Nucleotide sequence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysis of the complete alpha globin cluster.

In previous studies we identified and sequenced clones containing two adult alpha globin genes of the goat. Additional studies have revealed the presence of an embryonic alpha globin gene termed zeta. Sequence analysis of the gene shows that it is the largest mammalian or avian globin gene cloned to date. Its unusual size is mainly due to a 14 base-pair tandem repeat sequence in its first intron. A similar sequence is also found in the first intron of the human zeta gene. The goat zeta coding sequence differs greatly from that of the adult alpha, particularly at amino acid position 38, where it codes for the amino acid replacement of Gln for Thr. This change may confer a higher intrinsic O2 affinity on the zeta globin protein, ensuring a sufficient O2 supply for the developing goat embryo. The cloning and sequencing of this gene completes the alpha globin locus of the goat, composed of three genes in the following order 5'-zeta-I alpha-II alpha-3'. Evolutionary comparisons of the goat alpha locus with other amphibian, avian and mammalian loci reveal several interesting features. Statistical analysis confirms the hypothesis that the embryonic alpha gene is much older (400 million years) than the embryonic beta gene (200 million years), and that it is descended from a primordial gene, whose present-day counterpart is the Xenopus larval alpha globin gene. Our results also suggest that after the divergence of the avian line, the alpha A gene converted the alpha D gene during the evolution of the pre-mammalian line. The alpha D globin gene remains unconverted in the avian line, potentially because of insertion/deletion sequences that may prevent any gene conversion event. The divergence rates of specific globin genes have been analyzed and found to form an essentially straight line, in agreement with the neutralist view of evolution.

Structural organization of the alpha and beta globin loci of the goat.

Goats switch their hemoglobins during development in a manner similar to humans and thus provide a useful model system for studying the control of hemoglobin synthesis. Initially, goats synthesize embryonic hemoglobin, zeta 2 epsilon 2, which is replaced by fetal hemoglobin, alpha 2 beta F 2, as erythropoiesis moves to the liver and bone marrow. At birth, the fetal hemoglobin is replaced by juvenile hemoglobin, alpha 2 beta C 2, which in turn is replaced by adult hemoglobin, alpha 2 beta A 2, during the first year of life. In order to understand these switches, we have cloned the alpha and beta globin loci of goats. The alpha globin locus is composed of three genes, an embryonic and two adult genes, zeta-I alpha-II alpha. The beta globin locus is composed of twelve genes arranged in the following order, epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F. Close inspection of the beta globin locus indicates that it has arisen from a triplication of a four-gene set, epsilon-epsilon-beta-beta. Interestingly, the fetal globin gene has originated from an adult beta globin gene rather than from a second position gene as it has in humans. The gene at the end of the first four gene set, beta C, is expressed during pre-adult life while the gene at the end of the second set is the adult beta A gene. The last gene of the third set, beta F, is expressed during fetal development. Because the beta C, beta A and beta F genes have arisen quite recently during evolution, they have very similar nucleotide sequences. It is reasonable to assume that the few differences which are seen are important in developmental control. As one approach to defining regions involved in the regulation of the beta A, beta C and beta F genes their chromatin structure at different times of development has been characterized. Both DNase I sensitivity and accessibility to restriction endonucleases have been employed. While the entire beta globin locus is more sensitive to DNase in erythroid than non-erythroid cells, specific regions such as the 5' end of the genes are more accessible in cells expressing that particular gene.

Duplication of a four-gene set during the evolution of the goat beta-globin locus produced genes now expressed differentially in development.

Genomic clones which link the goat preadult (beta C) and adult (beta A) beta-globin genes have been isolated. These overlapping clones contain a previously unidentified embryonic like globin gene (epsilon III) and establish the following linkage map of eight genes in the goat beta-globin locus: epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A. This linkage map and the nucleotide sequence of the eight genes document a relatively recent duplication of a four-gene set: epsilon-epsilon-psi beta-beta. This duplication produced two genes (beta C and beta A) which are now expressed differentially during development. An embryonic like globin gene located downstream from beta A has also been isolated. The embryonic nature of this gene as well as the adult beta-like sequence of the goat fetal globin gene (gamma) suggest that a duplication of the four-gene set also produced the globin gene now expressed during fetal development.

Organization, structure, and expression of the goat globin genes.

Several hemoglobin switches occur during the development of the goat, making this a useful animal for the study of globin gene expression. In order to help understand the basis for these switches, we have isolated the beta-globin genes of the goat by recombinant DNA technology and characterized these genes with respect to linkage, nucleotide sequence, and expression. The linkage arrangement so far established is epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V. It is proposed that epsilon V is followed by epsilon VI-psi beta-gamma, but so far this linkage has not been established. Several conclusions can be drawn from our findings to date. First, the beta- and gamma-globin genes of the goat have a very different evolutionary history from the beta- and gamma-globin genes of humans. While the beta and gamma genes of the human can be traced to a duplication of the ancestral epsilon/beta-globin gene before the mammalian radiation, the goat beta and gamma genes have arisen much later, and are probably the results of a duplication of a four-gene set, namely the epsilon-epsilon-psi beta-beta primordial linkage group. The beta C gene probably arose from a similar, even later duplication of the non-gamma quadruplet. Because the beta C, beta A, and gamma genes of the goat have diverged much more recently in evolution, they are much more homologous than the equivalent genes in other species. In fact, there are large regions of these genes that share identical sequences. This is meaningful in that regions of sequence identity define areas that cannot be involved in the developmental regulation of these genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene conversion of two functional goat alpha-globin genes preserves only minimal flanking sequences.

We have determined the complete nucleotide sequence of the nonallelic adult goat I alpha- and II alpha-globin genes and, as is the case with the duplicated human alpha-to each other. Such high homology (99%) has most likely been preserved via a gene conversion mechanism. The conversion unit in goats is only about 9000 base pairs in length, and contained within this short region are all the known signals required for accurate and efficient transcription, with the CCAAT box adjacent to the 5'-boundary of the conversion unit and the poly(A) addition site adjacent to the 3' end. This conversion unit is also flanked by a 23-base-pair direct repeat "boundary sequence," vestiges of which are also observable in the human and mouse alpha-globin genes and pseudogenes. These direct repeats imply that a transposition-like event may have been responsible for the insertion of an ancestral alpha-like sequence into a new chromosomal locus, and that this insertion event and subsequent gene duplication may have predated the mammalian radiation.