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.     

Isolation and characterization of the histone variants in chicken erythrocytes.

Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone--histone interactions.     

Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone

A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labelled elastin mRNA (mRNAE). A recombinant containing a 9.9-kb (kilobase) insert was selected from several positive clones by secondary and tertiary screening for further characterization. Positive identification of this elastin clone, designated SE1, was made with radiolabelled mRNAE by hydridization-selected translation and Southern blotting of restriction-enzyme fragments of SE1 DNA. Hybridization of either mRNAE or elastin complementary DNA to restriction fragments of SE1 showed that most of these fragments of SE1 contained elastin-coding sequences. Orientation of the insert was established by preferential hybridization of a short complementary elastin DNA to restriction fragments adjacent to the right arm of Charon 4A. Reciprocal hybridizations of nick-translated SE1 and sheep genomic DNA on Southern blots showed that two restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclease-EcoR1 digest of total sheep genomic DNA. In the accompanying paper [Davidson, Shibahara, Boyd, Mason, Tolstoshev & Crystal (1984) Biochem. J. 220, 653-663], it is shown that a subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. Electron microscopy of SE1-mRNAE hybrids indicated the presence of at least seven large R-loops. Measurements of these structures indicated that SE1 is likely to contain less than 2 kb of coding sequence and more than 8 kb of intervening sequence, with an average exon size of 120 base-pairs. Thus the elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.     

Isolation and characterization of six different chicken actin genes.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.     

Isolation of a cDNA clone encoding the RNase-superfamily-related gene highly expressed in chicken bone marrow cells.

The RNase gene superfamily combines functionally divergent proteins which share statistically significant sequence similarity. Known members assigned to this family include secretory and nonsecretory RNases; angiogenin; eosinophil cationic protein; eosinophil-derived neurotoxin; sialic-acid binding lectin and anti-tumor protein P-30. We report the cDNA cloning of the chicken RNase Super Family Related (RSFR) gene that is specifically overexpressed in normal bone marrow cells and bone marrow-derived AMV transformed monoblasts. It codes for a 139 amino acid protein with a putative signal peptide and remarkable conservation of active-site residues, other residues known to be important for substrate binding and catalytic activity and half-cystine residues common for all RNase family members. Phylogenetic tree analysis shows that RSFR defines a new group of genes within the family. We also conclude that an amino acid sequence block CKXXNTF(X) 11C is a "shortest RNase superfamily signature" which is both necessary and sufficient to identify all previously recognized family members as well as chicken RSFR.     

Isolation of a chicken thioredoxin cDNA clone. Thioredoxin mRNA is differentially expressed in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts

We have isolated a cDNA clone for chicken thioredoxin by differential screening of a cDNA library. The protein product which this clone encodes is very similar to other thioredoxins, and it displays thioredoxin activity when expressed in Escherichia coli. This clone represents the first metazoan thioredoxin for which the protein or nucleic acid sequence is known. Comparison of the chicken thioredoxin protein sequence with those from bacteria and plants indicates structural features that appear to be essential for activity. Transformation of chicken embryo fibroblasts by Rous sarcoma virus elevates the level of thioredoxin mRNA whereas the level of thioredoxin mRNA in a nonproliferative tissue (brain) is much lower than in chicken embryo fibroblasts.     

Chromosomal organization of chicken histone genes: preferred associations and inverted duplications.

We present a detailed picture of the disposition of core and H1 histone genes in the chicken genome. Forty-two genes were located within four nonoverlapping regions totalling approximately 175 kilobases and covered by three cosmid clones and a number of lambda clones. The genes for the tissue-specific H5 histone and other variant histones were not found in these regions. The longest continuous region mapped was 67 kilobases and contained 21 histone genes in five dissimilar clusters. No long-range repeat was evident, but there were preferred associations, such as H1 genes with paired, divergently transcribed H2A-H2B genes and H3-H4 associations. However, there were exceptions, and even when associations such as H1-H2A-H2B we maintained, the order of those genes within a cluster may not have been. Another feature was the presence of three (unrelated) clusters in which genes were symmetrically ordered around central H3 genes; in one such cluster, the boundaries of a duplicated H2A-H4 gene pair contained related repeat sequences. Despite the dispersed nature of chicken histone genes, the number of each type was approximately equal, being represented as follows: 6 H1, 10 H2A, 8 H2B, 10 H3, and 8 H4.     

Isolation and characterization of two human H1 histone genes within clusters of core histone genes

Two human H1 histone genes, termed H1.3 and H1.4, were isolated from two cosmid clones. The H1.4 gene is associated with an H2B 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 EMBL3 library. In continuation of our previous analysis of two human H1 genes, this analysis raises the number of completely sequenced H1 histone genes within clusters of core histone genes to four.     

Isolation and characterization of two replication-dependent mouse H1 histone genes.

Mice contain at least seven nonallelic forms of the H1 histones, including the somatic variants H1a-e and less closely related variants H1 degrees and H1t. The mouse H1 degrees and H1c (H1var.1) genes were isolated and characterized previously. We have now isolated, sequenced and studied the expression properties of two additional mouse H1 genes, termed H1var.2 and H1var.3. Extensive amino acid and nucleotide sequence comparisons were made between the two genes and other mammalian H1 histone genes. A high degree of nucleotide sequence identity was seen between the H1var.2, rat H1d and human H1b genes, even well beyond the coding region, indicating that these genes are likely homologues. Unlike the previously characterized mouse H1var.1 gene which produces both nonpolyadenylated and polyadenylated mRNAs, the H1var.2 and H1var.3 genes produce only typical, replication dependent, nonpolyadenylated mRNAs.     

Patterns of histone modifications across the chicken alfa-globin genes’ domain

Using native chromatin imunoprecipitation (N-ChIP) followed by TaqMan RT-PCR quantitative analysis, we have determined the profiles of histone acetylation and histone methylation within the α-globin gene domain before and after switching of embryonic globin gene expression. The results obtained do not support a supposition that the inactivation of the embryonic α-type globin gene π in the erythroid cells of the adult lineage is mediated via formation of an inactive chromatin domain. On the other hand, we have demonstrated that suppression of the gene π activity in erythroid cells of adult lineage correlates with decrease of the histone acetylation level within the embryonic subdomain of the α-globin gene domain.     

Patterns of the histone modifications across the chicken alpha-globin genes domain

Using native chromatin immunoprecipitation (N-ChIP) followed by TaqMan RT-PCR quantitative analysis we have determined the profiles of histone acetylation and histone methylation within the alpha-globin gene domain before and after switching of embryonic globin genes expression. The results obtained do not support a supposition that the inactivation of the embryonic alpha-type globin gene pi in erythroid cells of the adult lineage is mediated via formation of an inactive chromatin domain. On the other hand we have demonstrated that suppression of the gene pi activity in erythroid cells of adult lineage correlates with the decrease of the histone acetylation level within the embryonic subdomain of the alpha-globin gene domain.     

Sequence variants of chicken linker histone H1.a

Two allelic isoforms (H1.a1 and H1.a2) of histone H1.a were identified within two conservative flocks (R11 and R55) of Rhode Island Red chickens. These proteins form three phenotypes: a1, a2 and a1a2. Birds with phenotype a1 were most common (frequency 0.825-0.980) while the a1a2 chickens appeared relatively rarely (0.017-0.175). The third phenotype a2, not detected in the tested populations, has only been revealed in progeny of the purpose-mated a1a2 birds. The polymorphism of histone H1.a was observed in all examined chicken tissues, so that the H1 preparations isolated from the lung, spleen, kidney and testis from the same individual exhibited identical phenotypes (a1, a2, or a1a2). This finding, together with inheritance data, supports the genetic nature of the H1.a polymorphism. As indicated by cleavages with alpha-chymotrypsin and protease V8, the H1.a1 and H1.a2 are two highly related proteins which differ within N-terminal part of their C-terminal tails. Only a single nonconservative amino acid substitution between both H1.a allelic isoforms was detected by Edman degradation: glutamic acid present at position 117 in histone H1.a1 was replaced by lysine in histone H1.a2. Furthermore, using microsequencing techniques we have found a sequence homology between the N- and C-terminal parts of an unknown minor protein H1.y, present in the phenotype a2, and similar regions of histone H1.b.     

Vertebrate histone genes: nucleotide sequence of a chicken H2A gene and regulatory flanking sequences.

The DNA sequence of a chicken genomal fragment containing a histone H2A gene has been determined. It contains extensive 5' and 3' flanking regions and encodes a protein identical in sequence to the histone H2A protein isolated from chicken erythrocytes. In the 5' flanking region, a possible "TATA box" and three possible "cap sites" can be recognised upstream from the initiation codon. To the 5' side of the "TATA box" is found an unusual sequence of 21 A's interrupted by a central G residue. It occupies the same relative position as the P. miliaris H2A gene-specific 5' dyad symmetry sequence and the "CCAAT box" seen in other eukaryotic polymerase II genes but is clearly different from both. A significant feature of the 3' non-coding region is the presence of a 23 base-pair sequence that is nearly identical to a conserved region found in sea urchin histone genes. The coding region is extremely GC rich, with strong selection for these bases in the third position of codons. Not a single coding triplet ends in U. No intervening sequences were found in this gene.     

Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene.

We have isolated and characterized two independent clones containing the chicken adult beta-globin gene. Each clone contains a 6.2-kilobase-pair Eco RI restriction fragment of chicken erythrocyte DNA inserted into the vector, lambda gtWES . lambda B. The orientation of the inserted fragment is opposite in the two clones. Characterization of the clones by electron microscopic R-loop studies, by restriction enzyme mapping, and by filter hybridization shows that the adult beta-globin gene is interrupted by at least one small and one large intervening sequence. In addition to the complete adult beta-globin gene, at least part of a second beta-globin-like gene was identified about 2.7 kilobase pairs from the 3'-end of the adult gene. The two independent clones, while very similar, do differ at two Msp I restriction endonuclease sites in regions flanking the adult beta-globin gene.     

Isolation and characterization of five subfractions of chicken erythrocyte histone H1

Chicken erythrocyte chromatin contains, besides the specific histone H5, a set of histone H1 subtypes. Five of them were isolated by ion-exchange chromatography and these very related proteins, called H1A, H1B, H1C, H1D and H1E, were characterized by their amino acid compositions.     

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.