Histone Genes Are Clustered but Not Tandemly Repeated in the Chicken Genome (Recombinant DNA/DNA Sequence Analysis/Development) JAMES DOUGLAS ENGEL* and JERRY B

Proc. NatI. Acad. Sci. USA Vol. 78, No. 5, pp. 2856-2860, May 1981 Biochemistry

Histone genes are clustered but not tandemly repeated in the chicken genome (recombinant DNA/DNA sequence analysis/development) JAMES DOUGLAS ENGEL* AND JERRY B. DODGSONt *Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201; and tDepartment of Microbiology and Public Health and Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 Communicated by Emanuel Margoliash, February 5, 1981 ABSTRACT: The recombinant chicken DNA library was MATERIALS AND METHODS screened for histone genes by using pSpl7, a recombinant sea urchin DNA probe containing the H2a and H3 genes of Stron- Isolation and Characterization of Histone Gene Recombi- gylocentrotus purpuratus. Three of the isolated A recombinants nants. Isolation of histone-encoding recombinants was accom- have been analyzed by restriction enzyme mapping and Southern plished by screening the previously described A Charon 4A/ blotting; one histone H3 gene-encoding recombinant was further chicken recombinant DNA library (14) by hybridization to the analyzed by DNA sequence determination. These studies reveal 1.9-kilobase pair (kbp) EcoRI fragment of the Strongylocentro- that the chicken histone genes are not tandemly reiterated, but tus purpuratus pSpl7 histone recombinant, which contains the that, atthe least, several histone genes are physically closely linked coding information for the H2a and H3 histone genes (15). in a nonrepetitive arrangement within the chicken genome. The Hybridization was done as described below for nitrocellulose evolutionary implications of this arrangement versus that seen in filters except that the NaCl concentration was 0.75 M, 10% Drosophila and sea urchins is discussed. dextran sulfate was included, and the temperature was 37°C. Competitor nucleic acids were poly(rA) at 10 ,ug/ml, poly(rC) The histone proteins are collectively responsible for the main- at 10 ,Ag/ml, heat-denatured Escherichia coli DNA at 5 ,Ag/ tenance of the primary eukaryotic nuclear DNA structure (1). ml, and E. coli rRNA or tRNA at 10 ,ug/ml. Washes were done The physical organization, replication, and expression of the as described below for nonhomologous hybridizations. Phages eukaryotic genome is controlled, to a greater or lesser degree, forming positive plaques were purified to homogeneity, grown by histone gene expression and histone organization in chro- in liquid culture, and further characterized by analysis ofmap- matin in concert with nonhistone chromosomal proteins (2). ping blots (16) of the individual A recombinants that had been Therefore, histone gene organization and its influence on the hybridized to specific S. purpuratus or D. melanogaster histone expression of those histone genes are of considerable impor- gene-coding fragments (6, 15). tance to an overall view of eukaryotic gene regulation. A recombinants containing pSpl7-complementary DNA se- Although our understanding of histone gene expression has quences were digested with commercial restriction endonu- been facilitated by elegant studies on the organization, arrange- cleases [New England BioLabs, Biotec (Madison, WI), or Be- ment, and developmental shifts of these genes in various sea thesda Research Laboratories (Rockville, MD)], electrophoresed urchin species (3-5) and in Drosophila (6), comparatively little on agarose gels, stained with ethidium bromide, photographed, is known about histone gene arrangement and expression in and blotted to nitrocellulose BA85 (Schleicher and Schuell) as the are re- described (14). Homologous probes (e.g., those isolated from higher eukaryotes. In the former organisms genes chicken DNA) were hybridized to DNA on the filters in a so- peated from about 100 to perhaps 1000 times per haploid ge- lution containing 1 M NaCl, 50 mM Tris-HCl at pH 8.2, 1 mM nome (6-9), whereas in higher eukaryotes the number of gene EDTA, lOx Denhardt's solution, 0.1% sodium dodecyl sulfate, repeats appears to be on the order of 10-50 copies (10-12). sonicated heat-denatured E. coli DNA at 50 ,ug/ml, and 50% The histone genes of the chicken are each represented ap- (vol/vol) formamide at 42°C for at least 3 X Cot,,, for filters (17) proximately 10 times in the genome, reportedly in a tandemly (Cot,12 being the product of DNA concentration and incubation duplicated array (10), as are those in Drosophila and sea urchin. time for 50% hybridization). The filters were then washed twice We were interested in how the normal chicken histone genes at ambient temperature for 5 min in 3 M NaCl/0.3 M sodium were arranged with respect to the erythropoiesis-specific hi- citrate, followed by four 15-min washes in 20 mM Tris1HCl, pH stone H5 genes [which also appear to be repeated 10 times 8.2/1 mM EDTA/1 x Denhardt's solution/0. 1% sodium do- within the genome (13)]. In order to address this question, we decyl sulfate/i mM sodium pyrophosphate at 60°C. Heterol- initiated experiments to isolate and investigate the arrangement ogous hybridizations (e.g., those performed using sea urchin or of the standard histone genes of the chicken. In this paper, we Drosophila clone probes) were performed identically, except describe the initial results ofthese studies and show that: (i) the that 1.5 M NaCl was used in the hybridization buffer and histone genes are clustered (i.e., present within definite groups 0.1-0.3 M NaCl was added to the second filter wash buffer. within the genome), and (ii) the histone genes of the chicken Positions of labeled complementary fragments were deter- are not repeated in tandem arrays. This substantial difference mined by autoradiography (16). between sea urchin and Drosophila as compared to chicken Restriction Enzyme Mapping. Ambiguities apparent in sin- histone gene arrangement may be reflective of some aspects of gle or combined digestion restriction enzyme cleavage maps early embryonic development in these various species. were resolved by a modification of the method of Smith and Birnsteil (18). Singly end-labeled fragments (isolated as outlined The publication costs ofthis article were defrayed in part by page charge below) were digested in the following reaction mixtures: 1-105 payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: bp, base pair(s). 2856 Downloaded by guest on October 5, 2021 Biochemistry: Engel and Dodgson Proc. Natl. Acad. Sci. USA 78 (1981) 2857

A .1, \I1TT1l 6- rX7 'I/ cpm of end-labeled fragment, 1 Aug of pBR322 DNA (19), plus A Abrlial v Xf o/////zZ//z/asss:A 2 (1-hr) units of restriction endonuclease in 30 A1l. Aliquots (5 ILx /.d) were withdrawn after 2, 4, 6, 8, 10, and 12 min ofdigestion, AUtizelou'- 41--vxx ejw+#ki#MvvL pooled, extracted once with CHClJisoamyl alcohol (24:1, volV 1 'II vol), and precipitated with 0.1 vol of 3 M sodium acetate/0. ACHMd k~ M EDTA. The sample was resuspended in 10 Al of loading ki 1\ buffer and electrophoresed. The agarose or polyacrylamide gels B Hybridization to H2a R\ H3 / were then dried and film was exposed to them (18). Exposure histone gene probes: H2b H4 times were between 15 min and 18 hr, depending on the initial specific activity of the end-labeled DNA fragment. FIG. 1. Restriction enzyme maps of three chicken histone recom- DNA Fragment Isolation. Individual sea urchin or Dro- binants. The maps and positions of hybridization to heterologous sea sophila histone gene DNA sequences to be used as probes were urchin and Drosophila histone gene probes are depicted. (A) Restric- isolated by excision of specific restriction fragments of pSpl7, tion enzyme maps of three separate recombinants. ', EcoRI linkers pSp2, or cDm 500 from gels and hybridized with the chicken/ (14); 1 ,EcoRI; ,BamHI; v,Hindfll; A,Kpn I. (B) Key forprobes used A recombinant blots (above). Specifically, the sea urchin probes to demonstrate the presence of histone genes on the mapped recom- from pSpl7 (15) di- binants shown in A. The determination of the loci of hybridization to prepared were as follows: the H2a gene specific histone gene probes is given in the text and exemplified by the gested with Hha I plus HindIII [yielding an internal (coding) data shown in Fig. 2. fragment], the H2b gene from pSp2 (15) (after subcloning the two Sp2 BamHI/EcoRI fragments in pBR322) digested with BamHI plus Kpn I (yielding a 456-bp 5' end plus internal frag- aged by prior chromosomal blotting data which indicated that ment), the H3 gene from pSpl7 digested with Hha I (yielding the 1.9-kbp EcoRI fragment of pSpl7 (the S. purpuratus re- a 562-bp 5' end plus internal fragment), and the H4 gene from combinant containing the sea urchin H2a and H3 genes) hy- pSp2 digested with Hha I (yielding a 1-kbp 5' end-internal-3' bridized to only a small number ofchromosomal chicken DNA end fragment). These fragments were isolated from the gels by fragments (approximately 10; data not shown). In the first electrophoresis into Whatman 3 MM paper (20), recovered, and screening attempt of the A/chicken library (2 X 105 plaque- then nick-translated (21). The Drosophila histone gene probes forming units) approximately 50 plaques were selected that con- were derived from cDm500 restriction fragments (6, 22) as fol- tinually yielded phage that hybridized to the pSpl7 probe lows: cDm500 DNA was digested with restriction enzyme, through the plaque purification procedures to phage homo- treated with gel-purified calfintestine alkaline phosphatase, and geneity. then labeled at the 5' ends with 32P by using phage T4 polynu- The putative histone gene recombinant DNAs were digested cleotide kinase. The digests were then electrophoresed on 5% with EcoRI, BamHI, HindIII, and Kpn I (as well as all paired polyacrylamide gels (23). Restriction enzymes used and desired combinations ofthese), electrophoresed, stained with ethidium fragments were: Taq I A fragment (containing histone H2a, in- bromide, photographed, and blotted to nitrocellulose filters. By tergenic, and histone H4 sequences), Mbo II C fragment (con- comparison of the pattern of cleavage in single and paired re- taining histone H2b internal sequence), and the Hha I E frag- striction enzyme digests, the restriction maps of the three re- ment (containing histone H3 coding sequence). The cDm500 combinants shown in Fig. LA were generated. Some maps that bands were visualized by autoradiography, excised, and then could not be resolved in this way (e.g., the relative positions isolated by electrophoresis into hydroxyapatite (24). The hy- of the five small EcoRI fragments in ACH3d) were defined by droxyapatite was withdrawn, layered onto a 4-ml Sephadex G- the kinase labeling/partial digest method ofSmith and Birnsteil 50 column preequilibrated with E buffer (40 mM Tris base, 1 (18). mM EDTA, adjusted to pH 7.4 with acetic acid) and washed These initial restriction mapping results (Fig. 1) implied that with 4 ml of E buffer. The DNA was then eluted from the hy- if the H2a and H3 genes (those presumably selected by the droxyapatite with 0.6 M sodium phosphate, pH 7.3. pSpl7 radioactive screening probe) of the chicken were ar- Subcloning. A recombinants were digested with various re- ranged in tandem arrays as previously reported, they were not striction enzymes, then ligated to either pBR322 or fdlO6 rep- repeated in any obvious arrangement with respect to neigh- licative form vector DNAs (19, 25) that had previously been boring restriction enzyme sites. We therefore continued to digested with the same restriction enzymes and subsequently probe the isolated chicken recombinants with specific histone treated with purified calf intestine alkaline phosphatase (26). coding-sequence fragments of the sea urchin and Drosophila The DNAs were ligated (27), then used to transform or transfect histone DNAs to determine whether or not some common ele- E. coli HB101 or JM101, respectively; recombinants were se- ments of a tandemly repeated gene structure could be demon- lected by drug resistance characteristics. Ten-milliliter lysates strated. ofsubcloned recombinants were prepared and analyzed directly A/Chicken Histone DNA Recombinants Contain Core His- for inserted recombinant fragment sizes by restriction endo- tone Genes. The same histone gene recombinants mapped nuclease digestion and gel electrophoresis of these small-scale above were analyzed by hybridization of internal fragments plasmid isolations (28). (containing primarily or exclusively histone-coding DNA se- Miscellaneous. Nick-translation, kinase labeling of 5' ends, quence) of H2a, H2b, H3, and H4 genes derived from both and DNA sequence analysis were performed as described (21, cloned sea urchin and Drosophila histone DNAs. One of these 29). All work with recombinant DNA molecules was performed blots is shown in Fig. 2. as prescribed by the current NIH Guidelinesfor Recombinant The example depicted is a blot of32P-labeled Mbo II C frag- DNA Research. ment of cDm500 (6) which contains DNA sequences entirely within the Drosophila histone H2b gene (22), hybridized to a RESULTS mapping blot of chicken histone recombinant ACH2e. As can Isolation and Restriction Enzyme Mapping of A/Chicken be seen in the autoradiogram (Fig. 2 Right) of the blot derived Histone DNA Recombinants. As a first effort to isolate histone from the ethidium bromide-stained agarose gel (Fig. 2 Left), gene-containing recombinants, we screened the chicken library specific hybridization is seen to only one segment of ACH2e, with heterologous histone gene probes, having been encour- which is defined by a 3.35-kbp EcoRI restriction enzyme frag- Downloaded by guest on October 5, 2021 2858 Biochemistry: Engel and Dodgson Proc. Natl. Acad. Sci. USA 78 (1981)

R B H K B/R H/R K/R H/B K/B K/H R B H K B/R H/R K/R H/B K/B KH kbp 22 -

7.6 6.0 5.6 - 4.9 -. 3.5

2.3- 2.1

1.44- 1.31 -

0.91 - 0.66 -

FIG. 2. Hybridization of Drosophila histone H2b probe to the mapping blot of ACH2e. Radioactively labeled Drosophila histone recombinant cDm500 Mbo C fragment, containing only histone H2b coding sequences, was isolated and hybridized to ACH2e DNA that had been cleaved with EcoRI (R), BamHI (B), HindIH (H), and Kpn I (K) (as well as all paired combinations) and then blotted to nitrocellulose (6, 22). External markers used (not shown) were: A DNA cleaved with EcoRI (partial), pBR322 DNA digested with EcoRI plus Pvu II, pBR322 DNA cleaved with Taq I, and pBR322 DNA cleaved with Alu I (19). (Left) Ethidium bromide-stained gel. (Right) Autoradiograph of the blot hybridized to the cDm500 Mbo II C 32P-labeled probe (22 hr, 1 screen, -70C).

ment (Fig. 2 Right, lane R). Similarly, the positions ofother core and their sequences were determined directly. The results histone genes were defined by using other internal histone shown for a portion of one of the sequencing reactions dem- gene-coding sequence DNA fragments that were derived from onstrates the existence of a chicken histone H3 gene within the cDm500, pSpl7, and pSp2 as probe in these blots (data not 1.28-kbp EcoRI fragment of ACH3d (Fig. 3). shown). As can be seen in the summary of these hybridization The DNA sequence derived from fH3dR6 predicts an amino data (Fig. 1), the genes are clustered but cannot be arranged acid sequence identical to that of amino acids 52 through 69 in into an array of tandemly reiterated histone gene sequences. the calf thymus histone H3 (30). The sea urchin H3 gene DNA Preliminary mapping ofseveral other chicken histone gene-con- sequence also predicts the identical conserved amino acid se- taining recombinants confirms the absence of a tandemly re- quence in this portion of the gene; of the 10 nucleotide base peated sequence (data not shown). differences in the chicken H3 and sea urchin H3 genes in this A Single Chicken Histone H3 Gene Sequence. Despite the region, all lead to conservative codon changes. Eight of the 10 several precautions taken in these experiments to reduce the differences are in the third position of the codon; two adjacent possibility that the recombinants isolated by using the pSpl7 nucleotide changes occur which conserve amino acid Ser-57 probe were not histone gene-containing recombinants, that for- (TCC in chicken versus AGC in S. purpuratus). mal possibility still existed. We therefore felt compelled to dem- As further identification ofthis sequence as a bonafide struc- onstrate that our A recombinants indeed contain bona fide tural gene for histone H3 transcription, we have performed chicken histone genes by primary DNA sequence determina- hybridizations ofthe fH3dR6 radiolabeled recombinant to total tion. The 1.28-kbp EcoRI fragment of ACH3d appeared to be cytoplasmic RNA immobilized on nitrocellulose filters. The re- the most amenable to direct sequence determination, because sult ofthis experiment is that fH3dR6 specifically labels a single both the heterologous Drosophila and sea urchin probes hy- RNA band approximately 600 to 650 bases in length (unpub- bridized strongly and specifically to this fragment on mapping lished observations). The result is consistent with the pre- blots. An fdlO6 subclone of ACH3d containing the histone H3- sumption that the subclone fH3dR6 contains the coding DNA complementary 1.28-kbp fragment (fH3dR6) was cleaved with sequence for an expressed chicken H3 histone gene. Msp I and 32P-labeled with polynucleotide kinase, and then the Histone Genes Are Not Tandemly Repeated in the Chicken three unique Msp I fragments arising from within the recom- Genome. The H3 gene whose sequence was partially deter- binant portion were isolated. These were secondarily cleaved mined (fH3dR6) was used to determine if the histone genes with Hha I, the six singly end-labeled fragments were isolated, actually were not tandemly arrayed in the chicken genome or Downloaded by guest on October 5, 2021 Biochemistry: Engel and Dodgson Proc. Natl. Acad. Sci. USA 78 (1981) 2859

fH3dR6 DNA CGG CGC TAC CAG AAG TCC ACG GAG CTG CTG ATC CGC AAG CTG CCC TTC CAG CGG sequence:

Histone H3 55 60 65 amino acid Arg Arg Tyr Gln Lys Ser Thr Glu Leu Leu Ile Arg Lys Leu Pro Phe Gln Arg sequence:

Histone H3 CGC CGC TAC CAG MG AGC ACT GAG CTT CTC ATC CGA AAA CTG CCA TTC CAG CGT DNA sequence:

FIG. 3. Partial DNA sequence ofhistone H3 gene in ACH3d. Depicted are: (Top) the DNA sequence of an Msp I/Hha I fragment derived from thefdlO6 subclone (fH3dR6) containingthe 1.28-kbpEcoRIfragmentofACH3d (adjacent to the left linker in Fig. 1); (Middle) the amino acid sequence of histone H3 of calf thymus for amino acids 52 to 69 (30); (Bottom) the DNA sequence of the same region of histone H3-coding DNA from the S. purpuratus recombinant pSpl7 (15). whether, for example, the tandem repeats were much larger library (14)], an observation that also does not appear to support than the segments isolated, giving rise to the nonoverlapping the earlier claim oftandem repetition ofthe histone genes (32). genomic segments represented by ACHla, ACH2e, and ACH3d With regard to the present findings, it is of interest that (Fig. 1). It was previously reported that the chicken histone whereas the histone genes are repeated 100 times in the Dro- gene cluster was repeated in a 15-kbp repeated tandem array sophila genome (6), the histone genes of the chicken are re- as detected by chicken histone cDNA-probed chromosomal peated in the genome only about 10 times (10). This is similar Southern blots (10). We repeated this experiment, using the same restriction endonucleases as those used in the previous A B C D E F G H study (EcoRI, BamHI, Bgl II, and HindIII) and probed with the partially sequenced subclone containing the histone H3 gene, kbp fH3dR6. As can be seen in the autoradiogram presented in Fig. 4, the chicken H3 gene probe hybridizes to anumber ofdistinct bands in chicken chromosomal DNA when each ofthe four restriction endonucleases is used. The clearest example of the dispersed 22 structure of the H3 genes is shown in Fig. 4, lane A. The H3 gene probe in this example hybridizes to nine distinct chromosomal EcoRI fragments, ranging in size between 1.3 kbp and 10.5- perhaps 30 kbp. This implies that (as a lower limit) there exist nine copies of this particular chicken histone H3-coding sequence (or one very closely related) in the genome, but further, 6.3- that the H3 genes are not tandemly arrayed (at least with respect 4.9- to the cleavage sites ofthe enzymes used). The histone H3probe fH3dR6 also hybridized specifically and with high affinity to the mapped locus within ACHla, implying that other H3 genes initially identified in this and other A recombinants by using heterologous probes are structurally quite similar to the histone H3 gene in ACH3d (data not shown). 2.1 - DISCUSSION The single most significant result of this work is the demon- stration, by direct gene isolation, mapping, and DNA sequence 1.3- analysis, and from Southern blot analysis, that the chicken histone genes are not repeated in tandem arrays. We have several possible explanations for this disparity between our results and those of Crawford et al. (10); we feel the most likely one arises 0.66 - from the fact that the previous study used cDNA derived from isolated chicken histone mRNA to probe the chromosomal blots, whereas in this study we used a probe ofknown sequence that was initially selected by using a sea urchin histone DNA sequence. If the sea urchin DNA-selected probe used in this FIG. 4. Hybridization of chicken H3 gene to chromosomal DNA. report and the embryonic histone mRNA used in the previous The ACH3d recombinant subclone containing the partially sequenced study are homologous to structurally different histone gene cod- H3 gene (fH3dR6; Fig. 3) was nick-translated (21) and then hybridized ing sequences (for example, adult and embryonic gene sets, to (lanes A-D) a blot ofchromosomal chicken erythrocyte'DNA cleaved respectively), this would cause a difference in the chromosomal with EcoRI (A), BamHI (B), Bgl II (C), and HindIH (D); lanes E-H are the same digests, respectively, hybridized to the adult ft3globin gene blotting pattern of the two different probes. However, we .chromosomal recombinant p,81BR15. Autoradiographic exposure times should note that, in a subsequent paper, the same laboratory were 30 hr and 2 hr, for lanes A-D and E-H, respectively. Sizes for that reported a tandem histone gene array in the chicken restriction fragments were determinedby hybridization tothe (-globin showed the map and sequence arrangement ofa single histone chromosomal recombinant (14, 31) and by comparison to published A gene recombinant similar to ACH2e [isolated from the identical and pBR322 DNA fragment sizes (see legend to Fig. 2). Downloaded by guest on October 5, 2021 2860 Biochemistry: Engel and Dodgson Proc. Natl.. Acad. Sci. USA 78 (1981) to the gene repetition frequency in mouse (11) and HeLa (hu- 5. Arceci, R. J., Senger, D. R. & Gross, P. R. (1976) Cell 9, 171-178. man) cells (12) and-therefore may be exemplary of a different 6. Lifton, R. P., Goldberg, M. L., Karp, R. W. & Hogness, D. S. kind ofgenomic organization ofthe histone genes ofhigher eu- (1977) Cold Spring Harbor Symp. Quant. Biol. 42, 1047-1056. karyotes as compared with that demonstrated in flies and sea 7. Kedes, L. H. & Birnsteil, M. L. (1971) Nature (London) New urchins; Biol. 230, 165-171. Why might this disparity in histone gene number exist in the 8. Weinberg, E. S., Birnsteil, M. L., Purdom, I. F. & Williamson, various species listed? One intriguing possibility is suggested R. (1972) Nature (London) 255, 240. various discussed above. 9. Grunstein, M., Shedl, P. & Kedes, L. (1973) in Molecular Cy- by the development of the organisms togenetics, eds. Hamkalo, B. A. & Papaconstantinou, J. (Plenum, Both Drosophila and sea urchins exhibit extremely rapid DNA New York), pp. 115-123. replication and cell division during early development (33, 34), 10. Crawford, R. J., Krieg, P., Harvey, R. P., Hewish, D. A. & whereas the latter organisms discussed (chicken, mouse, hu- Wells, J. R. E. (1979) Nature (London) 279, 132-137. man) have a much more moderate embryological cell division 11. Jacob, E. (1976) Eur. J. Biochem. 65, 275-282. time. Because normal histone synthesis is obligatorily coupled 12. Wilson; M. C., Melli, M. & Birnsteil, M. L. (1974) Biochem. DNA it that DNA Biophys. Res. Commun. 61, 404-410. to synthesis, follows rapid synthesis requires 13. Scott, A. C. & Wells, J. R. E. (1976) Nature (London) 259, rapid histone synthesis, which might therefore require manifold 635-640. duplication ofthe histone genes. We would therefore speculate 14. Dodgson, J. B., Strommer, J. & Engel, J. D. (1979) Cell 17, that the histone genes that have been studied in the greatest 659-668. detail in Drosophila and sea urchins (those that are most abun- 15. Sures, I., Lowry, J. & Kedes, L. H. (1978) Cell 15, 1033-1041. dant and tandemly repeated), are in fact, responsible for histone 16. Engel, J. D. & Dodgson, J. B. (1980) Proc. Natl. Acad. Sci. USA this cell division in 77, 2596-2600. gene expression during rapid early embry- 17. Engel, J. D. & Dodgson, J. B. (1978) J. Biol. Chem. 253, onic development, in analogy to the oocyte-specific 5S genes 8329-8355. ofXenopus (35). 18. Smith, H. 0. & Birnsteil, M. L. (1976) Nucleic Acids Res. 9, Finally, we note that we have not yet examined these re- 2387-2399. combinants for the presence of histone genes H1 or H5. Of 19. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. particular interest to us is the location and arrangement of the 43, 79-85. H5 histone of the pre- 20. Girvitz, S. C., Bacchetti, S., Rainbow, A. J. & Graham, F. L. erythropoiesis-specific genes chicken, (1980) Anal. Biochem. 106, 492496. viously reported to be present in the chicken genome at a copy 21. Maniatis, T., Jeffrey, A. & Kleid, D. G. (1975) Proc. Natl. Acad. number (about 10 per haploid genome) equal to the standard Sci. USA 72, 1184-1189. histone gene set (13). In a single preliminary experiment, we 22. Goldberg, M. (1979) Dissertation (Stanford-Univ., Stanford, CA). were not able to demonstrate the linkage of H5 to any of the 23. Maniatis, T., Jeffrey, A. & van de Sande, H. (1975) Biochemistry other histone gene clusters herein described. However, we 14, 3787-3796. to that H5 is or 24. Tabak, H. F. & Flavell, R. A. (1978) Nucleic Acids Res. 5, have not been able demonstrate unequivocally 2321-2330. is not physically linked to or interspersed within other isolated 25. Hermann, R., Neugenbauer, K., Pirkl, E., Zentgrof, H. & gene clusters. Schaller, H. (1980) Mol. Gen. Genet. 177, 231-236. 26. Lacy, E., Hardison, R. C., Quon, D. & Maniatis, T. (1979) Cell We thank N. Davison (California Institute ofTechnology), in whose 18, 1273-2381. laboratory the initial phage screening was carried out, for generous sup- 27. Dugaczyk, A., Boyer, H. & Goodman, H. M. (1975)J. Mol. Biol. port; I. Sures, M. Grunstein, and D. Hogness for strains and unpub- 96, 171-197. lished restriction maps; N. Heintz, R. Roeder, L. Kedes, and L. Here- 28. Klein, R. D., Selsing, E. & Wells, R. D. (1980) Plasmid 3, 88-99. 29. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, ford for critical and informative discussion; and E. Sikora for technical 499-536. assistance. J. B. D. was supported by the National Institutes of Health 30. Dayhoff, M. O., ed. (1972) Atlas of Protein Sequence and Struc- (Grant GM28837) and by the Leukemia Research Foundation. J. D.E. ture (National Biomedical Research Foundation, Washington, acknowledges support from the Leukemia Research Foundation and the DC), Vol. 5. National Institutes of Health (Grants HL24415 and CA15145). 31. Dolan, M., Sugarman, B. J., Dodgson, J. B. & Engel, J. D. (1981) Cell, in press. 1. McGhee, J. D. & Felsenfeld, G. (1980) Annu. Rev. Biochem. 49, 32. Harvey, R. P. & Wells, J. R. E. (1979) Nucleic Acids Res. 7, 501-549. 1978-1994. 2. Mathis, D., Oudet, P. & Chambon, P. (1980) Prog. Nucleic Acid 33. Rabinowitz, M. (1941)J. Morphol. 69, 1-21. Res. Mol. Biol. 24, 2-49. 34. Hinegardner, R. T. (1967) in Methods in Developmental Biology, 3. Kedes, L. H., Cohn, R. H., Chang, A. C. Y. & Cohen, S. N. eds. Wilt, F. H. & Wessells, N. K. (Crowell-Collier, New York), (1975) Cell 6, 359-369. 139-155. 4. Weinberg, E. S., Overton, G. C., Shutl, R. H. & Reeder, R. H. 35. Long, E. 0. & Dawid, I. B. (1980) Annu. Rev. Biochem. 49, (1975) Proc. Nati. Acad. Sci. USA 72, 4815-4819. 727-763. Downloaded by guest on October 5, 2021