Nucleotide sequence of the rat gamma-crystallin gene region and comparison with an orthologous human region

The sequences of a 51-kb region containing the cluster of five rat gamma-crystallin-coding genes (CRYG) and of a 7-kb region surrounding the sixth rat CRYG gene were determined. Approximately 78% of the total sequence represents intergenic DNA. We also sequenced 22 kb of DNA from the human CRYG gene cluster. All CRYG genes are associated with CpG-rich regions. The sequence similarity between the human and rat gene regions drops sharply (to 65%) in intronic and 3'-flanking regions but decreases only gradually in the 5'-flanking region. Highly conserved regions (greater than 80%) are found as far upstream as 1.5 kb. Overall intergenic distances are conserved. The human region contains much more repetitive DNA (24% vs. 10%) but less simple-sequence (sps) DNA (0.7% vs. 4%) than the rat region. Almost all repeats and spsDNA elements are located in the intergenic region. The location of repetitive and spsDNA differs between the orthologous regions and these elements were probably inserted after the evolutionary separation of rat and man. The Alu repeats in man and the B3 repeats in the rat are close copies of their respective consensus sequences and bordered by virtually perfect repeats. In contrast, the B1 and B2 repeats in the rat have diverged considerably from the consensus sequence and the surrounding direct repeats are usually imperfect. Thus the dispersion of the B1 and B2 repeats in the rat probably preceded that of the B3 repeats. Within the rat genomic region the spacing of Z-DNA elements is surprisingly regular, they are located about 12 kb apart. A search for putative matrix-associated regions suggests that the rat CRYG gene cluster is organized into two chromosomal domains.     

Comparison of the nucleotide sequence of cloned human and guinea-pig pre-alpha-lactalbumin cDNA with that of chick pre-lysozyme cDNA suggests evolution from a common ancestral gene.

Nucleotide sequence analyses of essentially full-length copies of human and guinea-pig pre-alpha-lactalbumin cDNAs contained within recombinant plasmids, (i) confirm the presence of 19 amino acid hydrophobic amino terminal peptide extensions encoded within each mRNA; and (ii) provides evidence for the existence of a minor variant of guinea-pig alpha-lactalbumin mRNA encoding a protein with a 36 residue carboxyl-terminal extension. Comparison of the nucleotide sequence within the coding region of the human, and the predominant guinea-pig pre-alpha-lactalbumin mRNAs, with the analogous region of hen pre-lysozyme mRNA provides compelling evidence that all have evolved from a common ancestral gene.     

Structural organization and complete sequence of the human alpha-N-acetylgalactosaminidase gene: homology with the alpha-galactosidase A gene provides evidence for evolution from a common ancestral gene.

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; EC 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter. The deficient activity of this enzyme results in Schindler disease, an autosomal recessive disorder characterized by the increased urinary excretion of glycopeptides and oligosaccharides containing alpha-N-acetylgalactosaminyl moieties. Recently, the 3.6-kb full-length alpha-GalNAc cDNA sequence was isolated and found to have remarkable nucleotide and predicted amino acid homology (55.8 and 46.9%, respectively) with the human alpha-galactosidase A (alpha-Gal A) cDNA. To investigate the possible evolutionary relatedness of the two glycosidases, the alpha-GalNAc chromosomal gene was isolated and characterized. Screening of a human genomic DNA cosmid library resulted in the identification of a clone, gAGB-1, with an approximately 35-kb insert that contained the entire alpha-GalNAc gene. A single approximately 15-kb EcoRI fragment of gAGB-1, which contained the complete 3.6-kb cDNA sequence, was digested and the subcloned fragments were sequenced in both orientations. The 13,709-bp alpha-GalNAc gene had nine exons ranging from 95 to 2028 bp and intronic sequences of 304 to 2684 bp. All exon/intron junctions conformed to the GT/AG consensus rule. Analysis of 1.4 kb of 5' flanking sequence revealed three Sp1 and two CAAT-like promoter elements. This region was GC-rich (56%), but no HTF island was identified. The gene contained six Alu-repetitive elements, all in the reverse orientation. Comparison of the structural organization of the alpha-GalNAc and the alpha-Gal A genes revealed that all six alpha-Gal A introns were identically positioned in the homologous alpha-GalNAc exonic sequence. Two additional introns, 1 and 8, were identfied in the alpha-GalNAc gene. The predicted amino acid sequences of alpha-GalNAc exons 2 through 7 and those of corresponding alpha-Gal A exons 1 through 6 were 46.2 to 62.7% identical. In contrast, there was little, if any, similarity between the deduced amino acid sequences of alpha-Gal A exon 7 and alpha-GalNAc exons 8 and 9. The remarkable amino acid identity and the identical exonic interruption by six introns of the alpha-GalNAc and alpha-Gal A genes suggest that this region in both genes is evolutionarily related and arose through duplication and divergence from a common ancestral gene.     

Estimating evolution of temporal sequence changes: a practical approach to inferring ancestral developmental sequences and sequence heterochrony

Developmental biology often yields data in a temporal context. Temporal data in phylogenetic systematics has important uses in the field of evolutionary developmental biology and, in general, comparative biology. The evolution of temporal sequences, specifically developmental sequences, has proven difficult to examine due to the highly variable temporal progression of development. Issues concerning the analysis of temporal sequences and problems with current methods of analysis are discussed. We present here an algorithm to infer ancestral temporal sequences, quantify sequence heterochronies, and estimate pseudoreplicate consensus support for sequence changes using Parsimov-based genetic inference [PGi]. Real temporal developmental sequence data sets are used to compare PGi with currently used approaches, and PGi is shown to be the most efficient, accurate, and practical method to examine biological data and infer ancestral states on a phylogeny. The method is also expandable to address further issues in developmental evolution, namely modularity.     

Contribution of homoplasy and of ancestral polymorphism to the evolution of genes in anthropoid primates

Molecular phylogenies of lineages that split from one another in short succession are often difficult to resolve because different loci and different sites within the same locus yield incongruent relationships. The incongruity is commonly attributed to two causes: differential assortment of ancestral polymorphisms and homoplasy. To assess the relative contribution of these two causes, sequences of 57 segments from 51 loci in six primate lineages (human, chimpanzee, gorilla, orangutan, macaque, and tamarin, abbreviated as H, C, G, O, M, and T, respectively) were subjected to "partitioning" analysis, in which phylogenetically informative sites were identified in all 15 pairwise comparisons of each of the 57 segments and tallied for their support or lack thereof for each of the theoretically possible phylogenies. The six lineages include one of the best known cases of a difficult-to-resolve phylogeny: the trichotomy (H, C, G), in which the three lineages may have diverged from each other within a short period of time. In this period many of the ancestral polymorphisms apparently persisted and yielded phylogenetically incongruent signals. By contrast, no ancestral polymorphism is expected to have survived during the interval separating the divergences of the O, M, and T lineages from the ancestor of the (H, C, G) group. Any phylogenetic incompatibilities at sites in the O, M, and T lineages relative to the (H, C, G) group are therefore presumably the result of homoplasy. The frequency of homoplasy estimated in this manner is unexpectedly high: 12% for the (H, C, G) clade and 19% for the (H, C, G, O) clade. At least three-quarters of the 48% incompatibility observed in the (H, C) clade is attributable to the sorting out of ancestral polymorphisms coupled with intragenic recombination. Possible reasons for this high level of homoplasy in the O, M, and T lineages are discussed, and a computer simulation has been carried out to produce a model explaining the observed data.     

Evidence that the ancestral haplotype in Australian hemochromatosis patients may be associated with a common mutation in the gene.

Hemochromatosis (HC) is a common inherited disorder of iron metabolism for which neither the gene nor biochemical defect have yet been identified. The aim of this study was to look for clinical evidence that the predominant ancestral haplotype in Australian patients is associated with a common mutation in the gene. We compared indices of iron metabolism and storage in three groups of HC patients categorized according to the presence of the ancestral haplotype (i.e., patients with two copies, one copy, and no copies of the ancestral haplotype). We also examined iron indices in two groups of HC heterozygotes (those with the ancestral haplotype and those without) and in age-matched controls. These analyses indicate that (i) HC patients with two copies of the ancestral haplotype show significantly more severe expression of the disorder than those with one copy or those without, (ii) HC heterozygotes have partial clinical expression, which may be influenced by the presence of the ancestral haplotype in females but not in males, and (iii) the high population frequency of the HC gene may be the result of the selective advantage conferred by protecting heterozygotes against iron deficiency.     

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.      

Regional localization of the fibronectin and gamma-crystallin genes to bovine chromosome 8

The genes for isocitrate dehydrogenase-1, fibronectin, and gamma-crystallin are syntenic in man, mouse, and cow. In an effort to assign this bovine syntenic group to a specific chromosome and to allow a cytological comparison of the conserved chromosomal region containing these genes in their respective species, we have localized the fibronectin and gamma-crystallin genes to bovine chromosome 8, region 1.1-1.4. This study incorporates the techniques of hybrid somatic cell analysis and in situ hybridization and the use of a Robertsonian-translocated marker chromosome from a related species for regional assignment of genes to a specific bovine chromosome. The regions on human chromosome 2q, mouse chromosome 1, and cow chromosome 8 that contain these genes are cytologically similar, perhaps representing evolutionary conservation at the cytogenetic level as well as at the gene level for this group of loci.     

Concerted evolution of primate alpha satellite DNA. Evidence for an ancestral sequence shared by gorilla and human X chromosome alpha satellite

To understand evolutionary events in the formation of higher-order repeat units in alpha satellite DNA, we have examined gorilla sequences homologous to human X chromosome alpha satellite. In humans, alpha satellite on the X chromosome is organized as a tandemly repeated, 2.0 x 10(3) base-pairs (bp) higher-order repeat unit, operationally defined by the restriction enzyme BamHI. Each higher-order repeat unit is composed of 12 tandem approximately 171 base-pair monomer units that have been classified into five distinct sequence homology groups. BamHI-digested gorilla genomic DNA hybridized with the cloned human 2 x 10(3) bp X alpha satellite repeat reveals three bands of sizes approximately 3.2 x 10(3), 2.7 x 10(3) and 2 x 10(3) bp. Multiple copies of all three repeat lengths have been isolated and mapped to the centromeric region of the gorilla X chromosome by fluorescence in situ hybridization. Long-range restriction mapping using pulsed-field gel electrophoresis shows that the 2.7 x 10(3) and 3.2 x 10(3) bp repeat arrays exist as separate but likely neighboring arrays on the gorilla X, each ranging in size from approximately 200 x 10(3) to 500 x 10(3) bp, considerably smaller than the approximately 2000 x 10(3) to 4000 x 10(3) bp array found on human X chromosomes. Nucleotide sequence analysis has revealed that monomers within all three gorilla repeat units can be classified into the same five sequence homology groups as monomers located within the higher-order repeat unit on the human X chromosome, suggesting that the formation of the five distinct monomer types predates the divergence of the lineages of contemporary humans and gorillas. The order of 12 monomers within the 2 x 10(3) and 2.7 x 10(3) bp repeat units from the gorilla X chromosome is identical with that of the 2 x 10(3) bp repeat unit from the human X chromosome, suggesting an ancestral linear arrangement and supporting hypotheses about events largely restricted to single chromosome types in the formation of alpha satellite higher-order repeat units.     

Nucleotide sequence of a mutation in the proB gene of Escherichia coli that confers proline overproduction and enhanced tolerance to osmotic stress

We determined the nucleotide (nt) sequence of a mutation that confers proline overproduction and enhanced tolerance of osmotic stress on bacteria. The mutation, designated as proB74, is an allele of the Escherichia coli proB gene which results in a loss of allosteric regulation of the protein product, gamma-glutamyl kinase. Our sequencing indicated that the proB74 mutation is a substitution of an A for a G at nt position 319 of the coding strand of the gene, resulting in a change of an aspartate to an asparagine at amino acid (aa) residue 107 of the predicted protein product. Rushlow et al. [Gene 39 (1984) 109-112] determined that another proB mutation (designated as DHPR), that resulted in a loss of allosteric inhibition by proline of the E. coli gamma-glutamyl kinase, was due to a substitution of an alanine for a glutamate at aa residue 143. Therefore, even though both the DHPR and the proB74 mutations caused a loss of allosteric inhibition of gamma-glutamyl kinase, they are due to different amino acid substitutions.     

Fine structure mapping of CIAS1: identification of an ancestral haplotype and a common FCAS mutation,L353P

Familial cold autoinflammatory syndrome (FCAS) is an autosomal dominant inflammatory disease with a high degree of penetrance that is characterized by episodes of rash, arthralgia, fever, conjunctivitis, and leukocytosis after generalized exposure to cold. FCAS was previously mapped to a 10-cM region on chromosome 1q44, and subsequently the gene ( CIAS1) responsible for FCAS was identified. In this paper, we describe the physical and genetic mapping of the FCAS locus, and we report a large ancestral haplotype and a new disease-causing mutation. A BAC contig of approximately 3 Mb was developed and subsequently used for high throughput sequencing. We identified a critical region of 4 cM using rare crossover events in four large North American FCAS families. An unusually large shared haplotype (40 cM) was identified in three of the four families. We found a single heterozygous missense mutation (T1058C=L353P) in exon 3 of CIAS1 in all four families that is responsible for the large majority of FCAS cases described in the literature. We also report a comprehensive list of intragenic single nucleotide polymorphisms. The data provided here will assist others researching the 1q44 region and will aid clinicians in the diagnosis of FCAS.     

Molecular footprints of human immunoglobulin gene evolution: a new sequence family.

Analysis of the human VK (ref. 2) gene locus led to the detection of a new sequence family (L sequences). Its copy number is in the range of 10(2). The L sequences, which are about 500 bp long, are found as part of the 3' flanking regions of a clustered set of human VKI genes but they occur also separate from the genes. Models are discussed in which L sequences are viewed as molecular footprints of amplification and transposition processes of VK genes.     

Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.