Evolution of the β-globin gene cluster in man and the primates

The arrangement of primate β-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to man. The arrangement of the entire ?γγδβ-globin gene cluster in the gorilla and the yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequence divergence of approximately 1% in 5 million years. A new world monkey (owl monkey) has a single γ-globin gene, suggesting that the ^Gγ-^Aγ-globin gene duplication in man is ancient, and occurred about 20 to 40 million years ago. The β-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 20,000 base-pairs) and contains single ?-, γ- and β-globin genes. The γ- and β-globin genes in this animal are separated by a curious gene containing the 3′ end of a β-globin gene preceded by sequences related to the 5′ end of the ?-globin gene.     

The sequence of the chromosomal mouse β-globin major gene: Homologies in capping,splicing and poly(A) sites

We have determined the entire nucleotide sequence of a cloned β-globin^maj gene derived from the BALB/c mouse. This sequence is 1567 bases long and includes the 5′ cap region as well as the presumptive poly(A) addition site of β-globin mRNA. The sequence establishes the fact that the gene is encoded in three discontinuous segments of DNA interrupted by two intervening sequences and precisely locates each. The smaller intervening sequence, 116 bases long, occurs between Arg and Leu codons at codon positions 30 and 31. The larger intervening sequence of 646 bases also occurs between Arg and Leu codons, but at codon positions 104 and 105. There is striking homology between the borders of the two intervening sequences, but no extensive dyad symmetry. Furthermore, the DNA region that just precedes and overlaps the 5′ cap structure of the mRNA shows homology to corresponding regions in other eucaryotic genes including the late adenovirus promoter. The 3′ untranslated sequence is closely homologous to that of the rabbit β-globin mRNA. The sequence thus allows us to identify several noncoding regions of potential importance for the expression and processing of genetic information. It also provides a basis for future comparison with other sequenced genes and a defined substrate for the development of direct tests of gene function.     

The adult β-globin genes of the “single” type mouse C57BL

A set of four clones containing the two adult β-globin genes of the “single” type mouse C57BL/10 (genotype Hbb^s/Hbb^s) were isolated from a library of cloned restriction fragments. The two genes, designated βs and βt, were physically mapped onto a 32 kb segment of the chromosome carried by the four clones. βs and βt form a stable heteroduplex 1850 bp long, indicating that they are intact and conserved at this level of resolution throughout their length, including their intervening sequences. The βs gene is allelic with the βdmaj gene of the BALB/ c mouse (genotype Hbb^d/Hbb^d). These two alleles, as well as their surrounding sequences, are highly conserved. In contrast, heteroduplexes of βt with its BALB/c allele, βdmin, revealed three extensive but localized rearrangements. One region of non-homology falls within the large intervening sequence, IVS2. To the 5′ side of the βt/βdmin gene position two unequal substitutions were observed; each results in the net insertion of about 1000 bp into the Hbb^d chromosome. The β/βdmin gene position is bracketed by a 1450 bp inverted repeat. One of the 1000 bp substitutions maps within this inverted repeat.     

Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNA^Asn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol?γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol?γ from yeast to human.     

Evolution of the mouse beta-globin genes: a recent gene conversion in the Hbbs haplotype

We have determined the complete nucleotide sequence of the two nonallelic adult beta-globin genes of the C57BL/10 mouse. These genes, designated beta s and beta t, show a sequence similarity of 99.6% over the region bordered by the translational start and stop codons. Both beta s and beta t encode functional polypeptide chains that are identical. A comparison of the C57BL/10 beta-globin haplotype, Hbbs, with that of the BALB/c mouse, Hbbd, suggests that the two haplotypes have distinct evolutionary histories. The two adult beta-globin genes of the Hbbd haplotype, beta dmaj and beta dmin, are 16% divergent at the nucleotide level and encode distinct polypeptides that are synthesized in differing amounts. Our analysis indicates that a gene correction mechanism has been operating on the Hbbs chromosome to keep beta s and beta t evolving in concert, whereas on the Hbbd chromosome, beta dmin has diverged considerably from beta dmaj. We suggest that gene conversion is responsible for the maintained similarity of the Hbbs genes. Furthermore, we attribute the divergence of the Hbbd genes in part to the absence of a region of simple-sequence DNA within the large intervening sequence of beta dmin. We propose that this region of DNA plays a role in facilitating gene conversion. The deletion of this area in beta dmin introduced a block of nonhomology between the beta dmaj-beta dmin gene pair and thus may have inhibited further gene correction within the Hbbd haplotype.      

Structure of the chicken interferon-γ gene,and comparison to mammalian homologues

The sequence of the chicken interferon-γ (ifn-γ) gene was determined, one of the first non-mammalian cytokine gene structures to be elucidated. Initial genomic clones were amplified from chicken genomic DNA and were used to isolate a cosmid clone covering the entire gene for sequencing. The exon:intron structure of chicken ifn-γ is very similar to those of its mammalian homologues, with the exception of the third intron, which is markedly shorter in the chicken. The first exon contains both 5′ UTR and signal sequence and the first 22 aa of the mature protein. The remainder of the coding region lies in exons 2–4. Exon 4 also encodes the stop codon and the 3′ UTR, including two possible polyadenylation signals. A number of potential regulatory sequences similar to those found in mammals have been identified, in the promoter, in each intron and in the 3′ UTR. In the promoter, these include the TATAATA- and CCAT-boxes, a consensus GATA motif in the reverse orientation and a potential NF-κB binding site. Other regulatory elements identified in the promoters of mammalian ifn-γ genes are absent. Internal to the gene structure, regulatory sequences identified include elements found in the DNase I hypersensitivity region of the first intron of the human ifn-γ gene and several potential NF-κB binding sites. The 3′ UTR contains an AT-rich sequence, including nine repeats of the `instability' motif ATTTA. As in mammals, chicken ifn-γ is a single copy gene. The gene is highly conserved, with no polymorphisms yet identified using either RFLP or SSCP in the coding region. However, promoter sequence polymorphisms between different inbred lines of chickens have been identified, with possible links to disease resistance.     

Engineering EGFP reporter constructs into a 200 kb human β-globin BAC clone using GET Recombination

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the β-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the β- and δ-globin genes also allowed the simultaneous generation of a δ-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the γ-globin genes and the termination codon of the β-globin gene enabled the generation of reporter constructs for both ^Aγ- and ^Gγ-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the - and β-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.     

Serum-mediated suppression of cell-associated plasminogen activator activity in cultured endothelial cells

Yeast strains harboring independent mutations within the SUP4 tyrosine tRNA gene have been selected by virtue of their inactivating effect upon the SUP4-o UAA suppressor. Three fourths of the mutations at SUP4 are point alterations; the rest resemble the deletions described by Rothstein (1979). A meiotic genetic fine structure map of the locus was made by crossing 69 of the mutants in all combinations and testing for the frequency of SUP4-o recombinants. The sequences of SUP4 genes cloned from 32 mutant strains were determined by the dideoxynucleotide terminator method, using as primer a synthetic oligodeoxynucleotide corresponding to a sequence adjoining the SUP4 3′ terminus. The positions of the DNA sequence alterations showed good colinearity with the positions of the mutations on the genetic map. One of the 26 mutant sites found by DNA sequencing lies within the intervening sequence. At this site three repeat mutations were found, each changing AT → TA. Whereas mutations were generally rather uniformly distributed throughout the tRNA^Tyr coding sequence, none occurred in the DNA sequences flanking the mature tRNA^Tyr sequence or in a 12 nucleotide sequence including the 10 bp which constitute the 3′ side of the intervening sequence.     

Comparison of the sequences of the nagE operons from Klebsiella pneumoniae and Escherichia coli K12: enhanced variability of the enzyme IIN-acetylglucosamine in regions connecting functional domains.

Summary The nagE operon, encoding the enzyme II specific for N-acetylglucosamine (EII^Nag), and adjacent DNA from the chromosome of Klebsiella pneumoniae were sequenced and compared with the corresponding sequence from Escherichia colt K12. The deduced EII^Nag sequences differ in 72 out of 651 amino acids, the K. pneumoniae sequence being three residues longer. The amino acid differences were distributed unevenly, and were most frequent in regions connecting the three functional domains of the protein. In the nagE-nagB intergenic region, two promoter, two operator, and one CAP consensus sequence with regulatory functions were highly conserved. The nag structural genes from both species were very similar (83% DNA similarity; 89% amino acid similarity) except for frequent AT to GC exchanges in the wobble base of codons in K. pneumoniae DNA relative to the E. coli DNA.     

The sequence of the gorilla fetal globin genes: evidence for multiple gene conversions in human evolution

Two fetal globin genes (G gamma and A gamma) from one chromosome of a lowland gorilla (Gorilla gorilla gorilla) have been sequenced and compared to three human loci (a G gamma-gene and two A gamma-alleles). A comparison of regions of local homology among these five sequences indicates that long after the duplication that produced the two nonallelic gamma-globin loci of catarrhine primates, about 35 million years (Myr) ago, at least one gene conversion event occurred between these loci. This conversion occurred not long before the ancestral divergence (about 6 Myr ago) of Homo and Gorilla. After this ancestral divergence, a minimum of three more gene conversion events occurred in the human lineage. Each human A gamma-allele shares specific sequence features with the gorilla A gamma-gene; one such distinctive allelic feature involves the simple repeated sequence in IVS 2. This suggests that early in the human lineage the A gamma-genes may have undergone a crossing-over event mediated by this simple repeated sequence. The DNA sequences from coding regions of both G gamma- and A gamma-loci, a comparison of 292 codons in the corresponding gorilla and human genes, show an unusually low evolutionary rate, with only two nonsilent differences and, surprisingly, not even one silent substitution. The two nonsynonymous substitutions observed predict a glycine at codon 73 and an arginine at codon 104 in the gorilla A gamma-sequence rather than aspartic acid and lysine, respectively, in human A gamma. Because only arginine has been found at position 104 in gamma-chains of Old World monkeys, it may represent the ancestral residue lost in gorilla and human G gamma-chains and in the human A gamma-chain. Possibly the arginine codon (AGG) was replaced by the lysine codon (AAG) in the G gamma-gene of a common ancestor of Homo and Gorilla and then was transferred to the A gamma-gene by subsequent conversions in the human lineage. DNA sequence conversions, similar to that attributed to the fetal gamma-globin genes, appear to be relatively frequent phenomena and, if widespread throughout the genome, may have profound evolutionary consequences.      

Evidence for Various Types of Synthesis of Human γ Chains of Haemoglobin in Acquired Haematological Disorders

AT first, the γ chain of human haemoglobin seemed to be a constant chemical species¹, but Schroeder et al. showed that two or possibly four of the γ chain loci which control the synthesis of the two types of chains, ^Aγ and ^Bγ, are active in new born infants². The two chains differ only at position 136, where ^Aγ has alanine and ^Gγ has glycine. Thus although they are inseparable by chromatography and electrophoresis, their relative concentrations can be estimated from the ratio of these two amino-acids in the third and last peptide (γ^CB3) produced by hydrolysis of the chains by cyanogen bromide. Schroeder et al. studied the variations in the levels of these two chains during the normal ontogenic development and in several cases of hereditary persistence of foetal haemoglobin (HPHF) and β thalassaemias^3–5. We have studied the γ chains synthesized in different types of acquired anaemia with increased amounts of foetal haemoglobin, of which haemoglobin F constitutes between 1 and 15%. Such anaemias include refractory anaemias with excess myeloblasts (RAEM), acquired sideroblastic idiopathic anaemias (ASIA), idiopathic aplastic anaemias and some varieties of leukaemias, especially juvenile myeloid leukaemias (JML).