoresis uas performed in vertical slab gels containing 1.4% agarose. The agarose uas melted in 40 mM Tris (pH 7.9) - 5 mM Na acetate - 1 mM Na^EDTA at 95° C. The solution uas coaled doun ta 50° C, 0.5 ug per ml ethidium bromide added, and the gel cast in pre-uarmed glass plates. After solidifying, the bottom spacer uas removed and f i l t e r paper soaked in electrode buffer (gel buffer plus 0.5 ug per ml ethidium bromide) uas placed snuggly at the bottom edges of the gel. Electrophoresis uas carried out at room temperature monitored by a Ul/ lamp. Photographs uere taken uith the gels in a UV box (Ultra-l/iolet Products Ltd.). X Tuo-Dimensional Analysis of IMucleosomal Proteins Nucleosomes uere separated on lou ionic strength gels and their histones analysed on an SDS second dimension gel by a modification of the method of uarshavsky et a l . (19.76). Nucleosomes labelled uith (^ H) lysine from uninduced and 26 hour DMSO-induced Friend c e l l s uere generated and separated on sucrose gradients as previously described. The monomer, dimer and trimer peaks uere pooled in a dialysis bag and concentrated by placing the bag in Sephadex G-100 pouder at 4° C u n t i l the volume uas sufficiently reduced. The induced nucleosomes were further concentrated in a 1 cm diameter dialysis bag (Fisher 14000. M. Ldt. cut o f f ) . A f i n a l OD^ gQ of 11 per ml uas obtained. Nucleosomes uere electrophoresed in tube gels (8 x 0.6 cm) containing the same lou ionic strength polyacrylamide gel as described above. Electro-phoresis uas alloued to continue u n t i l the Bromaphenol Blue tracking dye had been electrophoresed through a second time. Gels uere removed from their tubes and either stained uith ethidium bromide (as described above), or complexed uith SDS (Buffer 7). Gels uere complexed for % hour and electrophoresed on a 22% SDS gel uith marker histones run on one side. Electrophoresis uas into a standard 14 x 14 x 0.15 cm slab gel. The gels uere stained by the sil v e r nitrate method of Merril et al.(1979) or fluorographed by the method of Laskey and Mi l l s (1975). RESULTS I Protein Patterns of Uninduced and DMSO Induced Friend Cells As mentioned in the introduction, Peterson arid McConkey (1976) found very feu differences in the protein constituency between uninduced and DMSO induced Friend c e l l s using c e l l line 745. These experiments were repeated to determine i f their results hold true for c e l l line 745A. 35 Figure 1 and 2 show the protein patterns obtained from ( S)-methionine labelled Friend c e l l proteins using the two dimensional gel electrophoresis system of O'Farrell (1975). Figures A and B com-pare the protein patterns obtained from uninduced and DMSO induced whale c e l l preparations. It can be seen that the patterns obtained from these two samples are similar. Due to the large number of spots in these samples, the separation of many proteins was incomplete. In order to simplify the patterns, the cells were fractionated into preparations of purified cytoplasm and nuclei. The protein patterns of these frac-tions from uninduced and DMSO induced cells are shown in the four plates of Figure 2. The comparison D f uninduced and induced c e l l fractions again showed extensive s i m i l a r i t i e s . Most of the spots which cannot be seen in one plate when compared with i t s opposite were clearly seen in fluorographs of different expascire or containing sample with d i f f e r -ent labelling times. Qualitative or quantitative comparisons using this two dimensional technique must take into account the many pos s i b i l i t i e s for artifact generation. The f i r s t place artifacts may be generated i s during sample 31 Figure 1 Tuo-Dimensianal Gel Electrophoresis of Uninduced and DMSO-Induced Friend Cell Proteins. 35 Friend c e l l proteins uere labelled uith ( S)-methionine and separated by tuo-dimensional gel electrophoresis (first-dimension, isoelectric focusing; second-dimension, SDS gel electrophoresis) as described in Methods. Figure A, uninduced uhole c e l l proteins; 4.0 5 x 10 dpm uas applied to gel and then fluorographed for 1 ueek. Figure B, proteins from uhole cells uhich uere induced for 4 days uith 5 2% DMSO; 4.0 x 10 dpm applied to gel and then fluorographed for 2 weeks. 33 Figure 2 Fluarc-graphs of Tuio-Dimensionally Separated Proteins from Friend C e l l Cytoplasm and Nuclei. 35 Friend c e l l s uere labelled with ( S)-methionine and their nuclei and cytoplasm isolated by the TX-100 method. Each component was sub-jected to two-dimensional electrophoresis ( f i r s t dimension, isoelectric focusing; second dimension, SDS electrophoresis) as described in Methods. 5 Figure A, uninduced cytoplasm; 2.0 x 10 dpm loaded; fluorographed for 2; weeks. Figure B, cytoplasm from c e l l s induced with 2% DMSO for 4 days; 4.0 x 10 5 dpm loaded; fluorographed for 1 week. Figure C, un-5 induced nuclei; 2.0 x 10 dpm loaded; fluorographed for 2 weeks. 5 Figure D, nuclei from c e l l s induced with 2% DMSO for 4 days; 2.8 x 10 dpm loaded; fluorographed for 2 weeks. preparation where oxidation or reduction of a protein w i l l cause i t to migrate to a different position during isoelectricfocusing. P a r t i a l control of t h i s a r t i f a c t was accomplished by preparing the samples simultaneously using saturated urea solution. F i r s t dimension electro-phoresis was then performed at the same time using one tube gel electro phoresis chamber. Second dimension electrophoresis was also simultan-eously performed on a l l s i x samples inrorder to obtain consistent v e r t i c a l separation. After examination of a large number of fluorographs of varying degrees of exposure, few q u a l i t a t i v e differences were observed. This i s i n agreement with the results of Peterson and McConkey (1976), a l -though quantitative differences were more numerous than previously reported. These differences were judged i n r e l a t i o n to the intensity of nearby spots. I t was found that the X-ray f i l m was not evenly pressed against the fluaragraphs in some instances. This caused under-exposed areas, an example of which i s the bottom t h i r d of the gel pictured i n Figure 1 B. I t should be noted that spot intensity i s also dependent upon the time and length of l a b e l l i n g due to tr a n s l a -t i o n a l a c t i v i t y changes. I I Characterization of the IP-25 Molecule A) Molecular Weight Determination of IP-25 by SDS Electrophoresis. The molecular weight of IP-25 had previously been estimated to be 25,000 daltons using SDS gel electrophoresis (Keppel et al.,1977). Unfortunately, molecular weight determination using t h i s gel system i s affected by a number D f protein modifications. These include side group phosphorylation (Billings, 1979), amino acid substitutions ( IMoel et al.,1979), and overall charge of the molecules (Panyim and Chalkley, 1971). In an attempt to obtain a better weight estimate, the histones from Friend c e l l s were run in conjunction with calf thymus histones on SDS polyacrylamide slab gels. It was hoped that a similarity in structure between calf thymus H1 and Friend c e l l HI would compensate for migration abnormalities. Figure 3 shows the results of such an experiment. IP-25 can be seen as the band migrating just below the H1 doublet in lane c. when the molecular weights of standard pro-, teins (Figure 3, lane d) were plotted on a semi-log plot against dis-tance migrated (Figure 3), an apparent molecular weight of 26,500 was obtained for IP-25. If the molecular weight of calf thymus H1 i s taken to be 21,000 daltons (Teller et al.,1965), the molecular weight D f approximately 20,000 daltons would be obtained for IP-25. Since many factors can influence the migration of proteins, the 20,000 dalton weight i s probably the closer estimate. The reasons w i l l be expounded upon in the Discussion. B) Acid-Urea - SDS Two Dimensional Electrophoretic Analysis of IP-25. As was pointed out in the Introduction, there are multiple forms of the various histones. The proximity of IP-25 to the two H1 histones suggests that i t may be a histone H1 variant that appears during d i f f e r -entiation. This possibility i s strengthened by the fact that IP-25 i s soluble in 0.4 IM H2S0^, 5% HCIO^, and 0.5 M IMaCl. As a preliminary char-acterization of IP-25, the H^ SO^  soluble chromatin proteins were run on acid-urea gels. This electrophoretic gel system provides relatively good separation of a l l histone species but does not separate a l l variants 37 Figure 3 Molecular Weight Determination of IP-25 by SDS Polyacryla-mide Gel Electrophoresis. SDS electrophoresis uas carried out as described in Methods using a 22% gel. Top figure: lanes a and b) Sigma calf thymus histones, lane c) acid extracted histones from Friend c e l l s induced uith 1.8% DMSO for 5 days, lanes d) Sigma molecular ueight markers, e) trypsin, f) myoglobin, g) haemoglobin. The distances that the Sigma molecular ueight markers migrated uas plotted against their molecular weights on a logarithmic scale i n the bottom figure. The position of IP-25 on this curve gives i t s apparent molecular ueight. \ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ D i s t a n c e From O r i g i n ( c m ) 39 of histone species. The top gel in Figure 4 shows the separation obtained when his-tones from DMSO induced Friend c e l l s were run on long acid-urea tube gels. When the tube gels are run on an SDS second dimension gel, the individual bands can be more readily identified. The slowest migrating histone in acid-urea gels i s H1. Ldhile the two variants were not re-solved in the f i r s t dimension, a doublet can be seen co-migrating with the HT's of the standard histones run on the right side of the gel. Of the most interest i s the migration of IP-25 which can be seen migrating closer to histone H3 than H1. This migration i s not characteristic of normal H1 histones but i s similar to the migration patterns of histones H5 (Panyim et al.,1971) and H1D (Marks et al.,1975)(Panyim, and Chalkley, 1969b). The remaining histones migrate to their standard positions. The modification on the method of Panyim and Chalkley (1969) did not appear to have any effect on histone migration. The only effect this modifi-cation appears to have is to improve the resolution, especially when samples are applied in large volumes. C) TX-100 with Urea Gradient Gel Electrophoresis of Friend Cell Histones. A powerful separation method for histones i s the polyacrylamide gel system of Zweidler and Cohen (1972). It has been shown that histone species with a single amino acid substitution w i l l band at a different position using this gel system (Franklin and Zweidler, 1970). The presence of TX-100 appears to differentially retard the mobility of his-tone in the following order of increasing effect: H2a > H3 > H2b >H4 >H1. Figure k Acid-Urea - SDS Tuo Dimensional Gel Electrophoresis of DMSO Induced Friend Cell Histones. Histones uere extracted from Friend c e l l s by the method of Marushige and Bonner (1966) and run on an acid-urea tube gel (14 x 0.6 cm). The gel uas sliced longitudinally, one half uas stained uith Coomassie B r i l l i a n t Blue (top gel), and the other uas equilibrated in SDS buffer (pH 6.8). The gels uere trimmed to size and the equilibrated gel uas run uith marker histones on an 18% SDS gel and stained. It i s not clear hou TX-100 affects protein migration, but i t has been suggested that the effect i s dependent on the size of the hydrophobic region in the molecule (Zweidler, 1978). The detergent's effect can be altered by varying the urea concentration in the gel. Histones from DMSO induced Friend c e l l s uere separated using a urea gradient gel containing TX-100 (Figure 5). Due to the complexity of banding, the various bands had to be identified by removing strips from the gel (indicated by arrows at the bottom of the figure) and subjecting them to a second dimension SDS electrophoresis. The results can be seen in Figure 6. IP-25 was visible in gels pictured in plates 3 and k only, the position being indicated by c i r c l e s . A l l the histones could not definitively be identified, as H2a and H2b. had similar migra-tion patterns. The bands corresponding to these two histones were labelled in accordance with Zweidler (1978) and Kaster et al.(1979). Figure 5 shows IP-25 migrating slightly faster than the H1 histones. The effect of urea on IP-25 migration appears similar to i t s effect on the H1's as indicated by their parallel paths along the width of the gel. The migration characteristics of IP-25 are also similar to that of Xenopus laevis laevis histone H5 (Hoster et al.,1979) and the minor histone component MIM found i n mouse l i v e r (Zweidler, 1978). The two H1 components of Friend c e l l s cannot be separated on this gel system. The migration patterns of the core histones resembled previously pub-lished gels using histones obtained from different sources (Zweidler, 1978)(Koster et al.,1979). The H2a band appears several fold fainter than the rest of the core histones. whether this resulted from a staining arti f a c t 43 Figure 5 Urea Gradient Gel Electrophoresis of Histones. A urea gradient of 2.5 to 7.5 M uas cast uithin a TX-1D0 - AcH polyacrylamide gel as described in Methods. Histones from Friend c e l l s induced for 5 days uith 1.8% DMSO uere extracted by the method of Marushige and Banner (1966) and layered along the length of the gel. The figure shous the unique migration of each protein component.as i t passes through increasing urea concentrations. The arrous at the bottom of the figure indicate the area uhere gel slices uere removed and electro-phoresed in a SDS second dimension. (Figure 6) 45 Figure 6 Identification of Protein Bands from Urea Gradient Gel by SDS Electrophoresis. Sections from the urea gradient gel (Figure 5) were cut out, equilibrated in SDS buffer, and run on a 20% SDS slab gel. The numbering on each plate corresponds to the position marked on the urea gradient gel. IP-25 uas tao faint to be detected in the gels shoun in plates 1 and 2 but uas clearly visible in the gels depicted in plates 3 and k. uas not determined. The spl i t t i n g of the bands at higher urea concen-trations indicates that there are at least tuo variants D-f this H2a histone. The presence of tuo bands for histone H2b also indicates tuo variants are present. Other minor bands seen in the figure are either oxidation products or possibly other variants of the histone species in lou concentrations. The slouest migrating minor bands uere identi-fied as non histone proteins. D) Amino Acid Analysis Amino acids prepared as described in the Methods section uere analyzed on a Beckman model 118C amino acid analyzer. In a l l cases the elution profiles uere comparable to the factory prepared standards (Beckman). Figure 7 compares typical elution profiles of the three proteins analysed, H1a, H1b, and IP-25 uith a Beckman amino acid standard run. The figures uere obtained by joining points from the recorder print-out and photoreducing. These figures uere chosen to maximize the number of peaks uithin scale. The quality of baseline, separation, and peak sym-metry are comparable to the standards uith tuo exceptions, the extra peak between histidine and lysine, and the large ammonia peak. It i s not knoun why the extra peak appears. The large ammonia peak i s common to samples hydrolyzed with acrylamide and did not present problems with analysis. The amino acid composition of H1a, H1b and IP-25 is presented in Table 1. This data is derived from a minimum of two complete hydro-l y s i s sets consisting of at least three different times of hydrolysis for each protein. The analysis of each hydrolysis time was carried out a minimum of three times. Values were obtained by averaging the data with the exceptions of serine, methionine, and glycine. ka Figure 7 Amino Acid Analysis Elution Profiles. Protein bands uere cut out of SDS slab gels and hydrolyzed directly in 6 N HCl - rj.0.5% 2-mercaptoethanol. Procedures for amino acid analysis are described in Methods. Figures A to D are typical elution profiles for samples hydrolyzed for kZ hours* A) Beckman amino acid standards (5 nM of each amino acid except cysteine which is 2.5 nM, B) H1a, C) H1b, D) IP-25. 51 Table 1 Amino Acid Analysis of H1a, H1b, and IP-25. Amino acid analysis uas performed as described in Methods. Amino acids are expressed in mole %. Serine uas calculated by extrapolating the times of hydrolysis back to zero. Glycine values for H1a and H1b are 0.7 times the actual value to compensate for residual glycine uhich remains after SDS electrophoresis. The amino acid composition of protein A-24 uas calculated from the amino acid sequence as given by Busch et a l (1978). H1a H1b IP-25 A-24 Asx 2.8 3.3 4.6 7.9 Thr 5,3 4.6 5.0 5.9 Ser 9.0 8.8 12.0 3.7 Glx 4.3 4.7 6.3 11.3 Pro 7.3 6.5 6.6 3.9 G ly 7.4 6.4 7.4 8.9 Ala 22.3 19.8 13.5 9.9 Cys - - • - -Val 5.0 6.1 6.4 5.9 Met 0.5 0.7 0.8 03 III 1.3 1.7 2.8 6.4 Leu 4.4 4.5 3.7 12.3 Tyr 1.3 2.9 2.0 Phe 1.0 1.3 1.5 His 0.7 0-7 1.4 10.3 Lys 25.1 27.5 21.4 2.5 Arg 2.1 2.2 3.3 7.9 Trp — — -Serine i s known to degrade proportionately with hydrolysis time (DeLang, 1978). The serine concentration was determined by plotting percent serine against time of hydrolysis and extrapolating to zero time (Figure 8). Methionine breaks down due to oxidation caused by dissolved oxygen. The reducing agent 2-mercaptoethanol prevents same breakdown but not a l l . The methionine value far H1a was highly variable. The value presented was the shortest time of hydroly-s i s , which also gave the highest value. The methionine content of the other two proteins was averaged. As was shown earlier (Figures 4 and 5), the two H1 histones could only be separated by SDS electrophoresis. The running buffer of this system contains a high concentration of glycine a l l of which cannot be removed from the gels by washing. IP-25 can be separated Dn acid-urea gels but contains a modified form of H3 as an impurity when ex-tracted with H^SO^ (Figure 4). By extracting with perchloric acid, only the Hi's, IP-25 and a few high molecular weight proteins are SDI-ubilized. This fraction can be run on acid-urea gels and the amino acid composition of pure IP-25 can be determined. The composition of IP-25 from acid-urea gels was in agreement with that obtained from SDS gels with the exception of glycine. It was found that the true value of glycine was on the average 0.7 times that obtained from samples separated by SDS electrophoresis. The glycine concentrations for H1a and H1b was calculated by multiplying the experimental value by 0.7 to obtain a more accurate value. Table 1 shows the percent amino acid composition of proteins H1a, H1b, IP-25, and A-24. The two H1 histones are similar to most H1's 54 Figure 8 Degradation of Serine During Acid Hydrolysis. The serine concentrations for the different hydrolysis times oiere calculated as mole percent. By plotting these values and extrapolating to zero time,, an accurate estimation of serine content can be made. H1A (H) had a correlation coefficient of r=-D.996; H1B (••) r=-0.96D; IP-25 ( © ) r=-0.949. 20 40 60 80 Time of Hydrolysis studied to date. They are characteristic in their high lysine, alanine, and serine content as well as their lack of tryptophane. The presence of methionine i s not characteristic, but due to the heterogeneity of H1 molecules, i t i s not totally unexpected. Methionine has previously been found in Drasophila melanoqaster histone H1 (Alfageme, 1974). The similarities in composition between the two molecules i s evident. H1b differs from H1a by more than 25% in three amino acids, methionine, isoleucine, and phenylalanine. The reciprocal differences i s limited to methionine only. A l l these differences are found in amino acids which compose a small percentage of the to t a l . A single residue change in these three would account for the f u l l 25% difference. The amino acid composition of IP-25 has many similarities with the two H1 histones. It contains a high concentration of lysine, alanine and serine and also lacks tryptophane. The concentration of two of these amino acids, alanine and lysine, are significantly lower than either Friend c e l l H1 or H1 histones from other sources (Smerdon and Isenberg, 1976). The differences between amino acid composition of IP-25 and the two H1's are greater than the s i m i l a r i t i e s . IP-25 contains at least 11 amino acids whose composition differs from H1a by more than 25% : aspartate and/or asparagine, serine, glutamate and/or glutamine, alanine, valine, methionine, isoleucine, tyrosine, phenylalanine, histidine, and arginine. IP-25 i s closer in composition to H1b than Hla, but i t also varies from H1b by more than 25% in nine amino acids: aspartate and/or asparagine, serine, glutamate and/or glutamine, alanine, isoleucine, tyrosine, phenylalanine, histidine, and arginine. The amino acid analysis of the three proteins investigated i n d i -cates that the proteins labelled as H1 histones here and by others (Keppel et a l , 1977) i s indeed turn variants of histone H1. The compo-sition of IP-25 confirms that IP-25 i s "histone-like" in i t s high basic residue composition and lack of tryptophane, but does not prove that i t i s a true H1. The analysis of IP-25 when compared uith protein A-24 does show that they are tuo different protein species. This eliminates the possibility of IP-25 production being responsible for the decrease in H2a concentration observed in Figure 5. The compo-sition also indicates that IP-25 i s probably not an HMG protein. These proteins usually contain less basic and more acidic residues than IP-25. E) Peptide Maps of H3 Histones and IP-25. Three proteolytic enzymes uere employed for peptide mapping of the tuo H1 histones and IP-25. When digestion i s folloued by SDS electrophoresis, a peptide map based on molecular weights i s obtained. This method i s fast and sensitive using samples prepared by polyacryl-amide electrophoresis since digestion can be carried out without the need of eluting the sample. Figure 9A shows the peptide map obtained with protease S. aureus US. This enzyme has a high specificity far glutamate, although cleavage is also obtained at aspartate. The top two bands of H1a and H1b are undigested histones. A limited number of peptides were visualized due to the low specific activity of the protein sample. IMo homologies between the three proteins were obtained using induced Friend c e l l proteins. When uninduced c e l l H1 histones were digested, 6 extra minor bands were resolved due to a higher specific activity of these 58 Figure 9 Peptide Maps of the H1 Histones and IP-25. Protein fractionation and digestion followed the procedures outlined in Methods. Figure A shows the 5. aureus V8 digest obtained with 10 ug of enzyme loaded per slot and electrophoresed for 10 hours without halt. Figure B shows digestion with 10 ug of chymotrypsin per sl o t . Electro-phoresis was halted for 4.5 hours when marker dye reached the separation gel. The total running time was 14 hours. The arrow indicates a minor band not resolved in the photograph. Figure C shows a spectrophotometry scan of the fluorograph obtained with papain digestion. Running con-ditions were identical t D chymotrypsin with the exception of 100 ug of enzyme loaded per slot instead of 10 ug. The x-ray film was sliced and scanned at 600 nm using a Gilford 250 spectrophotometer. samples. There appears to be only one high molecular weight peptide that i s homologous in the H1 histones. It should be noted that the uninduced and DMSO induced H1 histones contain the same major peptides. This indicates that the same H1 histones are synthesized before and after induction. Cleavage specificity of chymotrypsin i s limited to phenylalanine, tyrosine, and tryptophane. The amino acid analysis would indicate that there are approximately 5 cleavage points in histone H1a, 6 in Hjb, and 10 in IP-25. The chymotrypsin peptide map presented in plate B shows that only a limited number of fragments were visualized due to the low specific activity of the sample. Although there are no homo-logous peptides between the H1 histones, a minor band (marked by the arrow) of IP-25 i s homologous with the bottom band of H1b. The papain peptide maps were optically scanned to clearly dis-tinguish between bands and the high background exposure pre sent on the autoradiography The presence of phenylalanine in a protein enhances the susceptibility of cleavage occuring one residue toward the carboxyl end (Glazer and Smith, 1971). While there are preferential cleavage sites, a high degree of non-specific cleavage also occurs. This accounts for the high background exposure present on such autoradiography. The optical scans indicate that there are three passible peptides with homo-logous molecular weights between H1b and IP-25. These have been marked a, b, and c in Figure 9 C. I\la homology was observed between the two H1 histones. It should be noted that homologies between bands do not necessarily indicate that the same or similar sequences exist. The enzymes employed 61 in this study are of limited use in mapping of the proteins studied here due to the small number of residues for which they are specific. The enzyme of greatest use cf H1 histones as well as IP-25, i s trypsin due to the large number of fragments that i t generates. Pep-tide maps using this enzyme have already been made with peptide homo-logies found between the H1 histones but not with IP-25 (Hieppel et al,,, 1979). F) Amino Terminal Group Determination. A useful method of characterization of proteins i s dansylation and identification of the amino terminal residue. The method i s rapid, consisting of a simple labelling procedure using the fluorescent compound DansylCl, acid hydrolysis, and identification of the labelled residue by i t s unique chromatographic or electropboretic mobility. In an attempt to label IP-25, proteins were isolated using three different methods; 1) H^ SO^  extraction as seen previously in Figure 1 a; 2) perchloric acid extraction as described in Section Bk; and 3) r^SO^ extraction followed by chromatographic fractionation. The last method gave the cleanest preparation of histones. Figure 10, Panel 1 shows the elution profile of proteins chromatographed on the molecular sieve matrix Bio Gel 60 (Bio Rad Lab.). The fractions were collected in 1 ml aliquots which were pooled (as indicated by the arrows), then dialyzed, and lyophilized. When the fractions were run on SDS polyacrylamide gels, the purity of the fractions .could be assessed. Figure 10, Panel 2 shows that a high degree of purity can be obtained for the H1 histones plus IP-25 fraction and probably for the other histones i f a narrower range of fractions were pooled. 62 Figure 10 Fractionation of Friend Cell Histones by Bio-Gel 60 Chromatography. Histones from Friend c e l l s induced uith 1.8% DMSO for 5 days uere extracted by the TX-100 method and chromatographed as described in Methods. Panel 1 shous the elution profile as measured at 220 nm. Arrows A to D represent the fractions pooled for lyophilization. Samples from the pooled fractions uere run on SDS polyacrylamide gels, as shoun in Panel 2, to locate and determine the purity of each histone species. Lanes A to D correspond uith chromatography peaks. B 30 40 50 60 70 80 90 100 110 120 130 140 Fraction Number 64 The elution of IP-25 with the two H1 histones indicates that they possess a primary and/or secondary structure which i s si m i l a r i n the elution buffer used. H.1 histones are eluted with the void volume which gives them an apparent molecular weight of 60,000 daltons. This anomalous migration of H1 histones has been attributed to t h e i r high unit charge. (Holt, 1978). Fraction A from the chromatograph column and the other two sample preparations were dansylated as described i n Methods. In the f i r s t attempt to determine which protein species had become la b e l l e d , the proteins were run on an SDS acrylamide gel. This method res u l t s i n a general f l u o r -escence throughout the gel. This i s probably due to the dansylation of glycine molecules within the running buffer. A l l subsequent separation was on acid-urea gels. The only protein species that contained appreciable amounts, of lab e l were i n the histone H2 region. Due to the poor resolution of the two H2 histones, i t could not be determined which species was la b e l l e d . The lack of l a b e l l i n g of the other histones i s probably due to the blacking of the amino terminal residue, possibly by acetate (Helinca, 1971). The lack of dansyl uptake by IP-25 may be due to the same blockage. I l l D istribution of IP-25 on Friend C e l l Chromatin A) Determination of Nucleosamal Fraction Purity. An important c r i t e r i o n for l o c a l i z a t i o n of chromosomal proteins i s the attainment of p u r i f i e d mono- and multimers of nucleosomes. The determination of purity was achieved by examining the DIMA from various nucleosomal fractions. Figure 11, top plate, shows the sucrose gradient 65 Figure 11 Sucrose.Gradient Fractionation of .Nucleosomes. Nuclei from Friend c e l l s induced far 5 days uith 1.8% DMSO uere digested at a concentration of 48 OD per ml uith 200 units (OD^gg) of micrococcal nuclease for 5 minutes at 37° C. Nuclei uere homogenized in 800 u l of 10 mM EDTA (pH 7.4) and the released nucleosomes (44 OD per ml) uere loaded onto a 5 to 30% sucrose gradient. Gradients uere spun at 120,000 g for 22 hours in a Beckman SLd 27 rotor. Fractions uere collected from the bottom. The top figure shous the gradient profile as scanned at A = 254 nm. Nucleosomes from each peak uere pooled as indicated by the arrous. The DNA from the fractions uas purified and run on a 1.4% agarose gel (bottom figure). A) tetramer fraction, B) trimer, C) dimer, and D) monomer. profile of nucleosomes obtained from DMSD induced Friend c e l l s . .The proportionality of the peaks provides a good estimate of the degree of digestion. The distribution and size of peak uas consistent among ex-periments uhere identical procedures uere used. It uas found that the area under these peaks usually represented only about half the concentra-tion (ODggg) that uas applied to the gradient. This discrepancy i s probably due to larger pieces of chromatin uhich uere not spun out by the lou speed preparative centrifugation. These large pieces uould then be spun doun to a pellet during ultracentrifugation and not recorded during gradient fractionation. Fractions uere pooled from as uide a range of fractions as possible, uithout excessive contamination, to obtain as much material as possible. IMucleosomes uere digested uith protease and the proteins associated uith DNA uere extracted uith chloroform:octanol. The bottom plate of Figure 11 shous the electrophoretic profile of purified DMA from four nucleosome fractions taken from the gradient. The monomer fraction i s highly purified uith only trace amounts of dimer and trimer size DNA fragments. Dimer fractions comtain a small amount of higher molecular ueight mono-mer derived DNA and a trace of trimer size DNA. The trimer fraction contains predominantly trimer size DNA but there i s appreciable con-tamination uith dimer. It uas not possible to obtain clean separation of tetra- and higher multimers using this gradient system. B) Nucleosomal Protein Analysis. Nucleosomes isolated on sucrose gradients uere complexed uith SDS and their proteins analysed by SDS polyacrylamide electrophoresis. Figure 12 shous one such experiment. The histones plus IP-25 could be clearly see 68 Figure 12 One Dimensional Analysis of IMucleosomal Proteins. . IMucleDSomes from various induction times uere isolated by sucrose gradient centrifugation (Figure 11), complexed uith SDS, and run on 2296 SDS polyacrylamide gels. Lanes: a) control, uhole chromatin induced 5 days uith 1.8% DMSO, b) monomer, 26 hour induction, c) monomer, uninduced, d) monomer, 5 days induction, e) dimer, 26 hour induction, f) dimer, uninduced, g) dimer, 5 day induction. b9 in a l l the fractions from monomer to tetramer on the original gel although they are faint in this photographic reproduction. At higher multimers, not enough material uas available from individual experiments to detect IP-25. The core histones uere in a ratio of 1 : 1 : 1 : 1 as has been reported exseuhere for other organisms (Isenberg, 1979). The H1 proteins varied from a lou of about 15% of H3 in monomers to about 40% of H3 in trimer and higher multimers. This variation i s expected due to the loss of H1 histones resulting from the cleavage of linker DIMA uith exzyme (Bakayev et a l . , 1977). IP-25 maintained a constant ratio uith the H1 histones, being approximately 4% in uninduced c e l l s , 7% in 26 hour induction, and 11% of total H1 in 5 day DMSO induced c e l l s irregardless of the size of multimer (Figure 13). The parallel increase in IP-25 concentration uith H1 suggests they have a similar position, uhich uould presumably be inter-nucleosomal. liJhile the H1 histones shoued an increase in concentration uith increased multimer length, i t can be seen in Figure 12 that the relative concen-trations of the tuo H1 proteins varied from sample to sample. This can be better seen in Figure 13. The f i n a l concentration of H1 never exceeded 40% of H3 histone and individual samples uere sometimes quite variable. The va r i a b i l i t y in concentration of total H1 and betueen H1 species suggests there uas selective loss of these proteins during sample preparation. Losses as mentioned above can lead to the possibility of protein rearrangements occurring. It should be noted that even though va r i a b i l i t y exists in H1 content, the IP-25 concentration did not vary significantly in relation to total H1, suggesting that a l l of these proteins behaved similarly to extraction a r t i f a c t s . 71 Figure 13 Spectrophotometry Scans of IMucleosomal Proteins Separated an SDS Polyacrylamide Gels. The nucleasomal proteins from uninduced and DMSD induced Friend ce l l s were separated on SDS gels as shown in Figure 12. Gels uere scanned at ^= 550 nm using a Gilford 25D spectrophotometer. Panels A and B represent scans of monomeric and dimer nucleosomal proteins respectively. The three times of induction are labelled: a) unin-duced control, b) 26 hour induction, c) 5 days of induction. The non-histone chromosomal 1 proteins (IMHP) were also briefly examined. There uere no consistent differences observed between uninduced and DMSO induced c e l l s . However, a striking difference was noted in one IMHP which i s labelled (a) in Figure 13. It i s present in high concentrations in monomeric particles but i s not detectable in dimer of higher multimers. IMHP (b) was found in a l l particle sizes. The classification of IMHP i s given to these proteins due to their slow migration on acid-urea gels (Figure 4). C) Two Dimensional Examination of IMucleosomal Proteins. The protein component of micrococcal digested chromatin from un-induced and DMSO induced Friend c e l l s were analyzed by two dimensional electrophoresis. The use of two dimensional electrophoresis allows for the direct visualization of proteins bound to different chromatin lengths on one gel. As can be seen in the ethidium bromide stained gels in Figure 14, the various chromatin sizes can be clearly separated on low ionic strength polyacrylamide gels. By complexing this gel with SDS and running on a second dimension SDS gel, the protein components of each chromatin size can be analyzed. Figure 14 shows the results of one set of two dimensional gels after being stained by the sil v e r nitrate technique of Merril et a l . (1979). The arrows in plate B indicates the bands that correspond to IP-25. It i s present in the monomer and dimer sized chromatin fragments. Apart from IP-25, no consistent differences could be seen in chromatin protein composition.between uninduced (Figure 14, plate A) and DMSO induced (plate B) Friend c e l l s . Figure 14 Tuo Dimensional Electrophoresis of Nucleosomal Proteins. Micrococcal nuclease generated nucleosomes were fractionated and applied to a low ionic strength acrylamide tube gel to separate them according to size. The top gels in Figures A and B were stained uith ethidium bromide to shou the DNA of different sized nucleosomes (monomer, dimer, trimer, and oligomer). Fi r s t dimension gels uere complexed uith SDS and run on 22% SDS gels to resolve the nucleosomes proteins components and s i l v e r stained. Figure A, proteins from unin-duced Friend c e l l s , Figure B, proteins from ce l l s induced uith 1.8% DMSO for 5 days. The arrows indicate the IP-25 bands. The monomer bands in the f i r s t dimension gels are udder than the rest. This i s due to the heterogeneity of monomer fragment sizes that are produced by micrococcal nuclease digestion. The slowest migrating monomers are probably over 200 base pairs, containing the nucleo-some core as well as the linker DNA uith associated proteins. The fastest migrating chromatin i s probably composed of sub-nucleosomal particles. This heterogeneity allous for the localization of proteins uithin the chromatin. It can clearly be seen in plate B that the H1 histones are associated uith monomer fragments that contain linker DNA. It i s also evident from this figure that IP-25 i s present only uhen linker region i s present. This indicates that IP-25 i s an inter-nucleosamal protein. During the staining of the gels in Figure 14, a dark precipitate formed uithin the gels. The reason for this i s not clear. In order to obtain a better visualization of the chromatin proteins, the proteins uere labelled uith (^H)-lysine as described in Methods. The specific activity of the DMSO induced Friend c e l l chromatin uas not high enough to obtain fluorographs of sufficient exposure to visualize IP-25. D) Examination of Nucleosome Repeat Lengths from Uninduced and DMSO Induced Friend Cells. The repeat lengths on uninduced and DMSO induced Friend c e l l s nucleosomes uere compared by agarose and polyacrylamide gel electrophoresis. The isolated nuclei from ce l l s uas subjected to brief digestion uith micrococcal nuclease. As can be seen in the tracing in Figure 15 (Top), this brief digestion results in a monomer peak uhich i s proportionately smaller than obtained uith longer digestions (Figure 11). By exposing 77 Figure 15 Comparison of DMA Lengths of Micrococcal Nuclease Digested Chromatin. Nucleosomes uere obtained from uninduced and 5 day 1.8% DMSO induced Friend c e l l s as described in Methods. Nuclei at a concentration of 55 0°2gQ P e r m l were digested for 2 minutes uith micrococcal nuclease, fractionated on a 5 to 30% sucrose gradient and fractions collected as indicated in the top figure. DNA uas purified and electrophoresed on a 1.4% agarose gel (Methods). The bottom figure shaus the ethidium bromide stained migration pattern of monomeric and dimeric nucleosomes. Lanes: A) uninduced monomer, B) DMSO induced monomer, C) uninduced dimer, D) DMSO induced dimer. 79 nuclei tc- shorter digestion, larger quantities of higher multimers were obtained. For DNA analysis, the DNA mas purified as described in Methods, and separated by electrophoresis on agarose gels. An example i s shown in Figure 15 (Bottom). The size of monomer and dimer length DNA appears to be the same, or very similar, in the uninduced and DMSO induced c e l l s . An examination of trimer and tetramer DNA gives the same results. When DNA from higher multimers was subjected to electrophoresis, a streak was produced. The separation of DNA fragments on the gel lacked the resolution required to distinguish between multimers higher than tetra-mers. The results from electrophoresis of purified DNA and whole nucleo-somes in acrylamide gels were identical to those obtained with agarose. A straight line was obtained when the distance of migration by multi-mers was plotted against the logarithm of the number of nucleosomes in each multimer (Figure 16). This supports the contention that each band represents a repeat of the unit nucleosome. It should be pointed out that the gel systems used for these experiments can only distinguish between differences in size of about 10% to 20% or larger. 11/ Relationship Between IP-25 and Differentiation A) IP-25 Accumulation During Differentiation. During differentiation, there i s an accumulation of chromosomal protein IP-25. In Figure 17, the graph illustrates the rate of accumu-lation during the differentiation process. The uninduced c e l l s contain some IP-25, though less than 1% of the H1 total. During the f i r s t 12 hours accumulation takes place at a high rate. The rate of increase tapers off after 12 hours, after which time there i s a slower but linear 80 Figure 16 Analysis of Micrococcal Nuclease Digest Products. DNA from uninduced Friend c e l l s and 5 day 1.8% DMSO induced ce l l s mere isolated and run on long agarose slab gels. The log of the suspected number of nucleosomes in each multimer uas plotted against the distance migrated to determine i f DNA bands uere a l l multiples of a nucleosome subunit. Due to the equal lengths of DNA from the tuo samples, their points superimpose on the graph. 81 Migration (cm) 82 Figure 17 Accumulation of IP-25 during Induction uith DMSO as a Ratio of Total H1's. Cells uere induced uith 1.8% DMSO and.cultured in r o l l e r bottles. Samples uere collected at times indicated, the histones extracted, and run on 22% SDS gels. Gels uere stained uith Coomassie B r i l l i a n t Blue R-250 and scanned at 550 nm using a Gilford 250 spectrophotometer equipped uith a Gilford 3AG gel scanner. Peaks uere integrated using the same methods that uere applied to the amino acid analysis (Methods), and the ration of IP-25 concentration to the combined H1's platted against time of induction. 83 7 50 TIME (Hrs.) 100 increase un t i l a f i n a l concentration of about 7% is reached after 60 hours of induction. In other experiments concentrations of up to 12% have been obtained. The results presented here are in general agreement uith those obtained by Keppel et a l . (1977). The accumulation kinetics they obtained using 2.5 mM hexamethylenebisacetamide as inducer is in better agreement than their DMSO inductions. The c e l l lines they used also produced higher concentrations of IP-25, reaching 40% of total H1 at terminal different-iation. B) Effect of Various Inducers and Non-inducing Agents on IP-25 Accumulation. It has previously been noted that IP-25 and differentiation (i.e., haemoglobin synthesis) is a coupled event uhen DMSO or butyric acid are used as inducers (Reeves and Cserjesi, 1979)(Reeves and Cserjesi, 1980). These studies uere extended by determining the effect of a number of different agents, some of uhich have been shoun to induce Friend cells (Marks and Rifkind, 1978), on the accumulation of IP-25 on chromatin. Table 2 summarizes the results obtained after inducing cells for 48 hours uith the agents l i s t e d . In a l l cases uhere haemoglobin uas syn-thesized, IP-25 uas also a component of the chromatin. Conversely, agents not producing the induction event did not stimulate IP-25 accumulation. Other acid soluble proteins uere also examined to determine whether there existed any differences betueen chromosomal proteins in cells cultured in the presence of different inducers. Only dibutyral 3', 5' cyclic adenine monophosphate (db cAMP) appears to affect the protein constitution of Friend c e l l chromatin. The most dramatic protein 85 Table 2 Haemoglobin and IP-25 Induction. Friend c e l l s uere cultured for 48 hours in the presence of various inducers and their histones extracted by the modified method of Marushige and Bonner (1966). Histones uere run on 18% BDS gels and stained uith Coomassie B r i l l i a n t Blue to screen for IP-25. Haemoglobin synthesis uas determined by visual inspection of the c e l l pellet. INDUCER HAEMOGLEIN IP-25 2% DMSO + + 1 mM db cAMP + + 5 mM butyric acid + + 1 mM isobutyrate + + 50 mM N-methylacetamidE + + 0.1 mM prostoglandin B + + 2% ethenol -0.1 mM cXMP* X = adenine, cytosine, guanine, thymine, uridine difference in db cAMP induced c e l l chromatin i s the appearance of a protein band labelled CP-23 (db cAMP induced protein, molecular weight 23,000) just below IP-25 (Figure 18). While there do appear to be quantitative differences within the high molecular weight protein, a band in the position of protein CP-23 has not been previously detected in undigested chromatin. The other agents tested did not produce, any new proteins as detected by mass band staining on one dimensional SDS gels. C) Effect of DMSO Induction on DMSO Resistant Cells. Resistant c e l l s were generated by culturing normally DMSO res-ponding c e l l s in 2 l i t r e r o l l e r bottles in the presence of DMSO. Induction was in stages, 0.556 DMSO being added every two days u n t i l a f i n a l concentration of 1.556 DMSO was achieved. During- induction, c e l l s were given equal volumes of fresh media whenever the pH dropped. After 1.556 DMSO had been obtained, c e l l density did not increase appreciably for 10 days. At this time the resistant c e l l population overtook differentiated senescent c e l l s and logarithmic growth was again re-established. Cells were passaged 6 times (1 part confluent c e l l s : 5 parts fresh media) in the presence of 256 DMSO. The passaging of DMSO-resistant cells.growing in DMSO back to media containing 256 DMSO did not produce a significant lag phase. Cell pellets obtained from resistant c e l l s growing in 1.856 DMSO for 5 days did not appear to contain haemoglobin. The acid extracted chromatin proteins were run on SDS gels to screen for the presence of IP-25 (Figure 19). There appears to be low levels of IP-25 in resistant cells not exposed to DMSO (Lane A). Low levels of IP-25 are also found 88 Figure 18 Effect c-f db cAMP on Friend Cell Chromosomal Proteins. Friend c e l l s were cultured in the presence of db cAMP for 48 hours. Acid soluble proteins were extracted by the modified method of Marushige and Bonner (1966) using 0.4 l\l H^ SO^  to solubulize the proteins. Proteins were run on an 18% SDS polyacrylamide gel and stained with Coomassie B r i l l i a n t Blue R-250. Lanes: a and c) proteins extracted from ce l l s induced with 2% BMSO for 48 hours, b) proteins from c e l l s cultured with 1 mM db cAMP for 48 hours. 89 90 Figure 19 SDS Electrophoresis of Chromosomal Proteins From DMSO Resistant FriBnd Cells. Friend c e l l s resistant to DMSO induction uere obtained as described in test. Resistant c e l l s mere grown in r o l l e r bottles, half being cultured in the presence of 1.8/6 DMSO for 5 days. Cells uere harvested and their chromatin proteins extracted by the TX-100 method. SDS gel electrophoresis was carried out in 2256 gels then stained with Coomassie B r i l l i a n t Blue R-250. Lanes: A) uninduced Friend c e l l proteins, B) proteins from DMSO induced c e l l s . 9 1 92 in non-resistant, uninduced c e l l s . The presence of IP-25 in c e l l s not exposed to DMSO. may be due to spontaneous differentiation of a small percentage of c e l l s . There is a considerable increase in the amount of IP-25 in resistant c e l l s induced uith DMSO (Lane B). The high level of IP-25 in Lane B cannot be explained by spontaneous differentia-tion due to the lack of haemoglobin seen in c e l l pellets cultured up to 10 days in the presence of 1.8% DMSO. These results suggest, as discussed belou, that in the DMSO-resistant c e l l s , IP-25 but not haemoglobin, accumulation can be induced by grouth of the c e l l s in this polar solvent. Furthermore, the accumulation of IP-25 in the resistant cells actively proliferating in the presence of 1.8% DMSO suggests that the accumulation of IP-25, per se, uithin the chromatin of Friend c e l l s i s not necessarily the result of cessation of active c e l l division. 93 DISCUSSION The composition and structure of chromatin i s not static during c e l l u -lar differentiation. Developmental changes in NHP constitution have been known for over a decade, but recently i t has become apparent that other changes are also involved. For example, recent findings suggest that stage specific developmental changes of histones, core as well as HI, are accurately timed events (Newrock et a l . , 1977). Of even greater surprise has been the recent findings of DNA rearrangements during development (Brack et a l . , 1978) (Ueigert et a l . , 1978). The appearance of IP-25 during the differentiation of Friend c e l l s may be another developmental change, one which occurs during mammalian erythropoeisis. In this thesis, investigation of IP-25 has been confined to three areas: 1) characterization of the IP-25 molecule 2) localization within the chromatin structure 3) determining i t s relationship with differentiation I) Characterization of IP-25 In a paper published by Keppel et a l . ,(1977), the molecular weight of IP-25 was estimated at 25,000 daltons by the use of SDS polyacrylamide electrophoresis. The marker proteins used for this estimation were not indicated, but i t was assumed here that they were not histones. When IP-25's molecular weight was calculated using standard molecular weight markers (Sigma Chemical Co.), a molecular weight of 26,500 daltons was ob-tained (Fig. 3). However, i t has been reported that histones, and especially H1, migrate with an anomalously high molecular weight value (Hnilica, 1972). This may be due to the high positive charge of these molecules. This prob-lem can, in part, be circumvented by using proteins of similar charge as standards, in this case calf thymus histones. For this reason the value of 2 D , 0 0 0 daltons presented in the results (Fig. 3) i s probably more accurate than the previous estimate. Friend c e l l histones were also subjected to different electrophoretic systems in an effort to classify IP-25. The acid-urea gel system (Fig. k) strongly suggested that IP-25 was a basic protein. Its migration also i n -dicated that i t mas not a typical H1 histone due to the large separation of IP-25 from the tuo H1 variants (Fig. 4). If IP-25 uere a typical H1 i t uould co-migrate uith or migrate closely uith the two H1 histones in the acid-urea gel systems employed. This gel also shoued that IP-25 uas not a high mobility group protein since these migrate slower than histone H1 on an acid-urea gel system. It uas also noted that the migration of IP-25 resembled that of histones H5 (Panyim et a l . , 1971) and H1 D (Marks et a l . , 1975). Further gel analysis of Friend c e l l histones uas carried out by em-ploying a TX-10D gel containing a urea gradient as the separating media. This gel system (Fig. 5) confirmed the acid-urea gel results. It showed that IP-25 migrated differently than H1, indicating i t was not a typical H1 histone. The effect of urea on IP-251s migration did show however that i t and H1 histones have certain s i m i l a r i t i e s . This conclusion i s based on the observation that both responded similarly to increased urea concentra-tions in gel electrophoreses. The migration Df IP-25 on TX-100 gels i s the same as has been reported for Xenopus laevis laevis histone H5 (Koster et a l . , 1979) and a minor histone species, MN, found in mouse liv e r (Zweidler, 1978). While the data is suggestive, i t i s premature to suggest that IP-25 i s a mammalian H5 histone. To determine i f IP-25 95 i s mare like a histone H5 or an H1 varient, the protein was further character-ized by determining i t s amino acid composition. The amino acid analysis (Table 1) confirms that IP-25 has a "histone-l i k e " composition. This statement is based on three c r i t e r i a : 1) IP-25 has a high lysine and alanine content similar to histones H1 and H5, 2) i t has a high basic to acidic residue ratio, 3) and i t lacks tryptophane. The amino acid analysis does not allou a definitive classification of IP-25 into any main histone fraction or clearly indicate i t i s similar to histone H5. This may be due to the fact that the variability of histone H1 and H5 are so great that a division of these into tuo different classes may be a r t i f i c i a l . The amino acid composition of a number of H1 and H5 histones has been determined (Table 3). The tuo amino acids uhich best characterize H1 are lysine and alanine in high concentrations. This also holds true for H5 histones, but tuo additional amino acids, serine and arginine, are generally also in high concentration, although they are highly variable (Table 3). The amino acid compositions in Table 3 shous the variations of these.four amino acids in the histone H1 and H5 groups. While there are variations in other amino acids, the four uhich are tabled constitute at least 50% of the total amino acids in the H1 and H5 molecules. Table 3 shous that the tuo Friend c e l l H1 histones are similar to calf thymus H1. The H1 variant >H1D also appears to f i t into the H1 group-ing. IP-25 on the otherhand does not f i t precisely into either the histone H1 or the H5 group. It contains too lou an alanine and lysine content to be a typical H1. The turtle H5 composition (Hnilica, 1972) is very similar to IP-25 but contains a higher lysine content. Chick and goose H5 is also very similar to IP-25, but they contain a higher arginine content. IP-25 appears to resemble an intermediate of these tuo groups. If histones H1 and H5 are classified as one group, as has previously been suggested (Yaguchi et al.,1977), IP-25 mould be considered a member. To determine i f the variations in composition between Friend c e l l H1 histomes and IP-25 i s localized to certain areas of the molecule, peptide maps were generated. The tryptic peptide maps of Keppel et al.(1979) did not show homology between IP-25 and the two H1 Friend c e l l histones. How-ever, the peptide maps produced for this thesis indicates that fragments of similar molecular weights are produced when H1b and IP-25 are digested with chymotrypsin and papain (Fig. 4). There are two alternative explana-tions for this result: 1) non-homologous fragments, of similar molecular weight are produced due to chance locations of digest susceptible residues in similar positions along the molecules; or 2) these fragments are indeed very similar or identical in primary structure. Both explanations are possible due to the limited number of peptide generated using these enzymes. It is the latter possibility that i s of interest. The amino acid analysis indicated that IP-25 is substantially different from H1b (Table 1). Therefore, production of similar fragments would inferentially indicate that any primary structure homologies between IP-25 and H1 histones would, i f they existed, be limited to restricted areas on the molecule. Such restricted variability i s found when H1 and H5 are compared (Yaguchi et al.,1977). To definitively determine i f the fragments are homologous in primary structure, amino acid sequencing of the peptides would have to be undertaken. II) Localization of IP-25 Attachment on Friend Cell Chromatin. Localization of IP-25 was achieved by the digestion of Friend c e l l nuclei with micrococcal nuclease (Figs. 11-14). This enzyme preferentially cleaves between nucleosome core particles. The products of digestion are 97 multiples of nucleosomes linked together by regions of DNA uith associated histone H1 and NHP proteins. It i s within monomers that the greatest heterogeneity of DNA length and protein constituency exists (Bakayev et al.,1979). This is due to the mode of action of the enzyme. Monomers are produced with linker DNA s t i l l attached and species where the linker region has been trimmed to leave only the 140 to 145 base pair core length DNA. Uith increased digestion even the core DNA is cleaved, giving subnucleo-somal particles (Bakayev et a l . , 1979)(l/arshavsky et al.,1976). Using this enzyme, nucleosomes were isolated and their proteins analysed. It uas found that the concentration of IP-25 uas proportionally the same relative to histone H1 concentration in monomer and higher multimer nucleo-some sizes (Fig. 13). This result uould only be expected i f IP-25 was located in the same position and had the same attachment specificity as histone H1 in chromatin. The results were not affected by the time of digestion or the length of time of DMSO induction of the c e l l s . Of course the overall quantities of IP-25 increased with increased DMSO induction time (Fig. 17). The ratio of the two H1 histones varied from nucleosome monomers to higher multimers (Figs. 12 and 13). In one experiment, the ratio of H1a to Hlb increased with the size of multimer in a linear fashion. Other experiments showed more var i a b i l i t y , though the trend was the same. These results can be explained in two ways: 1) It i s possible that H1a i s less strongly bound tD the chromatin than Hlb and therefore preferentially lost. It i s also possible that H1b adhers with greater a f f i n i t y than H1a and i s not completely removed by SDS treatment when the samples were prepared for analysis, although this possibility seems unlikely. 2) The second possibility i s that an unequal distribution of the two H1 species places H1b into nuclease susceptable areas Dn the chromatin. This 98 second explanation has recently been supported by the findings of Gorka and Laurence (1979). The constant ratio of IP-25 to total HT histones during digestion ex-periments suggests that IP-25 i s distributed equally and in the same area as histones H1a and H1b. A preferential loss of one of the H1 variants appears to produce a corresponding proportional loss in IP-25. The results presented here for the localization of IP-25 uithin chromatin structure are in partial disagreement uith those reported by Keppel et'al. (1979). They reported an absence of H1 histones in monomer fractions and an absence of IP-25 from monomer and dimers. It uas suggested that IP-25 protected the DNA from cleavage and thus only areas lacking IP-25 uould be extensively digested. This does not explain the lack of H1 histones from the monomeric fraction. For example, other uorkers (Bakayev et al.,1979)(varshavesky et al.,1976) have found that monomer fractions contain a heterogeneous mixture of nucleosome sizes after limited digest, some of uhich contain H1 histone. It therefore seems more reasonable to assume that in the uork of Keppel et al.(l979), the H1 loss i s due to extensive digestion uhich cleaved off a l l linker region DNA along uith the associated H1 histones from monomer nucleosomes. Tuo-dimensional nucleosome separation uas carried out by the procedure of varshavsky et al.(1976) to obtain more direct visualization of IP-25 localization an various multimer sizes of nucleosomes (Fig. 14). In these experiments the digestion of chromatin and the separation of nucleosomes prior to electrophoresis uas identical to the previous ex-periments involving one dimensional analysis of nucleosomal histones. If the differences in the experiments reported by Keppel et al.(1979) and of. those presented in this thesis are due to differences in these steps of methodology, the new two-dimensional seperation would not be expected to re-solve the discrepancies. However the results of the two-dimensional electro-phoresis do clearly show that IP-25 is indeed present in monomer and dimers (Fig. 14) when the methodology of this thesis is used. It also more clearly demonstrates that IP-25 is associated with the linker region hot the nucleosome core. However, the results of both one and two-dimensional elecrophoretic separations agree quite well and i t is therefore concluded that IP-25 i s indeed associated with some types of monomer nucleosomes. The effect of IP-25 on chromatin structure i s not clear. If i t acts as a mammalian histone H5, i t might protect the linker region as proposed by Keppel et al.(1979). This could result in larger DNA lengths i n micro-coccal digested chromatin. It has been reported that the onset of histone H5 production in chicken erythrocytes was followed by decreased suscept-a b i l i t y of digestion of linker region DNA leading to larger DNA digest products (Weintraub, 1978). The analysis of uninduced and DMSO induced Friend c e l l DNA did not show a major change in nucleosome repeat length DNA (Fig. 15 and 16). However, the experimental procedure used was not sensitive enough to detect minor differences in repeat lengths. The possibility s t i l l exists that IP-25 is protecting linker DNA in a similar manner as histone H5 but is not changing the repeat length appreciably. I l l ) The Relationship of IP-25 and Differentiation. The accumulation of IP-25 on chromatin appears to occur at a high rate soon after an inducing agent is added. About 5056 of the maximal concentration of IP-25 is reached by 24 hours of induction with DMSO. At this stage of induction, the amount of haemoglobin per c e l l has only climbed from 0.45 pg to about 1.5 pg (Reeves and Cserjesi, 1979). This constitutes an increase of less than 10% of maximum concentration. It appears that IP-25'accumulation on chromatin precedes differentiation as 100 measured by haemoglobin accumulation. These findings are in agreement uith previously published experiments (Keppel et a l . 1979). There are two possible methods for the rapid accumulation of IP-25: 1) Cell division may not be needed for IP-25 accumulation, and 2) cel l s which do divide shortly after induction accumulate large quantities Df IP-25 on their chromatin. If division i s not required, three possible control mechanisms exist to explain the rapid accumulation rate: 1) IMeui transcription of mRIMA for IP-25 (and i t s subsequent trans-lation into protein) commences without the need of c e l l division. This possibility has been demonstrated for other proteins in c e l l s when they are induced with butyric acid (Reeves and Cserjesi, 1980). 2) IP-25 mRIMA i s present in uninduced c e l l s but i s not translated into detectable protein due to post-transcriptianal suppression. Such control has been reported in Xenopus development for histone H1.(woodland et al.,1979). The presence of DMSO may cause the c e l l s to actively translate pre-existing IP-25 mRIMA. 3) IP-25 may be present in the c e l l s prior to induction but does not accumulate upon the chromatin u n t i l the ce l l s undergo DMSO induction. A l l three po s s i b i l i t i e s in themselves, or in conjunction, are feasible explanations for the rapid IP-25 accumulation, but they do not preclude the possibility that IP-25 gene expression occurs only after c e l l division. If division i s needed prior to the expression of IP-25 genes, a limited number of cel l s would be responsible for the high degree of IP-25 synthesis i n i t i a l l y . The synthesis of IP-25 would then lower in c e l l s that were induced early in their c e l l cycle and cel l s which divided after induction. If this were not the case, accumulation of IP-25 would increase at an Table 3 Partial Amino Acid Compositions of Lysine Rich Histones. Amino acid Chick Erythroyte Goose Fish H51 Frog Turtle Calf" ' Thymus Drosow phila^ Histone H1 Friend C e l l H1a Friend Cell H1b Calf H1° 5 IP-25 serine 13.1 10.3 8.8 7.8 8.6 5.6 10.3 9.0 8.8 8.5 12.0 alanine 15.2 17.0 16.2 17.0 20.5 24.3 18.1 22.3 19.8 16.8 13.5 lysine 24.9 22.6 23.9 28.7 30.3 26.8 20.5 25.1 27.5 31.3 21.4 arginine 11.4 11.5 7.1 9.9 3.3 1.8 2.3 2.1 2.2 2.6 3.3 Hnilica, 1972 Oliver and Chalkley, 1972 Panyim and Chalkley, 1969b 102 exponential rate for about 48 hours after induction, the time required for tuo c e l l divisions. While the rate of accumulation of IP-25 i s i n i t i a l l y more rapid than the rate of haemoglobin synthesis and accumulation, this does not necess-ar i l y mean that IP-25 accumulation i s a prerequisite far differentiation, are linked (Table 2). On the other hand DMSO resistant c e l l s do appear t D increase their chromatin bound IP-25 concentration uhen groun in the presence of DMSO, but they do not accumulated haemoglobin. Houever, a lack of haemoglobin synthesis in i t s e l f does not necessarily indicate that erythroid-specific differentiation i s not taking place. For example i t has previously been shoun that DMSO resistant c e l l s can differentiate as monitored by spectrin and glycophorin induction, and yet these c e l l s lack any increase in haemoglobin accumulation. In addition, uhen DMSO resistant cells groun in the presence of DMSO f a i l to accumulate IP-25, there i s a corresponding failure to induce haemoglobin, spectrin, and glycophorin (Heppel et a l . , 1979). Thus the relationship that exists betueen the various overt biochemical expressions of erythroid d i f f -erentiation in these c e l l s are very complex and do not f i t easily into any simple theoretical model. The data presented in this thesis supports the hypothesis that IP-25 i s a "histone-like" protein uhich can be clas s i f i e d as a member of a histone H1 and H5 superset. It appears to be associated uith the linker region of the chromatin but does not cause major alterations to the core DMA length upon cleaving uith micrococcal nuclease. Whether cr not i t i s required for the f i n a l steps of erythropoesis uas not determined, but the results presented here and by others (Keppel et a l . , 1979) strongly suggests that IP-25 i s linked uith the expression of erythroid character-i s t i c s in Friend c e l l s , but not necessarily responsible for erythro-poetic differentiation. 104 LITERATURE CITED 1. ftlfagene, C. R. Zweidler, A. Mahowald, A. Conn, L. H. 1974. Histones of Drosophila embryos: Electrophoretic isolation and Structural studies. J . B i o l . Chem. 249: 3729-3736. 2. Bakayev, V. V. Bakayeva, T. G. Uarshavsky, A. J. 1977. Nuc-leosomes and subnucleosomes: heterogeneity and composition. Cell J l : 619-629. 3. Bakayev, V. V. Shmatchemko, W. V. Georgiev, G. P. 1979. Sub-nucleosomes, HMG-type proteins, and active chromatin. Proc. Acad. Sci. USSR 245: 93-96. 4. Beckman Application Notes. 119C-AN-001. Pub. Spinco Division of Beckman Instruments Inc. Palo Alto, California. 5. Bennett, M Steeves, R. A. Cudkowiez, G. Mirand, E. A. 1968. Mutant S alleles of mice affect susceptability to Friend spleen Focus-forming virus. Science 162: 564-565. 6. Bil l i n g s , P. C. Ort, 3. hi. Palmer, D. H. Talmage, D. A. Pan, C. G. Blumenfeld, M. 1979. Anamalous electrophoretic mobility of Drosophila phosphorylated H1 histone: i s i t related to the compaction of s a t e l l i t e DNA into heterochromatin? Nuc. Acid Res. 6: 2151-2164. 7. Blumenfeld, M. Orf, 3. Id. Sima, B . J . Kreber, R. A. Callahan, M. A. Snyder, L. A. 1977. Satellite DNA, H1 histone, and heterochromatin in Drosophila v i r i l i s . C. S. H. S. Q. B. 42: 273-275. 8. Bonner, hi. M. Laskey, R. A. 1974. A film detection method far tritium-labelled proteins and nucleic acids in polyacryl-amide gels. Eur. 3. Biochem. 46: 83-88. 9. Brack, C. Hirama, M. Lenhard-Schuller, B . Tonegawa,^S. 1978. A complete immunoglobulin gene i s created by somatic recombin-ation. Cell J5: 1-14. 10. Bradbury.,1 E. M. Cary, P. D. Chapman, G. E. Crane-Robinson, C. Danby, S. E. Rattle, H. hi. E. Boublik, M. Palau, J. Aviles, F. 3m 1975. Studies on the role and mode of operation of the very-lysine-rich histone H1 (F1) in eukaryotic chromatine. Eur. 3. Biochem. 52: 605-613. 105 11. Busch, H. B a l l a l , IM. R. Busch, R. H. Choi, Y. C. Davis, F. Goldknopf, L. Matsui, S.-I. Rao, M. S. Rothblum, L. I. 1977. The nucleolus, a model for analysis of chromatin controls. C. S. H. S. Q. B. 42: 665-683. 12. Candido, E. P. M. Reeves, R. Davie, J . R. 1978. Sodium butyrate inhibits histone deacetylation in culture c e l l s . C e l l 14: 105-113. 13. Chapman, G.E. Hartman, P. G. Bradbury, E. M. 1976. Studies on the role and mode of operation of the very-lysine-rich histone-'l-H in eukaryotic chromatin. Eur. J. Bioch. 6_1: 69-75. 14. Cleveland, D. U. Fisher, S. G. Kirschner, M. W. Laemmli, U. K. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J . B i o l . Chem. 252: 1102-1106. 15. Cole, R. D. Lauson, G. M. Hsiang, M. U. 1977. H1 histone and the condensation of chromatin and DIMA. C. S. H. S. Q. B. 42: 253-263. 16. Conkie, D. Affara, IM. Harrison, P. R. Paul, 3. Jones, K. 1974. In situ localization of globin messenger RIMA formation. J. Ce l l B i o l . 63: 414-419. 17. Conscience, J . F. Miller, R. A. Henry, J. Ruddle, F. H. 1977. Acetylcholinesterase, carbonic anhydrase and catalase activity in Friend erythroleukemic c e l l s , non-erythroid mouse c e l l lines and their Bematic hybrids. Exp. Cell Res. 105: 401-412. 18. Davie, J . R. Candido, P. M. 1978. Acetylated histone H4 i s preferentially associated uith template-active chromatin. Proc. Natl. Acad. Sci. USA 75: 3574-3577. 19. Delange, R. J. 1978. Peptide mapping and amino acid sequencing of histones. Meth.. Cell B i o l . _18: 169-187. 20. Dick, C. Johns, E. hi. 1969. A quantitative comparison of histones from immature and mature erythroid c e l l s of the duck. , Biochim. Biophys. Acta T75: 414-418. 21. Elgin, S. C. R. Bonner, J. 1970. Limited heterogeneity of the major chromosomal proteins. Biochem. 3: 4440-4447. 106 22. Elgin, S. C. R. Serunian, L. A. Silver, L. M. 1977. D i s t r i -bution patterns Df Drosophila nonhistone chromosomal proteins. C. S. H. S. Q. B. 42: 839-849. 23. Felsenfeld, G. 1978. Chromatin. Nature 271: 115-122. 24. Finch, J. T. Lutter, L. C. Rhodes, D. Broun, R. S. Rushton, B. Levitt, M. Klug, A. 1977. Structure Df nucleosome core par-t i c l e s of chromatin. Nature 269: 29-36. 25. Franklin, S. G. Zueidler, A. 1977. Non-allelic variants of histones 2a, 2b and 3 in mammals. Nature 266: 273-275. 26. Fredrickson, T. Tambourin, P. lilendling, F. Jasmin, C. Smajda, F. J. 1975. Target c e l l of the polycythamia-inducing Friend virus: Studies uith Myleran. U. S. Natl. Cancer Inst. 55: ^3-446. 27. Friend, C. 1957. Cell-free transmission in adult Suiss mice of a disease having the charater of a leukemia. J . Exp. Med. 105: 307-318. 28. Friend, C. Preisler, H. D. Scher, W. 1974. Studies on the control of differentiation of murine virus-induced erythro-leukemic c e l l s . Curr. Top. Dev. B i o l . 8: 81-101. 29. Gates, D. M. Bekhor, I. 1979. DNA-binding activity of tightly-bound nonhistone chromosomal proteins in chicken l i v e r chromatin. Nuc. Acid Res. 6: 3411-3425. 30. Gehrke, C. kJ. Tabeda, H. 1973. Gas-liquid chromatographic studies of the twenty protein amino acids: a single-column seperation. J. Chromatog. 76: 63-75. 31. Glazer, A. N. Smith, E. L. 1971. Papain and other plant sulf-hydryl proteolytic enzymes. In "The Enzymes" Vol. I l l Ed. P. D. Boyer Academic Press N. Y. 32. Glover, C. V. C. Gorovsky, M. A. 1979. Amino-acid sequence of Tetrahymena histone H4 differs from that of higher eukaryotes. Proc. Natl. Acad. Sci. USA 76: 585-589. 33. Gorka, C. Laurence, J. J . 1979. The distribution of histone H1 subfractions in chromatin subunits. Nuc. Acid Res. 7: 347-349. 34. Gray, U. R. 1972. End-group analysis using Dansyl chloride. Meth. Enzymology 25: 121-138. 35. Gross, K. Schaffoer, Id. Telford, 3. B i r s t i e l , M. 1976. Mol-ecular analysis of the histone gene cluster of Psammechinus  F R l l i a r i s : III. Polarity and asymmetry of the histone-coding sequences. Cell 8: 479-484. 36. Hackett, P. B. Traub, P. Galluitz, D. 1978. The histone genes in HeLa c e l l s are on individual transcriptional units. 3. Mol. B i o l . 126: 619-635. 37. Hnilica, L. S. 1972. In "The Structure and Biological Func-tion Df Histones." Ed. L. S. Hnilica Pub. CRC Press Cleveland, Ohio. 38. von Holt, C. Brandt, Id. F. 1977. Fractionation of histones on molecular sieve matrecis. Meth. Cell B i o l . 16: 205-225. 39.. Huang, S. Y. 1977. Electrophoretic and amino acid analysis of amphibian and r e p t i l l i a n histones. Masters Thesis Univ-ersity of British Columbia. 40. Hunt, L. T. Dayhoff, M. 0. 1977. Amino-terminal sequence identity of ubiquitin and the nonhistone component of nuclear protein A24. Biochem. Biophysic. Res. Comm. 74: 650-655. 41. Ikawa, Y. Sugano, H. 1967. Spleen focus in Friend disease: An electron microscopic study. Gann 58: 155-160. 42. Isenberg, I. 1970. Histones. Ann. Rev. Biochem. 48: 159-191. 43. Jacob, E. 1976. Histane-gene reiteration in the genome of mouse. Eur. 3. Biochem. 65: 275-284. 44. Jamrich, M. Greenleaf, A. L. Bautz, F. A. Bautz, E. K. F. 1977. Functional organization of polytene chromosomes. C. S. H. S. Q. B. 42: 389-396. 45. Javaherian, K. Sadeghi, M. Liu, L. F. 1979. Nonhistone proteins HMG^  and HM^ unwind DNA double helix. Nuc. Acid Res. 3569-3581. 108 46. Johnson, A. Id. Hnilica, L. S. 1971. Cytoplasmic and nuc-lear basic protein^synthesis during early sea urchin dev-elopment. Biochim. Biophys. Acta 246: 141-154. 47. Kabat, D. Sherton, C. C. Evans, L. H. Bigley, R. Koler, R. D. 1975. Synthesis of erythroid-specific proteins in cultured Friend leukemia c e l l s . C e l l 5: 331-338. 48. Kedes, L. H. 1979. Histone genes and histone messenger. Ann. Rev. Biochem. 48: 837-870. 49. Kedes, L. H. B i r n s t i e l , M. L. 1971. Reiteration and clustering of DNA sequences complementary to histone messanger RNA. Nature New B i o l . 230: 165-169. 50. Keppel, F. Allet, B. 1979. Biochemical properties and l o c a l i -zation of the chromosomal protein IP-25. Eur. J. Biochem. 96: 477-482. 51. Keppel, F. Allet, B. Eisen, H. 1977. Appearance of a chromatin protein during the erythroid differentiation of Friend virus-transformed c e l l s . PSoc. Natl. Acad. Sci. USA 74: 653-656. 52. Kleinsmith, L. J. 1975. Phosphotylation of non-histone proteins in the regulation of chromosome structure and function. 3. Cell Physiol. 85: 459-476. 53. Kornberg, R. D. 1977. Structure of chromatin. Ann. Rev. Biochem. 46: 931-954. 54. Kornberg, R. D. 1974. Chromatin structure: A prepeating unit of histones and DNA. Science 184: 868-871. 55. Kost, T. A. Koiury, M. 3. Hankins, 111. D. Krantz, S. B. 1979. Target c e l l s for Friend virus-induced erythroid bursts in vitro. Cell 18: 145-152. 56. Koster, J. G. Kasinsky, H. E. Destree, 0. H. J. 1979. Newly sythesized histones in chromatin in early embryos of Xenopus  laevis. Cell Differen. 8: 93-104. 57. Kunkel, N. S. Hemminki, K. Ldeinberg, E. S. 1978. Size of histone gene transcripts in different embryonic stages of the sea urchin Stronqylocentrotus purpuratus. Biochem. _17: 2591-2598. 58. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. 59. Lasky, R. A. Mil l s , A. D. 1975. Quantitative film detection of and ^ C in polyacrylamide gels by fluorography. Eur. J . Biochem. 56: 335-341. 60. Levy-liJilson, B. Watson, D. C. Dixon, G. H. 1979. Multi-acetylated forms of H4 are found in a putative transcriptionally competent chromatin fraction from trout t e s t i s . Nuc. Acid. Res. 6: 259-274. 61. Lifton, R. P. Goldberg, M. L. Harp, R. Id. Hogness, D. S. 1977. The organization of the histone genes in Drosophila melanoqaster: Functional and evolutionary implications. C. S. H. S. Q. B. 42: 1047-1051. 62. L i l l y , F. Pincus, T. 1973. Genetic control of murine v i r a l leukemogenesis. Adv. Cancer Res. J7: 231-277. 63. Marks, D. B. Kanefsky, T. Keller, B. 3. Marks, A. D. 1975. The presence of histone H1D in human tissues. Cancer Res. 35: 886-889. 64. Marks, P. A. Rifkind, R. A. 1978. Erythroieukemic differen-tia t i o n . Ann. Rev. Biochem. 47: 419-448. 65. Marushige, K. Bonner, J. 1966. Template properties of l i v e r chromatin. J . Mol. Bi o l . J5: 160-174. 66. Merril, C. R. Suiitzer, R. C. UanKeuren, M. L. 1979. Trace polypeptides in cellular extracts and human body fluids detected by tuo-dimensional electrophoresis and a highly sensitive silver stain. Proc. Natl. Acad. Sci. USA 76: 4335-4339. 67. Melli, M. S p i n e l l i , G. Uyssling, H. Arnold, E. 1977. Presence of histone mRNA sequences in high molecular weight RNA of HeLa c e l l s . C e ll 11_: 651-661. 68. McDonold, G. A. Dodds, T. C. Cruickshank, B. 1978. In " Atlas of Haematology" 4 t h Ed. Churchill Livingstone. NY NY 110 69. Newman, S. A. No-cm, P. 1979. Urea-induced binding cf histone 1 to nucleosomes lacking linker DNA. Nuc. Acid Res. 6: 609-623. 70. Newrock, K. M. Alfagene, C. R. Nardi, R. V. Hohen, L. H., 1977. Histone changes during chromatin remodeling in embryogenseis. C. S. H. S. Q. B. 42: 421-431. 71. Noel, D. Nikaido, K. Ames, G. F.-L. 1979. A single amino acid sustitution in a histidine-transport protein drastically alters i t s mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochem. _18: 4159-4165. 72. Noll, M. 1977. Internal structure of the nucleosome: DNA folding i n the coserved 140-base-pair core particle. C. S. H. S. Q. B. 42: 77-85. 73. Noll, M. Kornberg, R. D. 1977. Action of micrococcal nuclease on chromatin and the location D f histone H1. 3. Mol. B i o l . 109: 393-404. 74. Nudel, U. Salmon, 3. Fiback, E. Terada, M. Rifkind, R. Marks, P. Bank, A. 1977. Accumulation of •<- and £-globin messanger RNAs in mouse erythroleukemia c e l l s . Cell J2: 463-469. 75. O'Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. 3. B i o l . Chem. 250: 4007-4021. 76. Oliver, D. Chalkley, R. 1972. An electrophoretic analysis of Drosophii.la histones. Exp. Cell Res. 73: 295-302. 77. Panyim, S. Chalkley, R. 1971. The molecular weights of vertabrate histones exploiting a modified sodium dodecyl sulfate electrophoresis method. J. Bi o l . Chem. 246: 7557-7560. 78. Panyim, S. Chalkley, R. 1969. High resolution acrylamide gel electrophoresis of histones. Arch. Biochem. Biophy. 130: 337-346. 79. Panyim, S. Chalkley, R. 1969b. A new histone found only in mammalian tissue with l i t t l e c e l l division. Biochem. Biophys. Res. Comm. 37: 1042-1049. 80. Panyim, S. Bilek, D. Chalkley, R. 1971. An electrophoretic comparison of vertebrate histones. 3. B i o l . Chem. 246: 4206-4215. 81. Peterson, 3. L. McConkey, E. H. 1976. Proteins of Friend leukemia c e l l s . 3. B i o l . Chem. 251: 555-558. 82. Randon, 3. F. Cotter, R. I. L i l l e y , D. M. J. Worcester, D. L. Campbell, A. M. Uooley, 3. C. Richards, B. M. 1977.. Scattering studies of chromatin subunits. C. S. H.. S. Q. B. 42: 11-22. 83. Reeves, R. 1977.Analysis and reconstruction of Xenopus ribosomal chromatin nucleosomes^ .Eur. J. Biochem. 75: 545-560. 84. Reeves, R. Cserjesi, P. .1979. Constitutive synthesis of globins uithin most of the members of an uninduced, proliferating pop-ulation of Friend erythroleukemic c e l l s . Dev. B i D l . 6_9: 576-598. 85. Reeves, R. Cserjesi, P. 1979b. Sodium butyrate induces new gene expression in Friend erythroleukemic c e l l s . 3. B i o l . Chem. 254: 4283-4290. 86. Reeves, R. Cserjesi, P. 1980. Reversibility of butyrate-induced neui gene expression in Friend erythroleukemic c e l l s . Manuscript in preperation. 87. Renz, M. Nehls, P. Hazier, 3. 1977. Histone H1 involvment in the structure of chromatin fiber. C. S. H. S. Q. B. 42: 245-251. 88. Rifkind, R. A. Bank, A. Marks, P. A. IMossel, H. L. 1976. In "Fundamentals of Haematology" Year Book Medical Pub. Inc. Chicago. 89. Ross, .3. Gielen, J. Packman, S. Ikauia, Y. Leder, P. 1974. Globin gene expression in cultured erythraleukemia c e l l s . J . Mol. Bi o l . 87: 697-714. 90. Sharp, P. A. Sugden, B. Sambrook, 3. 1973. Detection of two restriction endonuclease a c t i v i t i e s in Haemophilus para- influenzae using analytical agarase-ethidium bromide electro-phoresis. Biochem. J2.: 3055-3063. 91. Shelton, H. R. IMeelin, 3. M. 1971. Nuclear residual proteins from goose erythroid c e l l s and l i v e r , Biochem. JO: 2342-2348. 92. Smerdon, M. 3. Isenberg, I. 1976. Conformational changes in subfractions of calf thymus histone H1. Biochem. J5: 4233-4242. 112 93. Spadafora, C. Dudet, P. Chambon, P. 1979. Rearrangements Df chromatin structure induced by increasing ionic strength and temperature. Eur. 3. Biochem. 100: 225-235. 94. Steeves, R.A. Bennett, M. Mirand, E. A. Cudkouicz, G. 1958. Genetic control by the Id locus of susceptibility to (Friend) spleen focus-forming virus. Nature 218: 372-374. 95. Stein, G. Stein, 3. 1976. Chromosomal proteins: Their role in the regulation of gene expression. Biosci. 26: 488-498. 96. Stein, G. S. Stein, J . L. Thomas, 3. A. 1977. Chromosomal proteins and the regulation of gene expression in normal and neoplastic c e l l s . Leuk. Res. J_: 335-384. 97. Tambourin, P. E. Ldendling, F. 1975. Oxygen a f f i n i t y and allo s t e r i c effect of embryonic mouse haemoglobins. Nature 257: 333-334. 98. Tambourin, P. E. Gallien-Lartique, 0. ldendling, F. Huaulme, D. 1973. Erythrocyte production in mice infected by the poly-cythaemia-inducing Friend virus or by the anaemia-inducing Friend virus. Br. J. Haematalogy 24: 511-524. 99. Tarnouka, M. A. Baglioni, C. Basilica, C. 1978. Synthesis of H1 histones by BHH ce l l s in G1. Cell J5: 163-171. 100. Teller, D. C. Kinkade, 3. M. Cole, D. R. 1965. The molecular ueight og lysine rich histone. Biochem. Biophys. Res. Comm. 20: 739-744. 101. Tepperman, A. D. Curtis, J . E. McCulloch, E. A. 1974. Eryth-ropoietic colonies in cultures of human marrou. Blood 44: 659-669. 102. Trentin, J . 3. 1970. In "Regulation of Haematopoisis" Vol. 1 pg. 161-186. Appletan-Century-Crofts N. Y. 103. Traxler, D. H. Boyars, J . K, Parks, Id. P. Scolnick, F. M. 1977. Isolation of a fiberblast nonproducer c e l l line con-taining the Friend strain of the spleen focus-forming virus. 3. V i r o l . 22: 361-372. 104. \7arricchio, F. 1977. Postnatal increase in histone H1a in the rat pancreas. Arch, biochem. Biaphy. 179: 715-717. 105. Varshavsky, A. J. Bakayev, V. V. Georgiev, G. P. 1976. Hetero-geneity of chromatin subunits _in vitro and location of histone H1. IMuc. Acid Res. 3: 477-492. 106. Uidali, G. Boffa, L. Littau, V. C. Allfrey, K. M. Allfrey, V. G. 1973. Changes in nuclear acidic protein component of red blood cells during embryonic development. J. Biol. Chem. 248: 4065-4068. 107. Ideintraub, H. 1978. The nucleosome repeat length increases during erythropoiesis in the chick. IMuc. Acid Res. 5: 1179-1188. 108. Ideintraub, H. Groudine, M. 1976. Chromosomal subunits in active §enes have an altered conformation. Science 193: 848-856. 109. Woodland, H. R. Flynn, 3. M. Wyllie, A. 3. 1979. Utilization of stored mRIMA in Xenopus embryos and i t s replacement by newly synthesized transcripts: Histone H1 synthesis using interspecies hybrids. Cell 18: 165-171. 110. Yaguchi, Fl. Camille, R. Dove, M. Seligy, V. 1977. Amino acid sequence homologies between HI and H5 histones. Biochem. Biophy. Res. Comm. 76: 100-106. 111. Zweidler, A. 1978. Resolution of histones by polyacrylamide gel electrophoresis in presence of nonionic detergents. Meth. C e l l . B i o l . 17: 223-232. 112. Zweidler, A. Cohn, L. H. 1972. A new electrophoretic method revealing multiplicity, tissue specificity and evolutionary variation in histone F2a2 and F2b. Fed. Proc. 31: 926. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095240/manifest

Comment

Related Items