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.     

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.     

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.     

The rabbit beta-globin gene contains a large large insert in the coding sequence

We have used the rabbit β-globin DNA plasmid PβG1 (Maniatis et al., 1976) labeled with ³²P as a filter hybridization probe for DNA fragments containing the β-globin gene in restriction endonuclease digests of rabbit liver DNA. The β-globin DNA fragments we detect appear to contain the gene, present in PβG1 DNA, which codes for adult rabbit β-globin. These fragments have been ordered into a physical map of cleavage sites within and neighboring the structural gene in the rabbit genome (Jeffreys and Flavell, 1977). A detailed analysis of β-globin DNA fragments produced by cleavage with restriction endonucleases which are known to cut the β-globin gene has now shown that the β-globin structural gene is not contiguous in rabbit liver DNA, but is interrupted by a 600 base pair DNA segment inserted somewhere within the coding sequence for amino acid residues 101–120 of the 146 residue β-globin chain. Otherwise, the map of cleavage sites within the gene is co-linear with that deduced from the sequence of rabbit β-globin messenger RNA. Preliminary analysis indicates that this insert is also present in the β-globin gene in rabbit brain, kidney, spleen, bone marrow and sperm, and in erythroid cells isolated from the marrow of an anemic rabbit. The insert appears, therefore, to be a general property of the rabbit β-globin gene, even in tissues in which this gene is active, which suggests that the insert is not involved in inactivating the gene in nonerythroid tissues.     

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 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.     

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.     

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.