Concerted evolution of the cow epsilon 2 and epsilon 4 beta-globin genes

The nucleotide sequences of the cow epsilon 2 and epsilon 4 globin genes were determined. The sequences were 95% identical. These genes arose via a four-gene block duplication that also gave rise to the bovine fetal (gamma) and adult (beta) genes. Their deduced amino acid sequences are unlike any previously reported fetal or adult globins; rather, comparison to other mammalian globin genes indicates that they are embryonic in nature. The sequence data indicate that these two genes have converted each other during evolution. Pairwise comparison to the corresponding goat genes shows greater similarity between paralogues than between more directly related orthologues. This is in direct contrast to the situation between the cow and goat fetal and adult genes. These observations suggest that the frequency of DNA conversion or the fixation of conversion events may vary in different locations of the cow beta-globin cluster.      

Isolation of the chicken beta-globin gene and a linked embryonic beta-like globin gene from a chicken DNA recombinant library.

A library of random chicken DNA fragments, 15-22 kb long, has been prepared in the vector lambda Charon 4A. This library was screened with combined adult and embryonic globin cDNA, and several independent globin gene-containing recombinants were isolated. One of these recombinants, lambda Chicken beta-globin 1 (lambda C beta G1), contains the adult chicken beta-globin gene and a closely linked embryonic beta-like globin gene. Both genes are transcribed in the same direction with the adult gene located 5' to the embryonic gene. Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to lambda C beta G1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian beta-globin genes. The adult beta-globin gene also contains a minor (approximately 100 bp long) intervening sequence analogous to the one observed in mammalian beta-globin genes. Restriction enzyme analysis of the adult beta-globin gene on lambda C beta G1 is consistent with the hypothesis that its two intervening sequences occur in the same positions with respect to the beta-globin amino acid sequence as do the corresponding mammalian intervening sequences.     

Complete amino acid sequences of the major early embryonic alpha-like globins of the chicken

Vertebrate embryos contain hemoglobins composed of globin polypeptides structurally distinct from those of adults. Together with fetal and adult globin chains, these early embryonic globins are encoded by two developmentally regulated multigene families. To facilitate analysis of the structure and evolution of early embryonic alpha-globin genes, we have determined the complete amino acid sequences of the pi and pi' alpha-like globins of the chick embryo. While differing from each other by an alanine/glutamic acid interchange at position 124, this pair of sequences differs from the major and minor adult alpha-globins by 43%. The early embryonic and adult alpha-like sequences appear to have diverged following an ancient gene duplication. We discuss specific amino acid substitutions in functional positions as possible mediators of the reduced Bohr effect and elevated oxygen affinity, which are characteristic of early embryonic hemoglobins.     

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species

The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.      

The phylogeny of human globin genes investigated by the maximum parsimony method

Gene phylogenetic trees were constructed by the maximum parsimony method for various sets of ninety six globin chain amino acid sequences spanning plant and animal kingdoms. The method, executed by several computer programs, constructed ancestor and descendant globin messengers on tree topologies which required the least number of nucleotide replacements to account for the evolution of the globins. The human myoglobin-hemoglobin divergence was traced to a gene duplication which occurred either in the first vertebrates or earlier yet in the common ancestor of chordates and annelids, the alpha-beta divergence to a gene duplication in the common ancestor of teleosts and tetrapods, the gamma divergence from typical beta chains to a gene duplication in basal therian mammals, and the delta separation from beta to a duplication in the basal catarrhine primates. Evidence was provided by the globin phylogenies for the hominoid affinities of the gibbon and the close phyletic relationship of the African apes to man. Over the period of teleos-tetrapod divergence the globin messengers evolved at an average rate of 18.5 nucleotide replacements per 100 codons per 10⁸ years, a faster rate than most previous estimates. Very fast and very slow rates were encountered in different globin lineages and at different stages of descent, reducing the effectiveness of globins as molecular clocks. Rates increased with gene duplication and decreased after selection discovered useful specializations in the products of genes which had previously been freer to accept mutations. The early eutherian radiation was characterized by rapid rates of globin evolution, but the later hominoid radiation by extremely slow rates. This pattern was related to more complicated grades of internal organization evolving in human ancestors. The types of nucleotide replacements in the globin messengers over the long course of globin evolution did not seem indicative of any special mutational mechanisms.     

Sequence of rat alpha- and gamma-casein mRNAs: evolutionary comparison of the calcium-dependent rat casein multigene family.

The complete sequences of rat alpha- and gamma-casein mRNAs have been determined. The 1402-nucleotide alpha- and 864-nucleotide gamma-casein mRNAs both encode 15 amino acid signal peptides and mature proteins of 269 and 164 residues, respectively. Considerable homology between the 5' non-coding regions, and the regions encoding the signal peptides and the phosphorylation sites, in these mRNAs as compared to several other rodent casein mRNAs, was observed. Significant homology was also detected between rat alpha- and bovine alpha s1-casein. Comparison of the rodent and bovine sequences suggests that the caseins evolved at about the time of the appearance of the primitive mammals. This may have occurred by intragenic duplication of a nucleotide sequence encoding a primitive phosphorylation site, -(Ser)n-Glu-Glu-, and intergenic duplication resulting in the small casein multigene family. A unique feature of the rat alpha-casein sequence is an insertion in the coding region containing 10 repeated elements of 18 nucleotides each. This insertion appears to have occurred 7-12 million years ago, just prior to the divergence of rat and mouse.     

A conserved nucleotide sequence, coding for a segment of the C-propeptide, is found at the same location in different collagen genes.

The nucleotide sequence of a segment of the chick alpha 1 type III collagen gene which codes for the C-propeptide was determined and compared with the corresponding sequence in the alpha 1 type I and alpha 2 type I collagen genes. As in the alpha 2 type I gene the coding information for the C-propeptide of the type III collagen gene is subdivided in four exons. Similarly, the amino proximal exon contains sequences for both the carboxy terminal end of the alpha-helical segment of collagen and for the beginning of the C-propeptide in both genes. Therefore, this organization of exons must have been established before these two collagen genes arose by duplication of a common ancestor. In several subsegments the deduced amino acid sequence for the C-propeptide of type III collagen shows a strong homology with the corresponding amino acid sequence in alpha 1 and alpha 2 type I. For one of these homologous amino acid sequences, however, the nucleotide sequence is much better conserved than for the others. It is possible that a mechanism of gene conversion has maintained the homogeneity of this nucleotide sequence among the interstitial collagen genes. Alternatively, the conserved nucleotide sequence may represent a regulatory signal which could function either in the DNA or in the RNA.     

Duplication/deletion polymorphism 5' - to the human beta globin gene.

DNA sequence analysis of the human beta globin locus has identified an array of simple tandem repeated sequences upstream from the beta globin structural gene. Comparison of several cloned human beta globin alleles demonstrated a high frequency of sequence heteromorphism at this site apparently due to duplication or deletion of single units of the repeat array. At least two such duplication/deletion events are necessary to account for the observed variation. No other sequence variation was observed, suggesting that duplication/deletion events within the tandem repeat array may be at least 13 to 14 times more frequent than nucleotide substitutions in the surrounding DNA.     

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.     

Cloning and sequence analysis of two embryonic beta-like globin cDNAs (y and z) of hamster.

A cDNA library was prepared from poly(A) mRNA extracted from 9-day hamster-yolk-sac erythroid cells. Two clones containing inserts coding for embryonic beta-like z or y globin-chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNAs and (b) nucleotide sequence analysis of the inserts and comparison to the embryonic beta-like globin genes of Balb/c mouse. Availability of sequences for embryonic and adult globin cDNAs will aid in investigations of the molecular mechanisms of the globin switch in hamster YSEC.     

Genomic organization and primary structure of five homologous pairs of intron-less genes encoding secretory globins from the insect Chironomus thummi thummi

From a Chironomus thummi thummi genomic library we have isolated two distinct recombinant phages, CttG-1 and CttG-3, each carrying a cluster of five homologous globin genes. In addition to the previously reported nucleotide sequence of globin gene D (Antoine and Niessing, 1984) we present the chromosomal arrangement, primary structure and predicted amino acid sequence of nine globin genes. The divergently transcribed globin genes all lack introns, they encode secretory preglobins each containing a highly conserved signal peptide. The amino acid sequences deduced from the globin genes correspond to globin III and variants thereof, to globin IV, and to a novel globin, whose direct amino acid sequence has not yet been reported.     

The evolution and sequence comparison of two recently diverged mouse chromosomal beta--globin genes

We have determined the entire nucleotide sequence of a cloned mouse beta--globinminor gene and compared it to the closely related sequence of the betamajor gene. These two genes differ by nine amino acids and presumably evolved from a common ancestral gene as recently as 50 million years ago. Since these genes are closely linked and coordinately expressed, they provide an especially favorable opportunity to assess selection and mutation as these processes affect genes under similar constraints. We find that evolution has preserved these two genes in two short segments of DNA which include their immediately adjacent flanking regions. These regions presumably encode functions that are necessary for proper globin gene expression. In contrast, the more distal flanking sequences and major segments of the long intervening sequences have diverged much more sharply. The homology pattern in these genes also provides considerable insight into the mechanisms by which less constrained nucleotide sequences diverge rapidly. Change in such regions apparently occurs less by point mutation than by insertion, deletion and duplication of relatively short segments of the genome.     

Analysis of the evolution of angiotensin II type 1 receptor gene in mammals (mouse, rat, bovine and human).

The nucleotide and amino acid sequences for mouse angiotensin II (AII) type 1A and 1B receptors were deduced from their complementary and genomic DNAs. Evolutionary analyses based on the nucleotide sequences of the coding region of AII type 1 receptor genes indicated that the duplication event of the type 1 gene occurred 24 +/- 2 million years ago before the divergence between the rat and mouse but after the divergence between rodents and the human/artiodactyls couple. This conclusion was consistent with the results of genomic Southern blot analyses, which revealed that the mouse and rat possess 2 similar but separate genes, whereas the bovine and human have only a single class gene.     

flaA-like sequences containing internal termination codons (TAG) in urease-positive thermophilic Campylobacter isolated in Japan

AIMS: To demonstrate two flaA-like sequences containing two internal termination codons (TAG) in two Japanese strains of urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: A primer pair of A1 and A2, which ought to generate a product of approx. 1700 bp of the flaA gene for Campylobacter jejuni, was used to amplify products of approx. 1450 bp for two Japanese strains of UPTC, CF89-12 and CF89-14. After molecular cloning and sequencing, the nucleotide sequences of the amplicons from the two strains were found to be 1461 bp in length and to have nucleotide sequence differences in relation to each other at four nucleotide positions, respectively. CONCLUSIONS: Nucleotide and amino acid sequence alignment and homology analysis demonstrated that the polymerase chain reaction (PCR) amplicons from the two Japanese strains have approx. 83% nucleotide and 80% amino acid sequence homology to the possible open reading frame of the flaA gene of UPTC NCTC 12892. SIGNIFICANCE AND IMPACT OF THE STUDY: Surprisingly, both PCR amplicons from the Japanese UPTC have two internal termination codons (TAG) at nucleotide positions from 775 to 777 and 817 to 819, respectively.     

Cloning and sequence analysis of cDNA for rat liver uricase

We have isolated cDNA clones for rat liver uricase using an oligonucleotide corresponding to the N-terminal sequence of 8 amino acids. The nucleotide sequences of the cDNAs have been determined, and the amino acid sequence of the protein deduced. A 867-base open reading frame coding for 289 amino acids, corresponding to a molecular mass of 33,274 daltons, was confirmed by matching eight sequences of a total of 53 amino acids from peptide sequence analyses of the fragments generated by lysyl endopeptidase digestion of purified rat liver uricase. The deduced amino acid sequence of rat liver uricase shares 40% homology with that of soybean nodulin-specific uricase and has an N-terminal extension of 7 amino acids. In contrast, soybean uricase has a C-terminal extension of 12 amino acids, which is presumably the result of local gene duplication. Completely different N- and C-terminal structures of the two uricases suggest that the signals for targeting the proteins to the peroxisome are not located on the terminal continuous stretches of amino acids.     

Human apolipoprotein E mRNA. cDNA cloning and nucleotide sequencing of a new variant

The complete nucleotide sequences of three cloned cDNAs corresponding to human liver apolipoprotein E (apo-E) mRNA were determined. Analysis of the longest cDNA showed that it contained 1157 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 61 nucleotides, a signal peptide region corresponding to 18 amino acids, a mature protein region corresponding to 299 amino acids, and a 3'-terminal nontranslated region of 142 nucleotides. The inferred amino acid sequences from two cDNAs were identical and corresponded to the amino acid sequence for plasma apo-E3 that has been reported previously ( Rall , S. C., Jr., Weisgraber , K. H., and Mahley , R. W. (1982) J. Biol. Chem. 257, 4171-4178). The third cDNA differed from the other two cDNAs in five nucleotide positions. Three of these differences occurred in the third nucleotide position of amino acid codons, resulting in no change in the corresponding amino acids at residues Val-85, Ser-223, and Gln-248. The other two altered nucleotides occurred in the first nucleotide position of codons, leading to changes in the amino acids encoded. In the variant sequence, a threonine replaced the normal alanine at residue 99 and a proline replaced the normal alanine at residue 152. We have concluded that the human liver donor was heterozygous for the epsilon 3 genotype. The variant cDNA corresponds to a new, previously undescribed variant form of apo-E in which the amino acid substitutions of the protein are electrophoretically silent; it would probably be undetectable by standard apo-E phenotyping methods. The amino acid substitution at position 152 occurs in a region of apo-E that appears to be important for receptor binding, and it may have clinical significance.     

Xenopus laevis integrins. Structural conservation and evolutionary divergence of integrin beta subunits

We report the sequences of cDNA clones for two different integrin beta subunits isolated from a Xenopus laevis neurula cDNA library. mRNAs corresponding to both genes are first detected at gastrulation. We show that these two beta subunits are very highly related (98% identity in amino acid sequence) and probably arose at the time of tetraploidization of the X. laevis genome around 50 million years ago. Comparison of these sequences with those of various other vertebrate integrin beta subunit establishes that all species analyzed to date contain a highly conserved integrin beta subunit (beta 1). The interspecies homologies within this class of integrin beta subunits (82-86% identity in amino acid sequence) are much greater than those among the three different beta subunits which are known in humans (40-48% identity in amino acid sequence). Analysis of the homologies clearly indicates duplication and divergence of this multigene family more than 500 million years ago prior to the appearance of the vertebrates. We also observe cross-hybridization between cDNA probes for chicken integrin beta subunits and genomic DNAs of several invertebrate species. Despite the divergence in sequence among different integrin beta subunits, certain features of their structure are remarkably conserved.