Conservation and variation in the large scale organisation of the globin gene domains of duck and chicken

Summary The genomic DNA of cloned recombinants containing the duck globin genes was compared to that of the analogous domains of the chicken. A 36 kb insert including the three alpha-type globin genes was isolated from a newly prepared duck genomic library in the cosmid PJB8; another recombinant contained a 45 kb insert with the four beta globin genes. In the alpha globin gene domain, the relative positions of genes, of repetitive sequences, and of the A+T-rich segments (AT-rich linkers, ATRLs) which frame the gene cluster (Moreau et al. 1982), were found to be closely maintained between duck and chicken. Although ATRLs and repetitive sequences also frame the gene cluster in the beta globin domains of duck and chicken, there is more genetic drift in their relative positions than in the alpha domain. It is of interest that several repetitive DNA segments were detected in the chicken beta globin domain which do not exist in corresponding positions in the duck. In view of the strict conservation in both species of genes and their relative positions in the cluster, this observation seems to exclude a simple function of repetitive sequences in the control of individual genes. The data are discussed with regard to the possible significance of repetitive and AT-rich DNA segments in genome organisation and function.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.     

Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi

A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library. The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3'. We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions. Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes. For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes. A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.     

Identification by nucleotide sequence analysis of a goat pseudoglobin gene

We have recently prepared a recombinant library of goat genomic DNA and have isolated clones containing th known goat globin genes. These include the alpha, gamma, beta C and beta A genes. In addition to these, another beta-like sequence has been observed. In this communication we report the complete nucleotide sequence of this gene, excluding a portion of the large intervening sequence. Several features suggest that this is a non-functional or pseudoglobin gene. The alterations include a frameshift mutation, substitution of the heme-binding histidines, a mutated termination codon, a change in the GT/AG excision sequence of the 5' end of the first intervening sequence, an AT rich sequence in the 3' untranslated region, and a mutated Hogness-Goldberg box. We conclude that this gene cannot function in the synthesis of globin.     

Structure of pyruvate dehydrogenase kinase. Novel folding pattern for a serine protein kinase

The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is of interest because it represents a family of serine-specific protein kinases that lack sequence similarity with all other eukaryotic protein kinases. Similarity exists instead with key motifs of prokaryotic histidine protein kinases and a family of eukaryotic ATPases. The 2.5-A crystal structure reported here reveals that pyruvate dehydrogenase kinase isozyme 2 has two domains of about the same size. The N-terminal half is dominated by a bundle of four amphipathic alpha-helices, whereas the C-terminal half is folded into an alpha/beta sandwich that contains the nucleotide-binding site. Analysis of the structure reveals this C-terminal domain to be very similar to the nucleotide-binding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.     

Correlations of repetitive and AT-rich DNA segments within the chicken globin gene domains

The repetitive DNA segments were mapped within a 30 Kbp genomic domain including (in 5 to 3 order) the chicken embryonic pi and adult alpha D (minor) and alpha A (major) globin genes. Two repeats map 5 and 8 Kbp upstream from the embryonic pi gene and another 3 Kbp downstream of the adult alpha A gene. These repetitive DNA sequences are placed within, or immediately adjacent to the AT-rich DNA segments framing this domain. Similar correlations exist also within the chicken beta globin gene domain. The positions of these AT-rich and repetitive DNA segments framing the alpha globin gene domain also correlate with other already explored features of long range DNA organisation, as clusters of sites of DNAse I hypersensitivity and differential methylation, sites of Matrix-DNA attachment, and with the beginning and end of the transcribed domain.     

Cloning and amplification of rabbit alpha- and beta-globin gene sequences into Escherichia coli plasmids.

cDNA, synthesized from rabbit globin mRNA, was used in a self-priming reaction, with avian myeloblastosis virus DNA polymerase, for the synthesis of double-stranded DNA. Globin DNA ranging from about 400 to 650 base pairs was elongated with dG tails using deoxypolynucleotide transferase and was annealed to linear Escherichia coli plasmic pCR1, elongated with dC tails. Preparation of the plasmid DNA involved an enzymatic reconstruction of one EcoRI-specific site on each side of the molecule. After transformation of E. coli cells to kanamycin resistance with the hybrid molecules, bacterial clones harboring recombinant plasmids were studied for the presence of globin-specific DNA. Plasmids containing either alpha or beta rabbit globin gene sequences were obtained. There was a 4-fold excess of recombinant plasmids containing beta-globin sequences over those with alpha-globin DNA. The longest beta-globin sequences found in plasmids were about 550 to 600 pairs long, and correspond therefore to the entire beta-globin structural gene and to some of the untranslated regions. The alpha-globin sequences were 400 to 450 base pairs long. Treatment of clone pCR1betarG 19 with EcoRI endonuclease released two DNA fragments (410 and 210 base pairs) resulting from cleavage at two reconstructed external EcoRI sites and at one internal EcoRI site within the rabbit globin gene. The same treatment of pCR1alpharG 11 released one fragment. In most other recombinant plasmids studied however, no fragment was released by EcoRI digestion.     

Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.     

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.     

Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts.     

Restriction mapping of cDNA recombinants including the adult chicken and duck globin messenger sequences: a comparative study

A comparison of the organization of six avian adult globin messenger sequences is based on previously reported recombinant duck adult globin cDNA plasmids (Therwath et al., 1980) and the actual construction and characterization of pBR322 recombinant plasmids including the beta and the normal alpha A and alpha D chicken adult globin mRNA sequences. Identification of the cloned DNA was performed using hybridization-selection under conditions permitting complete purification in one step of the three globin mRNAs, and translation of the corresponding mRNA. Orientation of the globin insert in the vector was determined, taking into account the computer prediction of the restriction sites based on the known amino acid sequences of the three globin chains (Roizès and Pelaquier, 1980) and those actually observed, and by identification of restriction fragments using 3'-specific probes. Identification, orientation and restriction mapping of these cloned DNAs reveals extensive homologies in organisation of beta sequences between duck and chicken, as well as among the alpha sequences in every two possible combinations.     

Protein structure and cDNA nucleotide sequence of the Japanese quail alpha A globin.

The primary structure of the alpha polypeptide chain (alpha A) of the major component (QII) of Japanese quail hemoglobin was determined by protein and cDNA sequence analysis. The amino-acid sequences of all the soluble tryptic peptides were determined by the conventional protein sequencing technology. The sequence of the remaining portion, which contained an insoluble "core region", was determined through determination of the cDNA nucleotide sequence. The cDNA clones coding for the alpha A globin were isolated from the quail reticulocyte cDNA library, mapped by restriction enzyme digestion, and the nucleotide sequence was determined completely. The primary structure of quail alpha A globin shows a close similarity to that of chicken alpha A globin.     

Expression and characterization of a human mitochondrial phenylalanyl-tRNA synthetase.

Human mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) has been identified from the human EST database. Using consensus sequences derived from conserved regions of the alpha and beta-subunits from bacterial PheRS, two partially sequenced cDNA clones were identified. Unexpectedly, sequence analysis indicated that one of these clones was a truncated form of the other. Detailed analysis indicates that unlike the (alphabeta)2 structure of the prokaryotic and eukaryotic cytoplasmic forms of PheRS, the human mtPheRS consists of a single polypeptide chain. This protein has been cloned and expressed in Escherichia coli. Gel filtration and analytical velocity sedimentation centrifugation indicate that the human mtPheRS is active in a monomeric form. The N-terminal 314 amino acid residues appear to be analogous to the alpha-subunit of the prokaryotic PheRS, while the C-terminal 100 amino acid residues correspond to a region of the beta-subunit known to interact with the anticodon of tRNAPhe. Comparisons with the sequences of PheRS from yeast and Drosophila mitochondria indicate they are 42 % and 51 % identical with the human mtPheRS, respectively. Sequence analysis confirms the presence of motifs characteristic of class II aminoacyl-tRNA synthetases. KM and kcat values for ATP:PPi exchange and for the aminoacylation reaction carried out by human mtPheRS have been determined. Evolutionary origins of this small monomeric human mtPheRS are unknown, however, implications are that this enzyme is a result of the simplification of the more complex (alphabeta)2 bacterial PheRS in which specific functional regions were retained.