Isolation and sequence analysis of a hybrid delta-globin pseudogene from the brown lemur

The β-globin gene cluster of the brown lemur, a prosimian, is very short and contains a single ?-, γ- and β-globin gene, with an additional β-related gene sequence between the γ- and β-globin genes. Brown lemur DNA was cloned into the bacteriophage vector λL47.1 and a recombinant was isolated which contained an 11 × 10³ base insert including the β-globin gene and the additional putative β-globin pseudogene. The nucleotide sequence of this β-related gene was completely determined. A complete gene sequence was found, containing four frameshift mutations sufficient to establish its pseudogene status. The gene was interrupted by two intervening sequences with sizes and locations typical of mammalian β-related globin genes. The pseudogene sequence was compared in detail with human ?-, γ-, δ- and β-globin genes. The beginning of the pseudogene, from the 5′ flanking region to the second exon, was homologous to the corresponding regions of the human ?- and γ-globin genes. In contrast, the second intron, third exon and 3′ flanking region showed a remarkably close homology to the δ-globin, but not β-globin, gene of man. This suggests that the δ-globin gene is not the product of a recent gene duplication, but instead is present in most or all primates. This gene has been silenced on at least two separate occasions in primate evolution (in lemurs and in old world monkeys). In addition, the 5′ end of the lemur ψδ gene appears to have exchanged sequences with an ?- or γ-globin gene, and an analogous exchange with the β-globin gene seems to have occurred recently in the human δ-globin gene. The evolution and function of the δ-globin gene are discussed.     

Organization of human δ- and β-globin genes in cellular DNA and the presence of intragenic inserts

We have analyzed human cellular DNA for its δ- and β-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with ³²P-β-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3′ end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the δ and β genes and those containing sequences flanking either 5′ to the δ gene or 3′ to the β gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, δ- and β-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.     

Gene evolution in the chicken β-globin cluster

We have determined the cDNA sequence of the chicken embryonic β-like ?-globin gene. Comparison with the sequences of the chicken ρ-globin and β-globin genes reveals the presence of two regions that are identical or nearly identical in ? and ρ. The first contains the 5′ untranslated sequence and exon 1, while the second region includes the second half of axon 2. Outside these regions ρ and ? are less homologous to each other than to the adult β-globin gene. The embryonic ρ and ? genes are located at opposite ends of the β-globin-gene cluster, not contiguously as are all other known pairs of simultaneously expressed globin genes. We suggest a role for gene conversion in the synchronization of expression of two highly diverged genes.     

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.     

Partial sequence analysis of Xenopus alpha- and beta-globin mRNA as determined from recombinant DNA plasmids

Recombinant plasmids containing Xenopus globin mRNA sequences have been constructed using the mRNA:cDNA hybrid conditions of Zain et al. (1979, Cell16, 851–861). The partial nucleotide sequence of two of these recombinants has been determined. They have been identified as containing α- and β-globin-like sequences by homology to other amphibian globin proteins. The nucleotide sequence of these recombinants permits the comparison of conserved regions in both the coding and 3′ nontranslated regions of Xenopus globin mRNAs with the known sequences of other eukaryotic globin proteins and mRNAs. Among the features which have been conserved though evolution is the sequence AAUAAA close to the 3′ terminus of the nontranslated region. Extensive regions of homology occur between the 3′ nontranslated regions of Xenopus α- and β-globin mRNA.     

Restriction enzyme analysis of the β-globin gene in DNA from β°-thalassaemic subjects from Ferrara

The map of restriction sites including and surrounding the δ- and β-globin genes has been established for three Ferrara β°-thalassaemic subjects. The fragments obtained using nine restriction enzymes do not show any differences from normal DNA. Among others, restriction enzymes giving short fragments at the 5′ and 3′ ends of the β-globin structural gene have been employed. The results obtained for the thalassaemic DNA are identical to those for control DNA, thus excluding the presence of extensive deletions in or adjacent to the coding regions of the β-globin gene in Ferrara β°-thalassaemia.     

A molecular description of telometic heterochromatin in secale species

We report the nucleotide sequence of a rabbit β-globin pseudogene, ψβ2. A comparison of the ψβ2 sequence with that of the rabbit adult β-globin gene, β1, reveals the presence of frameshift mutations and premature termination codons in the protein coding sequence which render ψβ2 unable to encode a functional β-globin polypeptide. ψβ2 contains two intervening sequences at the same locations in the globin protein coding sequence as β1 and all other sequenced β-globin genes. An examination of the DNA sequences at the intron/exon junctions suggests that a putative ψβ2 precursor mRNA could not be spliced normally. We compare the flanking and noncoding sequences of ψβ2 and β1 and discuss the evolutionary relationship between these two genes.     

Secondary structure of mouse and rabbit α- and β-globin mRNAs: Differential accessibility of α and β initiator AUG codons towards nucleases

The nucleotide sequence from the 5′ terminus inward of one third of mouse α- and β^maj-globin messenger RNAs has been established. In addition, using 5′ ³²P end-labeled mRNAs as substrates and S1 and T1 nucleases as probes for single-stranded regions, the secondary structures of mouse and rabbit α- and β-globin mRNAs have been analyzed. Our results indicate that the AUG initiator codon in both mouse and rabbit β-globin mRNA is quite susceptible to cleavage with S1 and T1 nucleases, suggesting that it resides in a single-stranded exposed region. In contrast, the initiator AUG in the α-globin mRNA of both species is inaccessible to cleavage, indicating that it is either buried by tertiary structure or is base-paired. Since the rate of initiation of protein synthesis with β-globin mRNA in rabbit reticulocyte is 30–40% faster than for α-globin mRNA, these results imply a possible correlation between the differential rates of initiation with these two mRNAs and the accessibility of the respective AUG initiator codons.     

The primary structure of the human ϵ-globin gene

We describe the complete nucleotide sequence of the human ?-globin gene including 387 nucleotides of 5′ flanking sequence and 301 nucleotides of 3′ flanking sequence. The arrangement of coding, noncoding and intervening sequences in this gene is entirely consistent with its identification as the embryonic β-like globin gene.     

Tertiary structural analysis of the elongated part of an abnormal hemoglobin, hemoglobin Pakse

Hemoglobin variants in which a frameshift results in chain elongation are unusual. Hemoglobin Pakse (Hb Pakse) is an unstable hemoglobin with abnormal elongation, first described in Indochina. An alpha2-globin gene termination codon mutation, TAA -->TAT or Term -->Tyr, has been described in the pathogenesis of Hb Pakse. This abnormality causes a frameshift that elongates the alpha chain amino acids. Computer-based protein structure modeling was used in a bioinformatics analysis of the tertiary structure of these elongated amino acid sequences. The elongated part of Hb Pakse showed additional helices, which may cause the main alteration in Hb Pakse. Abnormalities in the fold structure of globin in Hb Pakse were identified, and helices additional to the normal alpha globin chains were shown in the elongated part of Hb Pakse.     

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.     

Linkage of adult α- and β-globin genes in X. laevis and gene duplication by tetraploidization

We have used cloned adult X. laevis α- and β-globin cDNAs to analyze globin genes in X. laevis DNA. We detected α¹- and β¹-globin genes which contain intervening sequences and code for the major adult globins, plus additional diverged α²- and β²-globin genes of unknown coding potential. Unlike the case in mammals, the X. laevis α¹- and β¹-globin genes are closely linked and occur in the sequence 5′-α¹-9 kb-β¹-3′. The α²- and β²-globin genes are also linked, and analysis of globin genes in X. tropicalis suggests that this duplication of an α-β-globin gene pair in X. laevis is the result of chromosome duplication by tetraploidization. The close linkage of α- and β-globin genes in Xenopus provides evidence that vertebrate α- and β-globin genes evolved by tandem duplication of a single primordial globin gene.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.