GENOMICS

10,940-948

Isolation

(1991)

and Characterization of Two Human HI Histone within Clusters of Core Histone Genes

WERNER ALBIG,*

EFTERPI KARDALINOU,

Genes

* BIRGIT DRABENT, * ANDREAS ZwdMm, 7 AND DETLEF DOENECKE*

‘Institut fiir Biochemie, Universitat Gottingen, Humboidtallee 23, O-3400 GcSttingen; and t Max-Pianck institut filr Experimenteiie Medizin, Abt. Moiekuiare Neuroendokrinoiogie, Hermann-Rein-Strasse, O-3400 Gbttingen, Federal Republic of Germany Received

December

27, 1990;

Press, Inc.

INTRODUCTION

The Hl class of histones contributes to the structure of chromatin at several levels of organization. It is a prerequisite for sealing two rounds of DNA around the nucleosomal histone octamer (Simpson, 1978; Allan et al., 1986) and it is needed for the formation of higher order chromatin structures above the level of nucleosomes (Thoma et aZ., 1979). Each histone class consists of several subtypes, with the probable exception of H4. The Hl group represents the most complex pattern of histone isoforms. Based on two-dimensional gel electrophoresis data, five main types of Hl histones were defined and termed Hla-Hle (Lennox and Cohen, 1983). Another subtype, Hl”, is restricted mainly to terminally differentiated tissues (Panyim and Chalkley, 1969), whereas a testicular Hl histone (Hit) is found only at specific stagesof sperm development (Seyedin et al., 1981; Cole et al., 1984, 1986). Varied patterns of Hl histones in different tissues suggest a functional role for individual Hl subtypes, which may influence higher order chromatin strucSequence data EMBL/GenBank M60752.

0888-7543/91

Copyright All rights

0 of

$3.00 1991

from this article have been deposited Data Libraries under Accession Nos.

by Academic Press, Inc. reproduction in any form reserved.

April

1, 1991

tures by modulating the cooperative interactions between Hl histones (Weintraub, 1984). Histone genes are organized as clusters (Hentschel and Birnstiel, 1981; Maxson et al, 1983). Such groups of histone genes can be highly regular tandem repeats of the five histone genes, or the individual histone classes can be randomly organized within clusters. Varied numbers of histone genes in the mammalian genome have been described (Stein et al., 1984). Mapping of histone gene clusters, hybridization analysis, and sequencing demonstrated the heterogeneity of histone gene clusters and the differences between nucleotide sequences of individual histone genes of the human and murine genomes. A lack of any defined histone gene repeat pattern has been similarly found in avian histone gene clusters. D’Andrea et al. (1985) demonstrated that the chicken histone gene complement is essentially distributed in three different clusters containing 40 histone genes including six Hl genes (coding for the subtypes Hla-d and two additional variants Hla’ and Hlc’; Shannon and Wells, 1987). A gene arrangement similar to those of the chicken histone gene clusters was found in the duck genome, where again all five histone genes were represented in this cluster of genes (Tiinjes and Doenecke, 1987). The number of human histone gene clusters is unknown. Chandler et al. (1979) hybridized cloned sea urchin histone gene probes to human metaphase chromosomes and found hybridizations only on the long arm of chromosome 7. Later, Triputti et al. (1986) screened a panel of mouse-human hybrid cells and found positive signals on chromosomes 1, 6, and 12. This chromosome assignment was confirmed by in situ hybridization of labeled histone gene probes to metaphase chromosomes. This localization on individual chromosomes describes the histone gene distribution in general, but individual dissimilar clusters may be found within these large regions. The information on human histone gene clusters is still in-

Two human II1 histone genes, termed H1.3 and H1.4, were isolated from two cosmid clones. The H1.4 gene is associated with an HZB gene, whereas genes coding for all four core histones are located in the vicinity of the H1.3 gene. This cluster arrangement was found both in the two cosmid clones and on overlapping bacteriophage clones isolated from an EMBLB library. In continuation of our previous analysis of two human Hl genes, this analysis raises the number of completely sequenced Hl histone genes within clusters of core histone genes to four. o 1~1 Academic

revised

with the M60746-

940

HISTONE

GENE

CLUSTERS

complete. Most portions of histone gene clusters previously described showed several core histone genes, but Hl genes were missing in most cases. Carozzi et al. (1984) and Zwollo et al. (1984) were the only authors to detect all five histone genes on continuous or overlapping genomic DNA clones. The Hl genes participating in these clusters were identified in one case by sequencing an N-terminal segment of the Hl gene (Carozzi et al, 1984), whereas Zwollo et al. (1984) showed only Southern blot hybridization data. In our attempt to characterize the human Hl gene complement, we first described the Hl” gene (Doenecke and Tiinjes, 1986) and, recently, we published the gene structures of two main-type Hl histones (Eick et al., 1989). Previously, the only known human Hl sequence was based on protein sequencing (Hlb; Ohe et al,, 1986), except for the partial sequence derived from a human Hl gene by Carozzi et al. (1984). Both newly described gene sequences differed from these data and from Hla, Hlc, and Hld, which have been published in the meantime (Ohe et al,, 1989). The nomenclature used by Ohe et al. was based on criteria other than the designation of subtypes Hla-e by Lennox and Cohen (1983). To avoid any confusion, we decided to term the newly discovered H1 genes with consecutive numbers as H1.l and H1.2 genes (Eick et al., 1989). Here, we describe the gene and the surrounding structures of two additional Hl gene sequences, termed H1.3 and H1.4, together with neighboring core histone gene sequences. MATERIALS

Screening of a Human Cosmid Library

AND

METHODS

Genomic Phage Library and a

A human genomic library in EMBL3 bacteriophage was obtained from Clontech (Palo Alto, CA). The library had been constructed from human granulocyte DNA and was screened using the in situ plaque hybridization technique described by Benton and Davis (1977). After three rounds of purification, five recombinant bacteriophage were isolated and the phage DNA was prepared (Sambrook et al., 1989). A cosmid library, which had been prepared in the laboratory of A. M. Frischauf (London) with DNA from human blood cells using pcos2EMBL as a vector (Ehrich et al., 1987), was screened as described by Herrmann et al. (1987). Two cosmid clones were isolated and analyzed as described (Herrmann et al.,

1987). In both cases, a DNA fragment prepared from the coding portion of the human H1.2 gene (Eick et al., 1989) was taken as a hybridization probe and labeled with [a-32P]dATP according to Feinberg and Vogel-

INCLUDING

Hl

941

GENES

stein (1983) using the Amersham random prime labeling kit.

Mapping and Subcloning Insert DNA

of the Phuge and Cosmid

DNA was prepared from the individual cosmid and phage clones, cut with several restriction enzymes and combinations thereof, separated electrophoretitally and analyzed by Southern blot hybridization (Southern, 1975). Hybridization probes were prepared from the coding portions of the human H1.2 gene (Eick et al, 1989) and individual core histone genes (Eick and Doenecke, unpublished). These probes were labeled with [u-~~P]~ATP by random priming (Feinberg and Vogelstein, 1983). Hybridizations were done at 64°C in 4X SSC (1X SSC: standard saline citrate, 0.15 M NaCl, 0.015 M Na citrate) in 5~ Denhardt’s solution (Denhardt, 1966). DNA fragments were isolated and subcloned in pUC19 plasmid, and the Escherichia coli K12 derivative DH5a was used as a host strain. Plasmid DNA was prepared as described (Birnboim and Doly, 1979; Sambrook et aZ., 1989).

DNA Sequence Analysis and Sl Nuclease Assay The subcloned fragments derived from the phage and cosmid clones were sequenced according to Sanger et al. (1977) using the T7-sequencing kit provided by Pharmacia (Uppsala, Sweden) under the conditions described by the manufacturer for sequencing short and long distances relative to the primer sites. The 35S-labeled sequencing reaction products were separated on 6% polyacrylamide gels (Maxam and Gilbert, 1977) and exposed to Kodak X-ray film. Each of the sequences presented in this paper was obtained by analyzing both DNA strands. The mRNA start sites of both Hl genes were determined by Sl nuclease mapping using the protocol of Weaver and Weissmann (1979). RNA was prepared from the human monocytic U937 cell line (Sundstriim and Nilsson, 1976) as described by Chirgwin et al. (1979) and hybridized to appropriate end-labeled, single-stranded DNA fragments, and the resulting hybrids were exposed to Sl nuclease. Protected DNA was analyzed by electrophoretic comparison with a G/A sequence ladder (Maxam and Gilbert, 1977).

Materials

and Enzymes

Restriction enzymes were from Boehringer (Mannheim, FRG), Bethesda Research Laboratories (BRL, Gaithersburg, MD), and New England Bio-

942

ALBIG EH

EEH

E

H

H EEB

ET

AL.

EE

HZB H2A

E

H3 cormi

‘

Ii

HH

E

Ill.3

E

H4

CS

J phage

L phage

,

BHHE

Hl2

012 lkb -

e

HSHII

BE

BE

H2B cormid

I

C3 phage

phage

I

F12

1

El4 phage

0

B

HI.4

Jll

FIG. 1. Chromosomal arrangement of histone genes within two gene clusters. Overlapping inserts of cosmid indicated below the restriction site maps. Coding sections and transcription directions are indicated by arrows. sites are abbreviated: E, EcoRI; H, HindHI; B, BarnHI; S, SaK

labs (Beverly, MA). DNA-modifying enzymes were from Boehringer and BRL and the T7-sequencing kit was from Pharmacia (Uppsala, Sweden). Radioactively labeled nucleotides were from AmershamBuchler (Braunschweig, FRG). Filter materials were from Schleicher & Schuell (Dassel, FRG) and Amersham-Buchler. Chemicals for electrophoresis were from Serva (Heidelberg, FRG) and Sigma (Munich, FRG). All other chemicals were from Merck (Darmstadt, FRG). RESULTS

AND

DISCUSSION

Isolation of Two Human Histone Containing HI Genes

Gene Clusters

We have isolated two groups of histone genes upon screening a human cosmid library and an EMBLS phage library with a human Hl histone gene probe (termed H1.2 in Eick et al., 1989). Five recombinant phage and two cosmids were isolated and the insert DNA segments were mapped using Hl and core histone probes (composite drawing in Fig. 1). The phage insert sizes ranged between 12 and 19 kb, whereas one of-the cosmid inserts was 30 kb in length. Surprisingly, the second cosmid insert measured just 6 kb. Since this insert size is too short to allow packaging of insert and cosmid vector in the bacteriophage particle, this short insert may have bypassed the packaging selection due to vector concatemerization. The large cosmid insert (designated C5) harbored one gene for each of the five histone classes, except an H4 gene. An H4 gene, however, was covered by an overlapping bacteriophage insert (phage H12), which had been isolated using the same Hl probe as that

and bacteriophage clones are Restriction enzyme cleavage

used for cosmid library screening. Comparison of the composite restriction map of these cosmid and phage inserts with the data of Zwollo et al. (1984) suggests that these clones may share common sites of the genome. Lack of sequence data and lack of fine mapping in the previous data, however, prohibit a formal proof. The small cosmid insert (C3) organization was identically found on three overlapping phage clones (B14, F12, and Jll; seeFig. 1). Thus, this surprisingly short cosmid insert covered an authentic genomic DNA fragment showing an Hl gene and, with the same transcriptional orientation, an H2B gene.

Sequences of Core and HI Histone

Genes

Each of the Hl and core histone genes indicated in Fig. 1 was sequenced and compared with published sequence data. Figures 2 to 8 show the sequences of the coding and flanking sequences of these seven genes. According to Franklin and Zweidler (1973), individual core histone subtypes may be discriminated on the basis of a few amino acid exchanges. According to this nomenclature, the H2A, H2B, and H3 genes described here may be defined as H2A.1, H2B.1, and H3.1 genes. The two H2B.l genes, however, which are located near the H1.3 and H1.4 genes, respectively, differ at one amino acid residue (Ala/Thr exchange at position 4). The amino acid sequence encoded by the H4 gene is identical to known mammalian H4 sequences (Wells and McBride, 1989). Each of the seven genes features the 3’ consensus sequence which is the site of histone mRNA precursor processing and formation of the mature histone mRNA 3’ end (Hentschel and Birnstiel, 1981; Hoffmann and Birnstiel, 1990). Several specific sequence motifs upstream of spe-

HISTONE

GENE

CLUSTERS

GAAAGAGAGT

TGGGGGAATA AGMAAGTTT

W~TGCCG

GTTTTAAAAC

TATGCATAGA

MCAMGGCA

GGAATGMGC

CCGMCTCTC

TCG~T~GT

TTCCCCCMG

TCTCTAATTA

TTTCATGCCC

AGATTTCTTA

T A T T T T T A C T CTTTTTTMG

GGGCMCAAA

CACAGCAGCG

CGGTAGATAC

GAGGAGTCCT

TTTCCAGCAG

1 GGAGCAAGGA

ACCMTCATC

ACTCAGCGTC

TCTCJTATSCCCTCAGC

GAGGA&TGC

TGTTCTGACA

GTTTGAGATT ACTTATTGlt

CGCCGCGCAT

GGG AAA CCC MA Gly Lys Arg Lys MG

AAA

Lys

GCA TCC GGA CCC CCA GTA TCT GAG CTT ATC ACC Ala Ser Gly Pro Pro Val Ser Glu Leu Ile Thr 45

GCA GTG GCA GCT TCT MC Val Ala Ala Ser Lys

GCG CTT MG MA Leu Lys Lys

Ala

MC

Asn

Comparison 15

AAG CCC AAG AAG GCA GGC GCA ACT GCT Lys Ala Lys Lys Ah Gly Ala Thr Ala 30

LyS Ala

GAG CGC AGC GGC G T T TCT CTG GCC GlU Arg Ser Gly VSl Ser Leu Ala 6o

GCG CTT GCG GCT GCT GGC TAC GAT GTA GM AAA Ala Leu Ala Ala Ala Fly Tyr Asp V&l Glu Lys '5

MC AGC CGT ATC AAG CTT GGC CTC MC Asn Ser Arg Ile Lys Leu Gly LB" Lys

AGC TTG GTG AGC AM Ser Leu Vdl Ser Lys

9o

GGT ACT CTG GTG CAG ACC AAA GGT ACC GGT GCT TCT GGC TCC TTC Gly Thr Leu Val Gln Thr Lys Gly Thr Gly Ala Ser Gly Ser PhP"' AM Lys

CTC MC Leu

Asn

GCT GGC GCA GCC MG CCT AGG MG Ala Gly Ala Ala Lys Pro Arg Lys

AAG MG Lys Lys

CCC AAG MG Pro Lys Lys

ATC MA Ile Lys

MG

ACT CCT MG Lys Thr Pro Lys

CAG CCA AAA AM MG

Lys

Lys

AAG GTA AAG AAG CCA GCA ACC GCT GCT

Lys

Lys

Pro

Ala

Ala

MG

GCA AAG MG Ala Lys Lys

Ala

Lys

MCTGGCGGG

ACGTTCCXT

TCTCAGTCAA AAGAGCTGAA

Val

Lys

Lys

Pro

Lys

Ala

Thr Ala

Ala"'

AGT GCG AAA AAG GTG AAA ACA CCT Ser Ala Lys Lys Val LyS Thr P=dao

GCT GCC AAG AGT CCA GCT MG Ala Ala Lys Ser Pro Ala Lys

CCC AAG GCG GCC MG

Lys

CCT GCT GGG GCA GCC Pro Ala Gly Ala Ala1'5

GTG GCT GGC GCC GCT ACC CCG MG AAA AGC Val Ala Gly Ala Ala Thr PP.3 LyS Lys SerI"

GGG ACC MG AAA GTG GCC MG Gly Thr Lys lys Val Ala Lys Pro

and the require subtypes subtypes 1984).

of Human

HI Histone

Genes

Comparison of the Hl amino acid sequences derived from the H1.3 and H1.4 genes shown in Figs. 2 and 3 with protein sequencing data published by Ohe et al. (1986,1989) indicates that these genes code for the subtypes Hlc (H1.3) and Hlb (H1.4) as defined by Ohe et al. (1989) on the basis of sequencing spleen Hl histones. By Sl nuclease analysis with RNA from the human mono&c cell line U937, we observed a full protection of the coding and 5’ flanking DNA up

AAG AAA GCG GCT TCC GGG GM GGC AAA CCC MG GCC Lys Lys Ala Ala Ser Gly Glu Gly LyS Pro LyS Ala"o

AM MG Lys Lys

Gln

943

GENES

CTCCCTCGTA

TTTTCTGGGA AGA-C

ATG TCG GAG ACT GCT CCA CTT GCT CCT ACC A T T CCT GCA CCC GCA GM Ser Glu Thr Ala Pm Leu Ala Pro Thr Ile Pro Ala Pro Ala Glu AAA ACA CCT GTG MG Lys Thr Pro Val Lys

Hl

ing and flanking sequences. The Hl genes derived protein primary structures, however, a detailed discussion, since individual Hl differ substantially and the pattern of Hl may be functionally important (Weintraub,

H1.3 CTTCGATTTG ACCGAAGTTT

INCLUDING

CCT MC Pro Lys

TCG GGG MG Ser Gly Lys

GCC AAA GCC CCT Ala Lys Ala Prd9' CCG MC

Pro

LyS

GTT ACA Val Thszo

TT;TCAGAGC

TGCTTCCCCT

TATMTTTGA

GAAAAACCGG

GCAGTTCGGT GTAGACAATT TTTATATTTT TGGCTTTTTT TGAGGTGTM

CAAACACMC 7 CGCACCAATC

ACGGCGCACG

TCCGCCCTAT ATAAACGGGC

2 GGGCGCAGCG CCGCGGCTCG

k&C

AGTGCCTCTG

CTTCCGGCTC

CGCTCACGCT

CACCCACAGG

GCTTTTTGGA GGGGGGAGTG GGGTGGAGAG GGGTGCTGCG

GTGTTGTGCG GCCACGGTCT ATCCTAGTCG TGCTGGTTGG GGGTCAGTTA TTAACAGCTC

FIG. 2. Nucleotide and.amino acid sequences of human histone H1.3 gene and protein, respectively. The mRNA-like strand is presented in 5’to I’direction (the same in Figs. 3-7). Consensus regulatory elements are marked: 1, Hl box; 4, CCAAT element; 5, TATA box; 6, 3’ stem-loop structure, followed by’ACCCA (mRNA terminus).. The cap site as determined by Sl nuclease mapping with RNA from the human cell line U937 is indicated by an open arrowhead. The conserved octapeptide TPKKVKKP starts at amino acid 154.

GTTCGGGGAT CATTATCT~

&ATTGCTCT

TGCCTTmc

GAG ACT GCG CCT CCC GCG CCC GCT GCT CCG GCC CCT GCC GAG Se= GlU Thr Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala GJ" 11

ATG WC MG

ACT CCC GTG MC

Lys

AAG MG

Lys

Lys

GCC CCC MG Ala Arg Lys

TCT GCA GGT GCG Gee Ser Ala Gly Ala Ala JO

MG CGC AAA GCG TCT GGG CCC CCG GTG TCC GAG CTC A T T ACT AM L!jS A=9 LY= Ala Ser Gly Pro Pro Val Ser Glu Leu Ile Thr LYS (5 GCT GTT GCC GCC TCC MG GAG CGC AGC GGC GTA TCT T T G CCC GCT Ala V*l Ala Ala Ser LyS Glu Arg Ser Gly Val Ser Leu Ala Ala 60 CTC MG AAA GCG CTG GCA GCC GCT GGC TAT GAC GTG GAG AAA MC L*U LY= LY= Ala Leu Ala Ala Ala Gly Tyr ASP Val Glu Lys Asn '5 MC AGC CGC ATC MG CTG GGT CTC AAG AGC CTG GTG AGC MC GW Asn Se= Arg II* Lys Leu Gly Leu Lys Ser Leu Val Ser Lys Gly 90 ACC CTG GTG CAG ACC MG

Thr LeU

Val

Gin

Thr Lys

GGC ACC GGC GCG TCG GGT TCC TTC AAA Gly Thr Gly Ala Ser Gly Ser Phe Lyso'

CTC MC MG AAG GCG GCC TCT GGG GM GCC MG CCT MG GCT AAA Asn LYS Lys Ala Ala Ser Gly Glu Ala Lys Pro Lys Ala Lyslo

Leu MG

Lys

GCA GGC GCG GCC AAG GCC AAG AAG CCA GCA GGA GCG GCG MG Ala Gly Ala Ala Lys Ala tys Lys Pro Ala Gly Ala Ala Lysll'

AAG CCC MG AAG GCG ACG GGG GCG GCC ACC CCC MG MO AGC GCC LyS P=O LyS Lys Ala Thr Gly Ala Ala Thr Pro Ltjs Lys Se= Ald"O MG

Lys

cific histone genes. have been described as binding sites for tram-acting factors involved in histone gene expression. Specifically, regulatory elements have been described upstream of Hl (Coles and Wells, 1985), HZB (Sive and Roeder, 1986), and H4 (Capasso and Heintz, 1985; Clerc et al., 1983) genes. Each of these elements was found in the respective genes and is underlined in the sequences. Coles and Wells (1985) have shown that a GC-rich element (Spl site) is frequently located upstream of vertebrate Hl genes. It is found in the promoter region of H1.4 (Fig. 3) and H1.l (Eick et al., 1989), but not in the H1.3 and H1.2 gene promoters. Thus, the seven genes described here essentially correspond to consensus histone gene cod-

GATCTGCGTG AAGCCTGAGG

TCGGGATCCG AGAGGACACT CTGCGGCTGC CAGCGAGGCG GGCTGaaG

LYS Th= P=O V*l

GCA GCT CCG MG AAA MG TGA Al& Pro LyP LyS Lys *I* TTGAAAATTT TAAACGGCTC

En.4

AAG ACC CCA MG AAG GCG MG LYS Thr Pro Lys LyS Ala Lys

MG

Lys

CCG GCT GCA GCT GCT GGA Pro Al& Ala Ala Ala Gly'65

GCC AAA AAA GCG AAA AGC CCG AAA AAG GCG AAA GCA GCC MG CCA LYS LYS Ala Lys Ser Pro Lys Lys Ala Lys Ala Ala LyS PrdsG

Ala

AAA AAG GCG CCC AAG AGC CCA GCG MG Lys Lys Ala PTO Lys Ser Pro Ala Lys MG

GCC AAA GCA GTT CCC Lys Ala Vdl Lys Prde5

Ala

GCG GCT AAA CCA AAG ACC GCC AAG CCC AAG GCA GCC MG Ala Lys Pro Lys Thr Ala Lys Pro Lys Ala Ala LyS

Lys Ala

AAG AAG GCG GCA GCC MG AAA AAG Lys Lys Ala Ala Ala Lys Lys Lys AAAGTTCCTT TGGCCMCTG ACCCACCGCT

CTCAGTAAAA

TGCTMGCAG

GG

CCA

Prd'O

TAG l

**

CTTAGAAGCC CAKACMCC

CAAAGGCTCT TTTCAGAGCC sGAGCTGTTGC ACTATTAGGG GGCGTGGCTC GGGAMACGC

FIG. 3. Nucleotide and amino acid sequences of human histone H1.4 gene and protein, respectively. Consensus regulatory elements are: 1, Hl box; 2, Spl site; 4, CCAAT element; 5. TATA box; 6, 3’ stem-loop structure, followed by ACCCA (mRNA terminus). Cap site: open arrowhead (determined as in Fig. 2 by Sl nuclease assay). The conserved octapeptide TPKKAKKP hrts at position 152.

944

ALBIG

H2A.I

TCG ACC MG TCC GTAS' TGA CTT AGC TGG TTC AGG CAT

)‘ACT

GM

GCTGTCAGAAA

ACAATAACAG

CAGTGAGMT

GAACGCACTT

MATAMAGC

TCGTGTCTAG AGTCTCTCCT

TTTATAGGCC

TTTCATGCM

ATAAAGMTT

CAMATATCC

AGCTCTGATT

GGGCMTGTG

TTAGTGACGC

ATACATGTM

AATAGCCTTC

ACCTTATTTC

CTTTCTMTT

GGTTGGCTCG TCMAGAACA

ATCAAATTGC GAGCCCATTC

GCCTTTCACA

ATTCTACCGA

TGACTATAAC

T T T T T C T T T A TTCAGTGGAT T G T T Z T

ATTTTMCCA a TAGCTTCTTA TTCCTCCATC TCTGCTGTTA GGAAGCCACT

ATG TCT GGA CGT GGA AAG CAA GGC GGC AAA GCT CGG GCA AAA GCT AAA Ser Gly Arg Gly Lys Gin Gly Gly Lys Ala Arg Ala Lys Ala Lys 15 ACG

Thr

CGT TCT TCC Arg Ser Ser

AGG Arg

GCC Ala

His

CCC Arg

CTC CTC CGC MA Leu Leu Arg Lys

GGC Gly

GCT Ala

CCA GTG TAC Pro Val Tyr

CAC

GGT CTT CAG T T T CCA G T T GGC CGT GTG Gly Leu Gin Phe Pro Val Gly Arg Val 30 GGC MC Gly Asn

TAC

Tyr

TCC GM Ser Glu

CTG GCA GCG GTG CTG GM Leu Ala Ala Val Leu Glu

GAG ATC T T A GAG CTA GCT GGC AAC GCG GCT Glu Ile Leu Glu Leo Ala Gly Asn Ala Ala

CGA GTC GGG GCC Arg val Gly Ala '5 T A T CTG Tyr Leo

ACG GCC Thr Ala (Q

CGC GAC MT AAG AAG Arg Asp Asn Lys LyS 6o

ACC CGC ATC ATC CCG CGC CAC CTG CAG CTA GCC ATC CGC MC Thr Arg Ile Ile Pro Arg His Leo Gln Leu Ala Ile Arg Am GAG GAG CTA GlU Glu Le"

MT AAG CTT CTA GGT Asn Lys Le" Le" Gly

GGT GTC CTG CCC MC Gly Val Leu Pro Asn

CGC GTG ACC Arg Val Thr

GAC Asp'=

ATC GCG CAG GGC Ile Ala Gin Gly 9o

ATC CAG GCC GTA T T G CTG CCT AAG AAG ACG Ile Gln Ala Val Leu Leu Pro Lys LyS Thrla5

GAG AGC CAC CAT AAG GCC AAG GGC AAG TGA Glu Ser His His Lys Ala Lys Gly Lys *** AATGATTACT

AGTCAAATCC

GAGCCACCCA

CCTTTTCTGT AAAGTGCTGG AATACACATA

GTCAGTGATC

CCGAGTCCCA

GAAACCAAAG

CGATGCCTGA

GCTCTTTTCA 6-AATCTCAATG

FIG. 4. Sequence of H2A.l gene. The mRNA-like strand of the H2A.l gene is shown in 5’ to 3’ direction, whereas the top line describes the complementary strand, beginning (5’) with the ATG initiation codon of the H2B.l gene described in Fig. 5. Numbering of consensus regulatory elements as in Figs. 2 and 3.

ET

AL.

sis does not depend on cell division. Its gene is not part of a histone gene cluster and the flanking parts of that gene differ from the respective portions of cellcycle-regulated histone genes. Comparison of the sequences encoded by H1.l to H1.4 reveals that the N-terminal and C-terminal domains of these four Hl subtypes differ in several segments. First, the second (basic) half of the N-terminal domain, which is involved in aligning the central domain at the nucleosomal DNA (Allan et al., 1986), varies in all four subtypes. Specifically, the H1.l sequence varies widely from the other three subtypes H1.2 to H1.4. This difference between H1.l and the other Hl histones is similarly evident in the C-terminal domain. Here, the alignment of H1.l to H1.4 needs several insertions (and some deletions) in the H1.l sequence compared with the other Hl sequences, even with the Hl” sequence. On the basis of a previous comparison of several Hl sequences with chicken Hl and H5 data (RuizCarrillo et al., 1983), we have shown that most Hl histones show the octapeptide T( S)PKKAKKP within their C-terminal domains. This sequence

HZB.l(a) X'AAC GM AGG TGC AGG TCT GTA5' T T G CTT TCC ACG TCC AGA CAT

to the mRNA start sites of both the H1.3 and H1.4 genes, as indicated in Figs. 2 and 3. This proves that both genes are expressed genes coding for the subtypes described by Ohe et al. (1989) as Hlc and Hlb. This nomenclature used by Ohe et al. differs from the assignment given by Lennox and Cohen (1983) to individual spots in two-dimensional electrophoresis. In that case, however, sequence data on human Hl subtypes are missing. Previously, we have published the sequences of two human Hl genes (Eick et al., 1989), which differ from the data presented here as well as from the protein sequence data on Hla-d published by Ohe et al. (1986, 1989) and from the partial Hl sequence presented by Carozzi et al. (1984). Because of the discrepancies described above, we decided to introduce a consecutive gene nomenclature (Hl.l-H1.4). In addition to the four main type Hl sequences, which are all associated with core histone genes, we have included the Hl” protein structure as derived from the respective gene (Doenecke and Tonjes, 1986) in the comparison of individual Hl subtypes (Fig. 9). The alignment of amino acid sequences shows that the subtype Hl” varies drastically from the other four subtypes. This may reflect the different role of Hl”, which is confined to terminally differentiated cells, and its synthe-

AGTGGCTTCCT

MCAGCAGAA

GAACTAACM

ATGGAGGAAT

AAGMGCTAG

TTATAGTCAT

CAATTTGATT G G T T - T T

GTTCTTTGAC

GAGCCMCCA

AAGGCTATTT TACATGTATG

CGTCACTGAC

TCCACTGAAT

AAAGARAAAG

MTGGGCTCG

CGGTAGMTT

GTGAAAGGCG

A~TAGAMGG

AAATMGGTG

@ZAGAGCTG

GATATTTTGA A T T C T T T A T T FGAMG

TAGACACGAG

C T T T T A T T T A AGTGCGTTCA TTCTCACTGC

ATG

CCT GM Pro Glo

CCA Pro

GCT

Ala

AAG Lys

TCA

Ser

a ACATTGCCCA

GCCTATAAAA GGAGAGAZ S T G T T A T T G T T TTCTGACAGC

GCT CCT GCT CCG MG Ala Pro Ala Pro Lys

AAG GGT TCC AAG Lys Gly Ser LyS I5

AAG GCT GTG ACC MG Lys Ala Val Thr Lys

GCG CAG Ala Gln

CGC AGT CGT MG

AGC TAC TCC GTG T A T GTG TAC AAG GTG CTA Ser Tyr Ser Val Tyr Val Tyr Lys Val Le"'s

Arg

Ser

At-g

Lys

AAA CAG G T T CAC Lys Gln Val His ATC ATG Ile "et

MT AS"

GAG GCT TCC Glu Ala Ser

GAG

Glu

CAC

His

GAC Asp

ATC TTC GM Ile Phe Glu

CGC ATC GCA Arg Ile Ala

GGC Gly '5

CTG GCC CAC TAC AAC AAG CGC TCG ACC A T T ACC Leu Ala His i"yr Asn Lys Arg Ser Thr Ile ThrgO

TCC AGG GAG ATC CAG Ser Arg Glu Ile Gln CTG GCC MG Leu Ala Lys

AAG CGC AAG Lys Arg Lys I0

CCC GAT ACT GGC ATC TCA TCC AAG GCC ATG GGC Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly 6o

TCC TTC G T T MC Ser Phe Val Asn CGT Arg

AAG AAG GAT GGC MG Lys Lys Asp Gly Lys

ACC

Thr

GCA GTG Ala val

T A T ACA AGC TCC AAG Tyr Thr Ser Ser Ly.s

GCC GTG Ala

Val

TCC GM Ser Glu

CGT Arg

CTG CTG CTT CCC GGA GAG Leu Let, Leu Pro Gly Glu'Os

GGT

ACC

Gly

Thr

AAG GCT GTC ACC AAG Lys Ala Val Thr LYS'~~

TM

f**

ATGTGTGCTT AGGTGCTTTA MACTCAAAG

AGTCTCACAT

MAGAGCTTT

AATATTGMT

GCTCTTTTCA GAGCCACTCA -6TTCACCGTTT TCTAGGGMT AAGGGMTTT

TTCGATTTTG

TAATCCCAGC

ACTTT

FIG. 5. Sequence of H2B.l gene (a), adjacent to H2A.l gene described in Fig. 4. The mRNA-like strand is shown in 5’ to 3’ direction. The top line shows the complementary sequence, beginning with the ATG initiation codon of the H2A.l gene described in Fig. 4. Underlined elements 4, 5, and 6 as in Figs. 2-4; 3, Ott-1 binding site (Ref. (34)).

HISTONE

GENE

CLUSTERS

H2B.l(b) AGCGAAGTGC

ACAGCTGTCT

TGCCTAAAGA

GGGTAGTCTG GTCTGGGCTT TCTCATTGGG TACAAGTAM

AGCCMTGM

GGAACGAAAT

GTTTAcATTG GCATTTGTGA CGACACTCTA

MGCATAAAC

TAACCTCATT

GAAATCTGAT

FMTACCGC

ATCET

-TTMTE

Pro

Glu

CCT ACC MG TCT GCT CCT GCC CCA AAG AAG GGC TCC MG PrO Thr LyS Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys

MG LyS

GCG GTG ACT MG GCT CAG MG Ala Val Thr LyS Ala GlR Lys

AAG Lys

GAC Asp

GTG

T A T GTG TAC Tyr Val Tyr

CGC AGC CGC AAG GAG AGC TAT Se= A=g LYS Glu Ser Tys

MG Lys

GTC CAT CCC GAC Val HIS Pro Asp

CAG

Gin

ATC ATG MT Zle Met Asn

TCA

Ser

ACC GGC ATC Thr Gly Ile

TCC TTC GTC MC Ser Phe Val Asn

GAC

Asp

GCT TCC CGC CTG GCG CAT TAC Ma Ser Arg Leu Ala His Tyr

GAG

Glu TCC

Val

GGG MG Gly tys

AAG CGC MG Lys Arg Lys

MC MG Asn Lys

Ser Arg

CTG GCC MG Leu Ala Lys

GCC GTG TCG GAG Ala Val Ser Glu

CAC

His

GGC

Gly

MG Thr Lys

CTTTGCCMG

TAAGCATCTT TACACCTAAT

TTTCMTXM

TGAGTTGTM

AGTACCMTC

TTTMTGTTA

FIG. 6. Regulatory

CTTTTMGAG

TCATTTCATT

CGTTAGTACT

GCAGAGAAAT

AACTTGGMG

Sequence of H2B.l sites are underlined

GGC Gly'5

TTACAGGGM

AAAATAAAYGTGMT

GATCACTMC

AGGGATCGTC

CACAAT~CAGAGTGAT

TTCCTAACTC

ATTTACTTTG CAGATGMCT

AAG CAG ACG GCT CGT Lys

Gin

Thr

Ala

Arg

AAATCCACA Lys

Ser

Thr

CTCTACGGCC

-

GGC GGT Gly Gly Lys

GTG GCT CTG CGC GAG ATC CGT CGC TAC CAG AAG TCT ACC GAG CTT Val Ala Leu Arg Glu Ile Arg Arg Tyr Gin Lys Se2 Thr Glu Leu 60 CGG AAG CTG CCG T T T CAG CGC CTG GTG CGA GAA ATA GCT Arg Lys Leu Pro Phe Gin Arg Leu Val Arg GIu Ile Ala 75

CAG GAC TTC AAG ACC GAC CTG CGC TTC CAG

AGT

Gln

Sef

Phe

Lys

GCG CTG CAG Ala Leu Gin

GAG

Asp

ACC

Thr

CCT MA Pro Lys

MC Asn

Glu

Thr

Asp

Arg

Phe

Gln

TCC GCG GTG ATG Se? Ala

Val

Met

9o

GCC TGC GAG GCC TAC T T G GTG GGG CTT TTC GAG Ala Cys Glu Ala Tyr Leu Val Gly Leu Phe G/do5

CTG TGC GCT Leu Cys Ala

GAC ATC Asp Ile

Leu

CAG

Gin

ATT

Ile

CAT GCC AAA CGC GTG ACC His Ala Lys Arg Val Thr

CTT GCC CGC CGC A T T CGT GGG GAG Leu Ala Arg Arg Ile Arg Gly Glu

ATC ATG Ile Hebzo AGG Arg

GCG Ala”5

TGA *** A T T G T T T T G A GTACAAACCT

TAAATCCAM

TGAAAGGGCT GTAATCCTTT

GAGACGCATT

CATTTGAAAT

FIG. ulatory

AGTGGCATTC AGTTCCCTCG

Gly

Gly

Lys

Gly

Leu

CGT GAC MC Arg Asp Asn

Gly

tys

GGA GGC GCC Fly Gly Ala I5

ATA CAG GGC ATC ACG Ile Gin Gly Ile Thrao

A T T TCG GGC CTC A T T T A T GAG GAG ACC CGC GGT O T T CTT MG GTG Ile Ser Gly Leu Ile Tyr Glu Glu Thr Arg Gly Val Leu LyS Val 6o TTC CTG GAG MT Phe Leu Glu Am

MG Lys

GTG ATA CGG GAC GCC GTA ACC TAC ACG GAG CAC Val 11e Arg Asp Ala Val Thr Tyr Thr Glu His"

CGT AAG ACA GTC ACT Arg Lys Thr Val Thr

CGC CAG GGA CGC ACT Arg Gln Gly Arg Thr

GCA Ala

ATG Met

GAT G T T GTC TAC GCG CTC Asp Val Val Tyr Ala LeU go

CTG TAC GGC T T T GGT GGC TGA Le" Tyr Gly Phe Gly Gly ***

GCCTCACCCC

G G C T T T T T A T TTMCAGCTC

CCTTCGTCAC

ACGAAGGGCT

GTAACTGATG

RCCCAT-

GGCCCTTTTC AGGGCCACCT -sACGACTTGGG T T T C G T T T T G TAAATTTGGG

ATTCTAACTG

AGTTAAACCG

AGCCGTTTTT AGCGATCTTC

CTAAGATGGC

GGATGTGCTA

of H4 gene (upstream of H1.3). The CCAAT, elements are labeled 4-6 as in Figs. 2-7. In H4-regulatory element (Ref. (8)) is indicated

--GGCTCTTCTC AGAGCCMCC ACTGCACTGA

ACTTTGTCCG

AGACCACTL

TCC-TAGA

ATGCTTCATT TAAATTTCCA

Mm"rMGCG

7. Sequence of H3.1 gene (between H2A.l and H1.3). sites are underlined and labeled as in Figs. 2-6.

this scheme (except for an Ala/Val amino acid exchange in H1.3). SPKK, i.e., the beginning of the conserved octapeptide, has been defined as a DNA-binding motif by Suzuki (1989). Langan et al. (1981) have described a growth-associated phosphorylation site at threonine 153 (followed by Pro, Lys) of an Ehrlich ascites tumor cell Hl histone. Since the threonine at the beginning of the conserved octapeptide is at position 154 of, e.g., H1.3, we may conclude that the conserved octapeptide may be a site of phosphorylation of the Hl protein and one of the Hl DNA binding sites.

1s

GCC ACG GGC GGC GTG AAG AAG CCC CAT CGC TAC CGC CCT GGC ACC Ala Thr Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly Thr's

Asp

Lys

GCA

Ala

CCG CGC AAA CAG CTG GCC ACT AAG GCA GCT CGC AA0 AGC GCT CCG Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg LyS Sef Ala Pro 30

CTA ATC Leu Ile

Gly

gene (b) in the vicinity of Hl.4. and labeled as in Fig. 5.

TTGCACTGM

Thr

Arg

FIG. 8. Sequence TATA, and stem-loop addition, a tripartite by dashed lines.

GTTATTGGAC TTAGCACCAC

AATCAGAATC

Arg

GTTGTTGTTT GTCTTCGATC

GCC MG Ala Lys

CCACGCATGT

ACTTCCGGAA TTTAGCAACC

iii%

MAGTTAAGA

AAAGGCGGAG

CCC GCC ATC CGT CGC T T G GCC CGA CGC GGC GGC GTG AAA CGC Pro Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val LyS Arg"

I-n.1

TCTGTTCCTA TATAGAGGGG CAMCCAATC

AGGTCCGCAT MTTGATATA

MG Lys

starts in all mammalian Hl species with a threonine and in all other sequences examined with a serine (Tsnjes and Doenecke, 1987; Eick et al., 1989). The comparison shows that all human Hl histones follow

CAAATTTACC

AGTCTCCAM

30

GCC GTC ACC AAG Ala val Thr Lysl2a

CCCAAAGGCT -sCAAAAGGGGG

CCTCCAATTC

CGC CAT CGC AAA GTG CTG Arg His Arg Lys Val Leu

ACC AGT TCC AAG TM Thr Ser Ser Lys ***

TAC Tyr

ACAGAGGTTT CGTTCCCGCC

MG Lys

CGC TCG ACC ATC ACC Arg Ser Thr Ile Thr 90

ACC

CCTCACCGCC

MGTTACGGA

15

GAG ATC CAG ACG GCC GTG CGC CTG CTG CTT CCG GGG GAG Glu Ile Gin Thr Ala "al Arg Le" Leu te" Pro Gly GlU105

AGG

TCAACTTGCA TTTCCCCTAC

TAGTGTAGGG GACGCCCAGT

Gly

AAG GTG CTG Lys Val Leu 45

CGC ATC GCA Arg Ile Ala

AGGAGTCAAA

Ser

TCT TCC AAG GCA ATG GGG Ser Ser Lys Ala Met Gly 60

ATC TTC GAG Ile Phe Glu

CAGGAAACGG

CCCTTTCCTT CCCAAGGCM

ATG TCT GGT AGA GGC AAA GGT GGT AAA GGT T T A GGA MG

TMCGCTACG

ATG CCT GM

GAC

945

GENES

TAAGGGGCTT CAGTGTGTAG CAA,&CA -

GMCAGGGCC

TCGGCGGGAG T G A T T A T T T T CTCAGGTGTT TGCAACAGTG TTCTAkTAT

Arg

CAGACCCMA

GGCCTATTTA TAGTAGCCTC

AAGGTAGACT TTTMGTGTC

Ala

Hl

H4

MCTGCTCCA

ATG GCG

INCLUDING

Reg-

CONCLUSIONS The data presented here show that the genes coding for the human Hl histone subtypes Hlc and Hld are associated with core histone genes coding for the subtypes H2A.1, H2B.1, H3.1, and H4. One amino acid exchange between the two H2B.l gene products suggests that even the core histone subtype classification will need a revision upon further sequencing of human histone genes. Hl histones are known as the most complex group of histones. In mammals, five subtypes have been defined first by two-dimensional electrophoresis (Lennox and Cohen, 1983) and subsequently by HPLC. In addition, Hlt and Hl” represent Hl histones that are confined to just one cell type (spermatocytes, early spermatids, Hlt) or to several terminally differentiated tissues (Hl”).

946 H1.l H1.2 H1.3 H1.4 H10

ALBIG

ET

AL.

-A;KKKPA.GPSV;

sET"PPAPA:sAAPEKPLA~KKA-KK-P*i***---SETAPAAPAAAPPAEKAPVKKKAAKK--AGGTP---RKAS-----GPPVS SETAPLAPTIPAPAEKTPVKKKA-KK--A-GATAGKRKAS-----GPPVS SETAPAAPAAPAPAEKTPVKKKA-RKS TENSTSAPAAKPK------RAKASKKS-T-DHPK--------------YS

_ _ - -

.~

A-GA--AKRKAS-----GPPVS

~~~~

ELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKG ELITKAVAASKERSGVSLAALKKALAAAGYDVEKNNSRIKLGLKSLVSKG DMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTG v TLVQTKGTGiSGSFKLNK.KASSVETKPGAV-----TLVQTKGTGASGSFKLNKKAASGEAKPKV----------KKAGGTKPKKP TLVQTKGTGASGSFKLNKKAASGEGKPKA----------KKAGAAKPRKP TLVQTKGTGASGSFKLNKKAASGEAKPKA----------KKAGAAKAKKP VLKQTKGVGASGSFRLAKSDEPKKSVAFKKTKKEIKKVATPKKASKPKKA

--S&'ATKTKA--V

T-GASKKLK~AT-GAS-KK~V-KTPKKAK~P--AAT--RP--KSSKNPK~ V-GAAKKPKKAAGGATPKKSAKKTPKKAKKPA-AATVTKKVAKS---PKK A-GAAKKPKKVAGAATPKKSIKKTPKKVKKPATAA-GTKKVAKSA---KK A-GAAKKPKKATGAATPKKSAKKTPKKAKKPAAAA-GAKK-AKS---PKK ASKAPTKKPKATPVKKAKKKLAATPKKAKKP------------------PKTVKPKKViKSPAKAKAVitPKAAKARVTiiP---KTAKPiKAAPKKK AKVAKPKKAAKS-A-AKAVKPKAAKP--------KVVKPKKAAPKKK VKTPQPKKAAKSPAKAKAPKPKAAKPKSGKPKV--T-KAKKAAPKKK AKAAKPKKAPKSPAKAKAVKPKAAKPKTAKPKAA---KPKKAAAKKK -KTVKAK-PVK--A-SKPKKAKPVKP-----KA-KSS-AKRAG-KKK

FIG. 9. Comparison alignment of sequences. highly conserved central lishing the three domain

(214 (212 (220 (218 (193

of human Hl histone sequences, presented in one-letter amino acid code. Dashes indicate gaps introduced for Every 10th position is indicated by an open arrowhead, and the highly conserved octapeptide is underlined. The domain starts around position 45 and ends around position 120 (borders indicated by solid triangles), thus estabstructure (Ref. (1)).

In 1986, Ohe et al. sequenced the Hlb protein. We have recently described two human Hl genes (H1.l and H1.2; Eick et al., 1989). Shortly thereafter, Ohe et al. (1989) published the sequences of human spleen Hlproteins Hla, Hlc, and Hld. This allows a comparison of the gene sequencesdescribed here with known protein and gene sequence data. Two of the four completely sequenced Hl genes (H1.3 and H1.4) code for proteins, which had been described at the protein sequence level as Hlc and Hld (Ohe et aZ., 1989). In addition to these four sequences, Carozzi et al. (1984) deduced a partial sequence from a human Hl gene, which was part of a histone gene cluster. This sequence fragment again differs from the four known protein (Hla-d; Ohe et al.) and gene sequences (this paper; Eick et al., 1989). This allows the conclusion that the number of Hl histone genes in the human genome exceeds the number of the five main types Hla-e by at least one or two additional Hl species. In addition to the analysis of two novel human Hl gene sequences, this paper describes the structure and arrangement of core histone sequences in the vicinity of these Hl genes. Thus, all human Hl histone genes (except Hl”; Doenecke and Tonjes, 1936) are associated with core histone genes. The arrangement of core histone genes varies in all these clusters, which is

in contrast to the genomes of lower eukaryotes showing tandem repeats of identical clusters. The number of human histone gene clusters is still unknown. According to Triputti et al. (1986), histone genes map to chromosomes 1,6, and 12. None of the human histone gene clusters published until now overlapped with each other. Only the two overlapping clones HHGl and HHG4 (Zwollo et al., 1984) appear to represent the same site as the segment covered by cosmid C5. Given the number of at least 6 Hl histone genes within histone gene clusters, we should conclude that more than one Hl gene may on the average be contained within each of the three major histone gene loci. ACKNOWLEDGMENTS This work was supported by the Deutsche schaft. We thank A. M. Frischauf for providing The technical assistance of Silvia Biittinger, Susanne Hellmold is gratefully appreciated.

Forschungsgemeinthe cosmid library. Christina Kunz, and

REFERENCES 1.

ALLAN, J., MITCHELL, T., HARBORNE, N., B~HM, L., AND CRANE-ROBINSON, C. (1986). Roles of Hl domains in determining higher order chromatin structure and Hl location. J. Mol. Bid. 187: 591-601.

HISTONE

GENE

CLUSTERS

2.

BENTON, D. W., AND DAVIS, R. W. (1977). Screening lambda recombinant clones by hybridization to single plaques in situ. Science 196: 180-182.

3.

BIRNBOIM, H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 6: 1513-1526.

4.

CAPASSO, O., AND HEINTZ, N. (1985). Regulated expression of mammalian histone H4 genes in vivo requires a trans-acting transcription factor. Proc. Natl. Acad. Sci. USA 82: 56225626.

5.

CAROZZI, N., MARASHI, F., PLUMB, M., ZIMMERMAN, S., ZIMMERMAN, A., COLES, L. S., WELLS, J. R. E., STEIN, G., AND STEIN, J. (1984). Clustering of human Hl and core histone genes. Science 224: 1115-1117.

6.

CHANDLER, M. E., KEDES, L. H., COHN, R. H., AND YUNIS, J. J. (1979). Genes coding for histone proteins in man are located on the distal end of the long arm of chromosome 7. Science 206: 908-910.

7.

CHIRGWIN, J., PRZYBYLA, A., MACDONALD, R., AND Ru~R, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299.

INCLUDING

19.

Hl

947

GENES

zation and expression of histone 313. HERRMANN, B. G., BARLOWE, (1987). A large inverted duplication bination between chromosomes mal t complex inversion. CeU 48:

gene families.

Cell 25: 301-

D. P., AND LEHRACH, H. allows homologous recomheterozygous for the proxi813-825.

20.

HOFFMANN, I., AND BIF~NSTJEL, M. L. (1990). pendent regulation of histone precursor mRNA modulation of U7 snRNA accessibility. Nature

21.

LANGAN, T. A., ?iEILIG, C., AND LEICHTLING, B. (1981). Characterization of multiple-site phosphorylation of Hl histone in proliferating cells. In “Protein Phosphorylation” (0. M. Rosen and E. G. Krebs, Eds.), Vol. 8, pp. 1039-1052, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

22.

LENNOX, R. W., AND COHEN, L. H. (1983). The histone Hl complements of dividing and non-dividing cells of the mouse. J. Bial. Chem. 258: 262-268.

23.

Mm, A. M., AND GILBERT, W. (1977). A new method sequencing DNA. Proc. Natl. Acad. Sci. USA 74: 560-564.

24.

MAXSON, R., COHN, R., KEDES, L., AND MOHUN, T. (1983). Expression and organization of histone genes. Annu. Rev. Genet. 17: 239-277.

25.

OHE, Y., HAYASHI, H., AND IWAI, K. (1986). histone Hl: Isolation and amino acid sequence ant, Hlb. J. Biochem. 100: 359-368.

26.

COLE, K. D., YORK, R. G., AND KISTLER, W. S. (1984). The amino acid sequence of boar Hlt, a testis-specific Hl histone variant. J. Biol. Chem. 269: 13695-13702.

OHE, Y., HAYASHI, H., AND IWAI, K. (1989). Human spleen histone Hl. Isolation and amino acid sequences of three minor variants, Hla, Hlc and Hld. J. Biochem. 106: 844857.

27.

10.

COLE, K. D., KANDALA, J. C., AND KISTLER, W. S. (1986). Isolation of the gene for the testis-specific Hl histone variant Hlt. J. Biol. Chem. 261: 7178-7183.

PANYIM, S., AND C HALKLEY, R. (1969). A new histone found only in mammalian tissue with little cell division. Biochem. Biophys. Res. Commun. 37: 1042-1049.

28.

11.

D’ANDREA, R., COLES, L. S., LESNIKOWSKI, C., TABE, L., AND WELLS, J. R. E. (1985). Chromosomal organization of chicken histone genes: Preferred associations and inverted duplications. Mol. Cell. Biol. 5: 3108-3115.

RUIZ-CARIULLO, A., AFFOLTER, M., ANII RENAUD, J. (1983). Genomic organization of the genes coding for the six main histones of the chicken: Complete sequence of the H5 gene. J. Mol. Biol. 170: 843-859.

29.

COLES, L. S., AND WELLS, J. R. E. (1985). An Hl histone gene-specific 5’ element and evolution of Hl and H5 genes. Nucleic Acids Res. 13: 585-594.

SAIVIBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

30.

DENHARDT, D. T. (1966). A membrane the detection of complementary DNA. Res. Commun. 23: 641-646.

SANGER, F., NICKLEN, S., AND COULSON, A. R. (1977). sequencing with chain-terminating inhibitors. Proc. Acad. Sci. USA 74: 5463-5467.

31.

SEYEDIN, S. M., COLE, R. D., AND KISTLER, W. S. (1981). Histones from mammalian testes: The widespread occurrence of Hlt. Exp. Cell Res. 136: 399-405.

32.

SHANNON, M. F., AND WELLS, J. R. E. (1987). Characterization of the six chicken Hl proteins and alignment with their respective genes. J. Bial. Chem. 262: 9664-9668.

33.

SIMPSON, R. T. (1978). Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histones. Biochemistry 17: 5524-5531.

34.

Srv~, H. L., AND ROEDER, R. G. (1986). Multiple sequence elements are required for maximal in vitro transcription of a human histone HZB gene. Mol. Cell. Biol. 6: 3329-3340.

35.

SOUTHERN, E. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Bial. 98: 503-517.

36.

STEIN, G. S., SIERRA, F., STEIN, J. L., PLUMB, M., -HI, F., CAROZZI, N., PROKOPP, K., AND BAWACH, L. (1984). Organization and expression of human histone genes. In “Histone Genes” (G. S. Stein, J. L. Stain, and W. F. Marzluff, Eds.), pp. 397-455, Wiley-Interscience, New York.

8.

9.

lla.

12.

13.

CLERC, R. G., BUCHER, P., STRUB, K., AND (1983). Transcription of a cloned Xerwpus gene in the homologous frog oocyte system lutionary conserved sequence motif in the cleic Acids Res. 11: 86418657.

BIRNSTIEL, M. L. Levis H4 histone depends on a evo-50 region. Nu-

filter technique for Biochem. Biophys.

DOENECKE, D., AND T~NJES, R. (1986). Differential distribution of lysine and arginine residues in the closely related histones Hl” and H5. Analysis of a human Hl” gene. J. Mol. Bid. 18’7: 461-464.

14.

EHRICH, L. J., CRAIG, A., FRISCHAUF, A. M., POUSTKA, AND LEHRACH, H. (1987). A family of cosmid vectors with multicopy R6K replication origin. Gene 5’7: 229-237.

15.

EICK, S., NICOLAI, M., MUMBERG, D., AND DOENECKE, D. (1989). Human Hl histones: Conserved and varied sequence elements in two Hl subtype genes. Eur. J. Cell Biol. 49: llO115.

16.

FEINnERG, A. P., &ND VOGELSTEIN, B. (1983). Atechnique radiolabeling DNA restriction endonuclease fragments high specific activity. Anal. Biachem. 132: 6-13.

17.

FRANKLIN, S. G., AND ZWEIDLER, A. (1977). Non-allelic ants of histones Za, 2b and 3 in mammals. Nature 266: 275.

18.

HENTSCHEL,

C. C., AND BIRNSTIEL,

M. L. (1981).

A., the

for to vari273-

The organi-

Cell-cycle deprocessing by 346: 665-668.

for

Human spleen of a main vari-

DNA Natl.

948 37.

38. 39.

40.

41.

42.

ALBIG SUNDSTR~~M, C., AND NILSSON, K. (1976). Establishment and characterization of a human histiocyte lymphoma cell line. Int. J. Cancer 17: 565-577. SUZUKI, M. (1989). SPKK, a new nucleic acid-binding unit of protein found in histone. EMBO J. 8: 797-804. THOMA, F., KOLLER, T., AND KLUG, A. (1979). Involvement of histone Hl in the organization of the nucleosome and of the salt-dependent superstructures of chromatin. J. Cell Biol. 83: 403-427. T~NJES, R., AND DOENECKE, D. (1987). A highly conserved sequence in Hl histone as oligonucleotide hybridization probe: Isolation and sequence of a duck Hl gene. J. Mol. Euol. 25: 361-370. TRIPPUTI, P., EMANUEL, B. S., CROCE, M., GREEN, L. G., STEIN, G. S., AND STEIN, J. L. (1986). Human histone genes map to multiple chromosomes. Proc. Natl. Acad. Sci. USA 83: 3185-3188. WEAVER, R. F., AND WEISSMANN, C. (1979). Mapping of

ET

AL. RNA by a modification of the Berk-Sharp procedure: The 5 termini of 15S-globin mRNA precursor and mature lOS-globin mRNA have identical map coordinates. Nucleic Acids Res. 7: 1175-1193. 43.

WEINTRAUB, superstructures 17-27.

43a.

WELLS, D. E., AND MCBRIDE, C. (1989). A comprehensive compilation and alignment of histones and histone genes. Nucleic Acids Res. 17: r311-r346. ZWEIDLER, A. (1984). Core histone variants of the mouse: Primary structure and differential expression. In “Histone Genes” (G. S. Stein, J. L. Stein, and W. F. Marzluff, Eds.), pp. 339-371, Wiley-Interscience, New York.

44.

45.

H.

(1984). Histone-Hl-dependent chromatin and the suppression of gene activity. Cell 38:

ZWOLLO, P., STEIN, in the organization taining Hl histone 124: 988-993.

G. S., AND STEIN, J. L. (1984). Variation of human genomic DNA segments congenes. Biochem. Biophys. Res. Commun.