Structural change of a particular H2B histone gene possibly results in differences in its transcriptional regulation in different chicken tissues.

The chicken H2B histone gene family consists of eight highly homologous members (H2B-I to H2B-VIII) belonging to two major histone gene clusters. Seven of these genes have been sequenced and shown to encode three different H2B protein variants. Northern analysis with a probe, which mainly consists of the 5'-flanking region containing the sequence for the mRNA leader of H2B-V encoding a particular H2B protein variant, revealed that the mRNA level transcribed from this particular gene was higher in the kidney than in the oviduct and lung. To elucidate whether the structure of the H2B gene differs in the three different tissues, we analyzed DNAs from the oviduct, lung, and kidney. On Southern analysis, various H2B gene-specific probes hybridized with two particular H2B genes (H2B-IV and H2B-V), which are located in close proximity within a 12 kb EcoRI fragment, from the oviduct and lung with an intensity of about one quarter of that from the kidney. These findings suggest that some difference of DNA structure of the H2B-V gene may result in its relatively higher expression in the kidney.     

The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences.

Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X. laevis as hybridization probes. A 2.6 kb HindIII-generated N. crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector. Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4. Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N. crassa. The two genes are physically linked on the genome. DNA sequencing of the N. crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene. The amino acid sequences of the N. crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions. Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes.     

Recent and rapid amplification of the sperm basic nuclear protein genes in winter flounder

The high molecular weight basic nuclear proteins (HMrBNPs), which are tightly bound to sperm chromatin in winter flounder, are made up of imperfect reiterations of simple peptide sequences that contain phosphorylatable DNA-binding motifs. Genomic Southern blots hybridized with probes to the coding and non-coding regions of HMrBNP mRNA showed that HMrBNP sequences form a complex multi-gene family. Previously, one gene (2B) was used to establish an evolutionary link between histone H1 and the HMrBNPs. Further examination of this complex, multi-gene family has now revealed that the majority of the HMrBNP genes are linked as 4.5 kb direct tandem repeats that each contain a 2.8 kb coding region and a 1.7 kb intergenic region (IR). These findings, combined with the cloning of the IR, established that the tandemly repeated genes lack introns and code for the abundant 3 kb HMrBNP mRNAs that produce the prominent 110 kDa HMrBNP. Southern blotting of DNAs from other righteye flounder species showed that HMrBNP multi-gene families were present in closely related species, though with substantial differences in restriction patterns and band intensities, but were not detected in more distantly related flounders. These observations are consistent with recent and rapid elaboration of the HMrBNP gene family.     

Isolation of a clone containing human histone genes.

A recombinant clone containing human histone genes has been isolated. The clone, lambda HH-01, was selected from a genomal library using chicken histone cDNA and a cloned fragment containing chicken histone genes as probes. Sub-clones from lambda HH-01 have been mapped and coding regions located with cDNA. The human H3 gene has been identified by DNA sequence analysis.     

Structure and organization of the chicken H2B histone gene family.

The results of Southern blotting experiments confirm that the chicken H2B histone gene family contains eight highly homologous members. One or two more sequences which are considerably divergent from the others appear to exist in the chicken genome. Seven of the eight H2B genes have been cloned and sequenced. All seven genes fall in two histone gene clusters, but no common arrangement exists for the clusters themselves. Three different H2B protein variants are encoded by these seven genes. The nucleotide sequence homology among the genes within their coding sequences appears to exceed that required for the corresponding protein sequences, suggesting that histone H2B mRNA sequence and structure are both selected during evolution. An analysis of the 5' flanking sequence data reveals that these genes possess CCAAT and TATA boxes, elements commonly associated with genes transcribed by RNA polymerase II. In addition, these genes all share an H2B-specific element of the form: ATTTGCATA. The 3' sequences of these genes contain the hyphenated symmetrical dyad homology and downstream purine-rich sequence shared by histone genes in general.     

Isolation of a genomal clone containing chicken histone genes.

We have used enriched chicken histone cDNA to select genomal clones from a chicken library. Because the cDNA probe also contained other sequences, a further screening of positive plagues with negative probes eliminated most non-histone gene clones. One 'positively-selected' genomal clone, lambda CH-01, hybridised with cloned sea-urchin histone genes and also detected histone genes in EcoRI-digested genomal sea-urchin DNA. Limited DNA sequencing of HaeIII fragments identified two sequences within the coding region of chicken histone H2A. A third fragment predicted an amino acid sequence with strong homology to an H1 histone sequence.     

S(T)PXX motifs promote the interaction between the extended N-terminal tails of histone H2B with "linker" DNA.

The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): Polarity and gene organization

We have analyzed the histone genes from the sea urchin Lytechinus pictus. Examination of native DNA from individuals reveals four major Eco RI restriction endonuclease histone gene DNA fragments which have been labeled A (6.0 kb), B (4.1 kb), C (3.1 kb) and D (1.2 kb). The fragments A, B and C have been cloned into E. coli plasmids (pLpA, pLpB and pLpC). These histone gene fragments display length and sequence heterogeneity in different individuals. The plasmid pLpA contains the coding regions for H1, H4, H2B and H3 histones, and we determined that the DNA fragment D is tandem to A in native DNA and that it contains the H2A gene. The plasmids pLpB and pLpC contain the histone genes H2A-H1-H4 and H2B-H3, respectively, and together contain the sequences for the five major histones. Restriction analysis of native L. pictus DNA reveals that B and C are tandem to each other but not intermingled with the A-D-type repeat units, and are thus in separate clusters with a repeat length of 7.2 kb. Since the two cluster types do not segregate, they are not alleles. Hybridization of histone mRNA to exonuclease III-digested linear DNA demonstrated an identical polarity of the histone genes in the A-D- and B-C-type repeat units. This result revealed that the L. pictus histone genes have a polarity which is the same as other sea urchin histone genes examined to date—that is, 3′ H1-H4-H2B-H3-H2A 5′. Restriction endonuclease cleavage patterns of the cloned segments indicate that considerable sequence heterogeneity exists between the two types of histone gene repeat units.     

A rat histone H4 gene closely associated with the testis-specific H1t gene

A rat histone H4 gene closely associated with the testis-specific H1t gene was isolated by screening the Sargent-Bonner rat genomic library using cloned human histone genes as probes. Both the H4 gene and the H1t gene are located on a 7-kb EcoRI genomic DNA fragment. Although the deduced amino acid sequence of the rat H4 histone is identical to that of the sequence of human histone H4, the nucleotide sequence of the coding region differs significantly from the coding region of the human H4 gene. Moreover, the relative spacing between the 5'-consensus sequence elements is unique for an H4 gene. S1-nuclease protection analyses reveal that both the H4 and H1t mRNA species are present in a fraction of rat testis cells highly enriched in pachytene spermatocytes, while only the H4 mRNA species is present in a rat myeloma cell line (Y3-Ag1.2.3). During a 1-h hydroxyurea treatment of the Y3 cells, which produces a 99% inhibition of DNA synthesis, the level of this H4 mRNA drops by only 50%, indicating that the stability of this mRNA is only partially coupled with DNA synthesis.     

An H3-H4 histone gene pair in the marine copepod Tigriopus californicus, contains an intergenic dyad symmetry element.

Histone genes are one of the most widely studied multigene families in eucaryotes. Over 200 histone genes have been sequenced, primarily in vertebrates, echinoderms, fungi and plants. We present here the structure and genomic orientation of an H3-H4 histone gene pair from the marine copepod, Tigriopus californicus. These histone gene sequences are the first to be determined for the class Crustacea and among the first to be determined for protostomes. The H4 and H3 genes in Tigriopus are shown to be adjacent, to have opposite polarity, and to contain a 26 bp region of dyad symmetry centrally located within the spacer region between the two genes. A similarly located dyad element has been found in yeast which contributes to the coordinated cell cycle control of the adjacent histone genes. The Tigriopus H3-H4 histone gene pair is clustered with one H2A and two H2B histone genes on a 15 kb genomic Bam H1 fragment. The H4 gene sequence predicts an H4 protein with an unusual serine to threonine substitution at the amino terminal residue. The H3 gene sequence predicts an H3 protein which is identical to the vertebrate H3.2 histone.     

The chicken H5 gene is unlinked to core and H1 histone genes

An H5 cDNA clone was used to select H5 genomal recombinants from a chicken Charon 4A library. DNA sequence analysis shows that the H5 gene contains no introns. Putative 5′ promoter elements and a 3′ polyadenylation site are present within the 1.8 kb of DNA examined. Analysis of 41 kb of DNA surrounding the H5 gene shows that it is not closely linked to either H1 or core histone genes.     

Histone genes from Xenopus laevis: molecular cloning and initial characterization

Histone DNA sequences, were detected in Eco RI fragments of total Xenopus laevis DNA, by hybridization with 32P-labeled h22-DNA, a histone gene repeat unit of the sea urchin Psammechinus miliaris. The about 6 kb-size class, which was found to hybridize, was subsequently integrated into the E. coli plasmid pCR1. A clone was isolated that contains a 5.8 kb EcoRI fragment hybridizing with h22-DNA. A physical map was constructed using the restriction endonucleases BamHI, PstI, HincII, BglII, XbaI, PvuII, XhoI, AvaI, SmaI, HinfI and HpaII. The fragment was not cleaved by KpnI, AvaI, SalI and HindIII. Using this restriction map we were able to determine the gene order by hybridization with purified gene probes derived from h22-DNA. The gene order was found to be H3, H4, H2A and H2B. The localization of the H1 gene was not possible, probably due to its greater evolutionary divergence. Part of the sequence of the H3-gene is presented providing unambiguous evidence on the identity, map position and polarity of this gene.     

The reconstitution of a hybrid histone octamer containing avian 110Cys-des-thio-histone H3 and sea-urchin 73Cys-histone H4

A hybrid histone octamer was reconstituted from erythrocyte H2A and H2B, avian [110 Cys-des-thio]histone H3 and the sea-urchin sperm [73Cys]H4 variant. [110Cys-Des-thio]histone H3 was prepared by reaction of natural H3 with Raney nickel. The ability of the hybrid octamer to crystallize to the same form as the natural octamer demonstrated that the chemical modification of cysteine to alanine in H3 and the mutation from threonine to cysteine in sperm H4 do not alter histone-histone interactions in the octamer. Since the sulfhydryl groups of both H4 molecules are fully accessible to 5,5'-dithiobis(2-nitrobenzoate) these residues provide suitable sites for the introduction of a single cysteine-specific label per H4 molecule in the octamer.     

Isolation of yeast histone genes H2A and H2B

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.     

Organization of the Chicken Histone Genes in a Major Gene Cluster and Generation of an Almost Complete Set of the Core Histone Protein Sequences

We present a detailed picture of the disposition of the histonegenes in the chicken genome and an almost complete set of thecore histone protein sequences. Thirty-nine histone genes, sixH1, nine H2A, eight H2B, eight H3 and eight H4, were locatedwithin a histone gene cluster of 110 kb, which was covered byfive cosmid clones and two clones. Results of our sequenceanalyses, together with those reported previously, generateda set of the core histone amino acid sequences as follows: threeH2A variants, four H2B variants,two H3 variants and an H4 protein.     

Genomic organization and nucleotide sequences of two corn histone H4 genes

The sea urchin histone H4 gene has been used as a probe to clone two corn histone H4 genes from a lambda gtWES X lambda B corn genomic library. The nucleotide (nt) sequences of both genes showed that the encoded amino acid sequences were identical to that of the H4 of pea and one variant of wheat. The nt sequences of the coding regions showed 92% homology. 5'- and 3'-flanking regions do not show extensive nt sequence analogies. Southern blotting of the EcoRI digested genomic DNA suggests the existence of multiple H4 genes dispersed throughout the genome.