Immunofluorescent study of histone H5 in chick erythroid cells from developing embryos and adults

Mechanisms of Ageing and Development, 7 (1978) 109-122

109

©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

IMMUNOFLUORESCENT STUDY OF HISTONE H5 IN CHICK ERYTHROID CELLS FROM DEVELOPING EMBRYOS AND ADULTS

CASILDA MURA* and P. C. HUANG t Department o f Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205 (U.S.A.)

SUSAN W. CRAIG Department of Physiological Chemistry, School o f Medicine, Johns Hopkins University, Baltimore, Maryland 21205 (U.S.A.)

(Received May 8, 1977)

SUMMARY Indirect immunofluorescence has been used to detect the presence of histone H5 in single cells during avian erythropoiesis. Evidence is presented which demonstrates that this method specifically detects histone H5. The results show that the increase in the amount of H5 which can be extracted from nuclei during erythropoiesis is accounted for both by an increase in the amount of H5 per cell and by an increase in the total number of cells containing histone H5. Of particular interest are the observations that histone H5 is arranged within the nucleus in several distinct, reproducible patterns and that different patterns predominate at different stages of erythroid cell development. In addition to suggesting new possibilities for the role of histone H5, these findings indicate that immunofluorescence may be used profitably to explore the supramolecular organization o f chrom atin.

INTRODUCTION Unlike mammalian erythrocytes which lose their nuclei during maturation, the senescent erythrocytes of birds retain their nuclei in an inactive state. As avian erythroblasts mature to erythrocytes, the chromatin becomes highly compacted and incapable of DNA replication and RNA transcription. These inert nuclei contain a unique histone, H5, (F2c or KSA), which is absent from all other avian tissues [1, 2]. The tissue specificity of H5 coupled with the observation that the proportion of H5, with respect to total histone content, increases during embryonic development [3, 4] *Present address: Department of Biology, Carleton University, Ottawa, K1S5B6 (Canada). tTo whom correspondence should be addressed.

110 has led to the suggestion that the accumulation of H5 might cause the gradual condensation and inactivation of erythrocyte chromatin [5]. Another observation crucial to consideration of the function of H5 is that the histone appears early in the maturation of the erythrocyte. In anemic adult chickens, erythroblasts contain 20-25% of the final amount of H5 found in the mature cell [6]. At a subsequent stage of development, the early polychromatic erythrocytes contain 30-40% of the final level of H5 [7]. Studies on separated subpopulations of erythroid cells differing in their stage of maturation (defined by metabolic activity) have also shown that H5 increases gradually during maturation (see paper by Enea, Gottesman and Vidali pp. 97 of this issue). Since H5 is present in erythroblasts, a population which is still capable of division and in early polychromatic erythroblasts, which still synthesize RNA, it could be concluded that the presence of H5 is not in itself sufficient to inactivate the genome. In fact, it has recently been postulated that two concomitant events, namely a decrease in turnover rate of histone H5 during erythrocyte maturation [8, 9] and an increase in the level of dephosphorylation [ 10, 11] are responsible for the gradual inactivation of the erythrocyte nucleus rather than the simple presence and accumulation of H5. Previous analyses of H5 during development, although very informative, suffer from a common drawback in that they were performed on heterogeneous populations of erythroid cells. Therefore, it is not known whether the metabolically active cells are also the ceils which contain H5. In addition, the exact cellular basis for the increase in H5 during erythropoiesis is undetermined; i.e., does the increase represent the accumulation within individual cells, the accumulation of cells containing H5, or is it due to both processes? Finally, it is not known if all mature, metabolically inactive erythrocytes contain H5. Therefore, to obtain more information on the possible role of H5 in erythrocyte maturation, we have analyzed the appearance of H5 during erythropoiesis in single cells using indirect immunofluorescence. The reactivity of histone H5 with anti-H5 prepared from guinea pig sera was detected by sheep anti-guinea pig IgG coupled with rhodamine. MATERIALS AND METHODS

Preparation o f histone H5 and its antibody Histone H5 was prepared from circulating red blood cells of adult white leghorn chickens. Anti-histone H5 antibody was prepared from sera of guinea pigs immunized with purified H5 as reported previously [12]. The gamma globulin fraction of this serum was precipitated by sodium sulfate and further purified by chromatography on Sephadex G-200 with 0.05 M phosphate, pH 7.3 containing 2.2% NaC1 and 0.02% sodium azide as the eluting buffer. The gamma globulin fraction was identified by immunodiffusion using anti-guinea pig gamma globulin. A gamma globulin fraction from a non-immunized guinea pig was prepared in the same way. The IgG fraction of rabbit anti-guinea pig light chain antisera conjugated with rhodamine isothiocyanate (A28o/As~5 = 4.1) was prepared according to the method of Cebra and Goldstein [13] and was a gift of Dr. John Cebra.

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Affinity chromatography Sepharose 4B was activated with cyanogen bromide [14]. 1 g of cyanogen bromide was added to 10 ml of Sepharose 4B at 4 °C. The pH of the solution was maintained at 11 by the addition of 4 N NaOH. The reaction was allowed to proceed for 3-4 minutes at 4 °C with constant stirring. Following activation, the Sepharose was washed immediately in a sintered glass funnel at room temperature, with 50 ml of cold 0.1 N NaHCO3 and resuspended in 30 ml of cold 0.1 N NaHCOa. The Sepharose was then allowed to react with the protein (2 to 10 mg per 10 ml of the gel). The reaction mixture was stirred gently at 4 °C for 20 h. Individual proteins coupled to Sepharose included histone H5 from adult avian erythrocyte, and normal guinea pig IgG. The Sepharose-protein material was washed extensively with 1 M NaCI to remove unbound protein and then equilibrated with phosphate buffered saline, pH 7.3 (PBS). To each of the columns, (1 ml of packed Sepharose), 1 ml of anti-F2c immunoglobulin, prepared as indicated above, containing approximately 1 mg of protein (A2ao = 1.5) was added and then eluted at 4 °C using PBS as the eluting buffer. The fractions containing protein were pooled and concentrated until A2so = 1. The concentrated effluents from the different columns were used for staining cells.

Preparation of avian erythroid cells Circulating red blood cells from 3 to 18 day old embryos were collected in Hank's solution containing 4% fetal calf serum and 5% antibiotic-antimycotic (100 times concentrated). Cells were washed 3 times in the same solution and slides were prepared. After brief drying, the slides were either fixed in 95% EtOH in preparation for immuno. fluorescence staining or were directly stained with May-Griinwald followed by Giemsa stain. Bursa and thymus cells were prepared from fresh tissues obtained from 10 day old chickens. The tissues were minced in Hank's solution and released cells were treated in the same way as red blood cells. Cells were fixed in 95% ethanol for 15 min at 4 °C, and stored in a moist chamber at 4 °C. Adult blood was obtained from the Dover Poultry Products, Baltimore. Chick embryos were staged by wing bud length and width measurements [15].

Estimation o f cell number (a). A measured volume of blood from embryos of different ages was diluted in Hank's solution. Cells were counted in a hemocytometer. The total number of cells per embryo was calculated using estimates of the total embryo blood volume reported earlier [ 16]. (b). Total blood from embryos at different ages was collected in a measured volume of Hank's solution. Samples were taken for counting and total number of cells per embryo was calculated.

Immunofluorescent staining of the cells Histone H5 was detected by indirect immunofluorescence staining, which required two sequential steps. (1) One or two drops of anti-H5 immunoglobulin (A2ao = 0.8)were

112 added to the slide and spread with a large coverslip. The slides were incubated at room temperature for 45 minutes. After incubation the coverslip was rinsed off with Hank's balanced salt solution and the preparation was washed twice with PBS for 10 minutes each time; (2) one or two drops of rhodamine-conjugated IgG fraction of rabbit anti-guinea pig IgG light chain (1.5 mg/ml) were added to the preparation from step (1). The reagent was spread with a large coverslip and incubated at room temperature for 45 minutes. After incubation the coverslip was rinsed off with PBS and washed as in step (1). After the last wash the slides were fixed in 95% ethanol for 5 minutes. The preparation was mounted in 9:1 (v/v) glycerol/PBS, pH 7.4. Examination of the cells was done with a Zeiss fluorescence microscope which was fitted with a darkfield condenser (N.A. = 1.2) and an Osram HBO 200/W4 high pressure mercury lamp. Rhodamine fluorescence (red light, )kma x = 680) was observed using a green interference filter ( ~ ' m a x = 546 nm; Schott) for excitation and a red glass (RG1, Schott) Barrier filter. Photographs were taken on GAF (ASA 500) color film.

RESULTS

Specificity of anti-H5 IgG Data on the specificity of anti-H5 prepared against histone H5 isolated from adult chicken erythrocytes were reported in an earlier paper [12]. By immunodiffusion and complement fixation the guinea pig anti-H5 reacted with H5, but not with HI, H2a, H2b, H3, or H4 from embryo or adult erythroid cells. However, the use of antibody for immunofluorescent localization of antigen in cells requires additional and more stringent specificity controls [17]. Therefore, in the present study, the specificity of the anti-H5 for localization of intracellular H5 was tested by reacting preparations of chick erythroid cells with anti-H5 previously absorbed on insolubilized H5 or insolubilized normal IgG. When chick erythroid cells are reacted with guinea pig anti-H5 followed by rhodaminelabelled anti-guinea pig light chain IgG, red fluorescence is visible in the nucleus of some of the cells (Fig. la). If the anti-H5 IgG is absorbed with insolubilized H5 (Sepharose-H5), it fails to react with any of the cells (Fig. lb). The activity of anti-H5 is specifically absorbed by the Sepharose-H5 since Sepharose-normal IgG fails to remove the ability of anti-H5 IgG to stain the cells (Fig. lc). If lgG is used instead of anti-H5 as the first step in the indirect immunofluorescence stain, no fuorescent cells are observed (Fig. ld). The fluorochrome-conjugated antiguinea pig globulin used as indirect stain did not by itself react detectably with the chick erythroid cells (Fig. le). Finally, if chick bursa or thymus cells, which do not contain a biochemically detectable amount of H5, were stained with anti-H5, no fluorescence was detected in cells other than in occasional red cells which contaminated the population (Figs. I f and 1g). From the above results we conclude that the immunofluorescence stain for H5 is specific for H5 and does not react with any other histone or non-histone components of chick erythroid cells.

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Fig. 1. Specificity of the indirect staining procedure for detection of H5 in chick erythroid cells. The left hand panel shows fields of cells observed in ordinary dark field illumination; the right hand side presents the same fields observed using the appropriate excitation and barrier filters for selective visualization of rhodamine fluorescence (see experimental procedure). (a) 18 day erythroid cells indirectly stained with unabsorbed anti-H5 IgG; (b) 12 day erythroid cells indirectly stained with antiH5 previously absorbed with H5 covalenfly coupled to Sepharose. (c) 12 day erythroid cells indirectly stained with anti-H5 previously absorbed with normal IgG covalently coupled to Sepharose. The decrease in intensity relative to Fig. 1(a) reflects the difference in the stage of embryo from which the cells were taken. Twelve day cells have much less histone H5 than 18 day cells; (d) cells stained indirectly with guinea pig lgG; (e) 12 day cells stained directly with rhodamine-conjugated anti-guinea pig gamma globulin; (f) bursa and (g) thymus chick cells stained indirectly with unabsorbed anti-H5 IgG. Magnification 800 x.

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Immunofluorescent analysis o f the number o f avian erythroid cells which contain detectable H5 during embryonic development The quantitative distribution o f erythroid cells containing nuclear fluorescence during embryonic development is shown in Fig. 2. The number of cells containing H5 is expressed b o t h as percentage o f total cells and in absolute number o f cells. The number of cells per embryo was previously determined (see Table I). The data show that there is an increase both in the absolute number and in the proportion of cells containing H5. The transient maximum in the number o f cells with nuclear fluorescence at 4 days correlates well with the time o f maturation o f the primitive cell line, as described by others [18]. The decrease in the proportion o f cells with H5 between 4 and 6 days might i

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Fig. 2. Immunofluorescent analysis of the number of avian erythroid cells which contain detectable histone H5 during embryonic development. Results are expressed in % of total cells and in total number of cells (t) which contain H5. The values plotted were derived from Table I and represent the mean of 5-7 separate experiments ± S.E.M. TABLE I TOTAL NUMBER OF ERYTHROID CELLS PER EMBRYO

Age of embryo (days} 3 4 5

This study (number of cells X 107)

Literature a (number of cells X 107)

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116 be explained by an increase in immature definitive cell types in the circulation. The overall increase in absolute number of cells with nuclear fluorescence throughout development correlates well with the accumulation of circulating mature definitive red cells. Surprisingly, in adult chicken erythrocytes only 80% of the cells contain detectable amounts of H5. The presence of fluorescence in cells from 3-5 day old embryos indicates the presence of histone H5 in the primitive cell type. We observed that the intensity of nuclear fluorescence is greater in later stages of embryogenesis (12 days or later) than in the early states (day 3-5). As all the experiments were done under the same conditions, this observation suggests that the increase in H5 detected biochemically on the total cell population during development can be explained not only by an increase in the percentage of cells containing H5, but also by an increase in the amount of H5 per cell. Patterns o f H5 organization in erythroid cell nuclei

Several distinct and characteristic patterns of nuclear fluorescence were observed (Fig. 3). These patterns were highly reproducible from experiment to experiment and were striking in their clarity of detail. Analysis of the quantitative distribution of the different patterns through stages of development (Table II) shows that certain patterns tend to predominate at different times. Many of the patterns evident in the embryonic stages are completely absent in the adult erythrocyte. The nuclear H5 fluorescence in mature erythrocytes is present almost exclusively in an intense halo pattern whereas nuclear fluorescence in the embryonic erythroid cell nuclei is present in three or four different patterns in approximately equal representation. The proportion of H5 positive cells which did not fit one of the six different fluorescent patterns did not exceed 5% at any stage of development examined.

TABLE II PATTERN OF H5 FLUORESCENCE(PERCENTAGEOF POSITIVECELLS) IN EMBRYONICAND ADULT CHICK RED BLOODCELLS Age of No. H5 positive cells embryo [days/ No. cells counted

Type of H5 nuclear fluorescence pattern dots

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25 37 40 54 36 64 0

0 6 1 19 5 24 0

6 7 16 10 0 3 28

0 14 5 2 27 8 73

3 5 4 3 2 0 0

160/1963 680/2738 156/1802 221/2372 890/2236 837/1713 513/639

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Fig. 3. Patterns of nuclear fluorescence obtained by indirect immunofluorescent staining of chick red blood cells with antibody against histone H5. (a) dots: multiple, small, discrete speckles; (b) web: pattern varies from distinctly organized multiple fluorescent rings to a less organized latticework structure; (c) solid circle or oval: can occupy either the whole nucleus or only a very small portion thereof; (d) diffuse: dim stain occupying entire nucleus; (e) partial halo; (f) full halo. The nuclei in patterns (a)-(d) are in embryonic ceils (day 3-18); those in (e) and (f) are found in nuclei of adult erythrocytes (21 days post-hatching).

DISCUSSION The presence o f histone H5 in chick e r y t h r o i d cells has been d e t e c t e d in this study by indirect i m m u n o f l u o r e s c e n c e using a c o m b i n a t i o n o f anti-H5 IgG f r o m guinea pig sera

118 and a rhodamine-coupled anti-guinea pig light chain IgG from rabbit sera. This is the first report describing changes in the distribution of histone H5 in the nucleus within the cell and in different cells within the erythroid population at various developmental stages. The validity of the results and conclusions presented in this study depend ultimately on the specificity of the anti-H5 antibody. Therefore, considerable attention has been given to the characterization of this reagent. In an earlier study [12] it was shown by complement fixation that the anti-H5 reacts with H5 but not with H2a, H2b, H3, H4, or HI. As many immune sera contain antibodies reactive with heterotypic cells [17], it was important to establish (see Fig. 1) that neither the anti-H5 IgG nor the rabbit antiguinea pig light chain IgG contained such anti-tissue antibodies. The specificity of anti-H5 immunofluorescence localization was demonstrated by showing that anti-H5 absorbed with insolubilized H5 failed to stain avian erythroid cells, whereas anti-H5 absorbed with IgG from a non-immunized guinea pig was highly reactive with these same cells. Since the rhodamine-labelled rabbit anti-guinea pig light chain did not by itself stain the avian erythroid cells, it can be safely concluded that the indirect stain which we have employed is highly specific for H5. Further support for the tissue specificity of the anti-H5 stain is given by the observation that bursa and thymus cells, which contain only a very low level of H5, are completely non-reactive with anti-H5 as shown in Fig. 1. As suspected [19], the low levels of biochemically detectable H5 can be accounted for by contamination by the white cell preparations with erythrocytes. The results obtained from single cell analysis of the appearance of H5 during development show that the number of erythroid cells containing H5 as well as the proportion of such cells increases during embryogenesis in parallel with the increase in mature, metabolically inactive erythrocytes. It was also observed that there is an accumulation of H5 (greater fluorescence intensity) in the erythroid cells from chicken in later stages of development, i.e., 12-18 days and adult. These observations establish the cellular basis for the increase in H5 which can be extracted during development [3, 4]. The finding that only 80% of erythrocytes from adult chicken react with anti-H5 (see Table II) was unexpected. Since H5 is thought to be a repressor and to be responsible for the quiescence of adult erythrocytes, we expected that all adult erythrocytes would contain H5. Since in this study the proportion of anti-H5 negative cells was not decreased by using a 1000-fold increase in the ratio of anti-H5 to avian erythroid cells in the staining procedure, the presence of negative cells is not likely to be the result of subsaturating amounts of anti-H5 in the assay. There are several possible explanations for the presence of H5-negative adult erythrocytes. One might be that some adult circulating erythrocytes are still metabolically active. Indeed, the common assumption that all adult erythrocytes are metabolically inactive has been challenged [20], with the finding that RNA, but not DNA, synthesis takes place in circulating adult red blood cells. Possibly the H5 negative cells are the ones which can still incorporate [3H] -uridine into RNA. An alternative explanation for the presence of H5 negative adult erythrocytes is that in some cells the H5 is arranged in the chromatin in such a way that it is inaccessible to the antibody. It has recently been shown that antibody to native rat liver and thymus

119 chromatin does not react with any histones with the exception of H1 [21 ]. This observation suggests that the antigenetic determinants of some histones may be masked when the histones are associated with chromatin. Whether H5 determinants in the chromatin of some erythroid ceils may also be hidden is not yet known. Studies with cell fusion reactivated nuclei or poly (A-U) swollen chromatin may help in assessing this prospect. A third possibility is that H5 is really present in all adult erythrocytes but that in some cells it is present at such a low level that it" cannot be detected by indirect immunofluorescence. From studies comparing the number of [~2sI] cholera toxin molecules bound per cell at a given concentration of rat thymocytes with the percentage of cells stained with the same concentration of fluorochrome labelled cholera toxin, it has been estimated that roughly 30,000 molecules of cholera toxin per cell can be visualized by direct fluorescent staining methods (Craig and Cuatrecasas, unpublished data), Indirect fluorescence measurements are estimated to give 10 fold greater sensitivity than other methods [22]. Therefore, the lower limit of detection in our study is of the order of 3000 molecules ofhistone H5 per cell. At this time, we cannot distinguish between the various alternatives, but if 20% of mature erythrocytes are indeed H5 negative cells, then it is unlikely that histone H5 has a unique role in repression of avian red cell chromatin. Further experiments will be needed to unequivocally establish the presence of a subpopulation of mature red blood cells which lack histone H5. Another observation made in this study is the fact that there are very distinct patterns of H5 fluorescence which vary depending on the stage of the embryo from which the red cells originate. Although changes in the quantity of H5 per nucleus may at least partly explain the variation in pattern, it is also possible that these patterns reflect changes in the organization of chromatin which may have functional significance. It is, of course, already well established from both electron microscope [23, 24] and physical chemical studies [25] that the chromatin of avian erythrocytes undergoes structural changes (i.e., condensation) during maturation of the cells. There is also substantial evidence that the interaction of histories with chromatin plays a role in determining the structure of chromatin and, in this respect, the different classes of histones appear to have distinctly different functions. For example the structural subunit of chromatin, "nu body" [26] or "nucleosome" [27], composed of H2a, H2b, H3 and H4, and approximately 700-800 A of DNA which is condensed 5-7 fold in the particle structure has been described. H1 and H5 do not contribute to the formation of these particles but appear to be involved in condensing the superhelix structure of chromatin, into an even more compact state [28]. Removal of H5 and H1 does not change the X-ray diffraction pattern of chromatin [29] but does lead to dramatic loosening of chromatin structure [25, 30]. Significantly, H1 and H5 must be removed before the chromatin assumes a sufficiently open structure for the nucleosomes to be readily visualized in the "beads on a string" pattern. Thermodynamic studies with purified H5 have also shown that this protein is more effective in binding to DNA than H1 [31]. These observations support the idea that histones play a role in determining the conformation of chromatin and that histones H1 and H5 have a different structural role

120 from histone H2a, H2b, H3 and H4. The fact that secondary modifications such as phosphorylation and dephosphorylation of histone H5 can modify the interaction of this histone with chromatin [10, 11] suggests that such modifications may be one way alteration in the structure of chromatin could be achieved. In the light of this information, the highly ordered and intricate patterns of H5 localization which we observe in maturing erythroid cells might reflect H5 modulation of chromatin structure. The variety of patterns observed would then indicate that the chromatin is in a highly dynamic state during erythropoiesis and that it undergoes several structural changes which may be far more subtle than simple "progressive condensation" which is the only structural change heretofore described. It is possible that these patterns of H5 organization reflect particular functional states of chromatin. The observation that the inert nuclei of mature erythrocytes exhibit predominantly one pattern of H5 fluorescence is consistent with this notion. At a minimum, the existence of H5 patterns indicates that H5 associates with chromatin in a non-random, non-homogeneous fashion and that the organization of H5 in chromatin during avian erythropoiesis is subject to radical alterations. In this regard, it is pertinent to point out that recent immunofluorescence studies have indicated that histone H1 is organized in discrete bands on Chinese hamster chromosomes [32]. Furthermore, the patterns of immunofluorescence are definitively different for H1 and H4 in nuclei from Syrian hamster, human cancer and rat embryonal cells in culture [33]. These results suggest that the immunofluorescent pattern of each histone is reflective of the arrangement of this histone and of its relation to the overall chromatin conformation. Finally, the detection of H5 patterns suggests that immunofluorescence may prove to be a very useful probe into the organization of chromatin on a supramolecular level. Even more promising is the possibility that immunofluorescence may be able to detect structural changes which take place in chromatin in response to hormonal or other types of cellular activation or inactivation.

ACKNOWLEDGEMENTS This study was supported by grants from the Jane Coffin Childs Fund (S.W.C.) and the National Foundation/March of Dimes (P.C.H.).

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