Showing papers on "Pseudogene published in 1996"

P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature

Abstract: We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18,1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far descr

The Complete 685-Kilobase DNA Sequence of the Human β T Cell Receptor Locus

Abstract: The human beta T cell receptor (TCR) locus, comprising a complex family of genes, has been sequenced. The locus contains two types of coding elements--TCR elements (65 variable gene segments and two clusters of diversity, joining, and constant segments) and eight trypsinogen genes --that constitute 4.6 percent of the DNA. Genome-wide interspersed repeats and locus-specific repeats span 30 and 47 percent, respectively, of the 685-kilobase sequence. A comparison of the germline variable elements with their approximately 300 complementary DNA counterparts reveals marked differential patterns of variable gene expression, the importance of exonuclease activity in generating TCR diversity, and the predominant tendency for only functional variable elements to be present in complementary DNA libraries.

High intrinsic rate of DNA loss in Drosophila

Abstract: Pseudogenes are common in mammals but virtually absent in Drosophila. All putative Drosophila pseudogenes show patterns of molecular evolution that are inconsistent with the lack of functional constraints. The absence of bona fide pseudogenes is not only puzzling, it also hampers attempts to estimate rates and patterns of neutral DNA change. The estimation problem is especially acute in the case of deletions and insertions, which are likely to have large effects when they occur in functional genes and are therefore subject to strong purifying selection. We propose a solution to this problem by taking advantage of the propensity of retrotransposable elements without long terminal repeats (non-LTR) to create non-functional, 'dead-on-arrival' copies of themselves as a common by-product of their transpositional cycle. Phylogenetic analysis of a non-LTR element, Helena, demonstrates that copies lose DNA at an unusually high rate, suggesting that lack of pseudogenes in Drosophila is the product of rampant deletion of DNA in unconstrained regions. This finding has important implications for the study of genome evolution in general and the 'C-value paradox' in particular.

Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1.

Abstract: Over 100 distinct disease-associated mutations have been identified in the breast-ovarian cancer susceptibility gene BRCAI. Loss of the wild-type allele in >90% of tumors from patients with inherited BRCAI mutations indicates tumor suppressive function. The low incidence of somatic mutations suggests that BRCAI inactivation in sporadic tumors occurs by alternative mechanisms, such as interstitial chromosomal deletion or reduced transcription. To identify possible features of the BRCA1 genomic region that may contribute to chromosomal instability as well as potential transcriptional regulatory elements, a 117,143-bp DNA sequence encompassing BRCA1 was obtained by random sequencing of four cosmids identified from a human chromosome 17 specific library. The 24 exons of BRCAI span an 81-kb region that has an unusually high density of AJu repetitive DNA {41.5%), but relatively low density [4.8%) of other repetitive sequences. BRCAI intron lengths range in size from 403 bp to 9.2 kb and contain the intragenic microsatellite markers DI7S1323, DI7SI322, and DI7S855, which localize to introns 12, 19, and 20, respectively. In addition to BRCAI, the contig contains two complete genes: RhoT, a member of the rho family of GTP binding proteins, and VAT1, an abundant membrane protein of cholinergic synaptic vesicles. Partial sequences of the IAI-JB B-box protein pseudogene and lip JS, an interferon induced leucine zipper protein, reside within the contig. An L21 ribosomal protein pseudogene is embedded in BRCA1 intron 13. The order of genes on the chromosome is: centromere-lFP JS-VATI-P, hoTBRCAI-IAI-JB- telomere.

Capture of retrotransposon DNA at the sites of chromosomal double-strand breaks

Abstract: Non-homologous repair of broken chromosomes in Saccharomyces cerevisiae can be studied at a defined location by expressing the site-specific HO endonuclease that cuts the mating-type (MAT) locus. When homologous recombination is prevented, most double-strand breaks are repaired by non-homologous end-joinings similar to those observed in mammalian cells. About 1% of non-homologous repair events were exceptional, having 'captured' approximately 100 base pairs of DNA within the HO cleavage site. In each case, the insertion came from yeast's retrotransposon Tyl element. Four of the five contained the R-U5 region, which is the first part of Tyl messenger RNA to be converted to complementary DNA. The capture of cDNA fragments at the sites of double-strand breaks may account for the way that pseudogenes and long and short interspersed sequences (LINES and SINES) have been inserted at many locations in the mammalian genome.

Retrotransposon reverse-transcriptase-mediated repair of chromosomal breaks

Abstract: The abundance of short and long interspersed nuclear sequences (SINEs and LINEs) and pseudogenes in eukaryotic genomes indicates that reverse transcriptase (RT)-mediated phenomena are important in genome evolution. However, the mechanisms involved in their spread are largely unknown. We have developed a selection system in the yeast Saccharomyces cerevisiae to test whether RT-mediated events could be linked to the repair of double-strand breaks (DSBs). Here we show that DSBs can be fixed by the insertion of complementary DNAs at the break site. In the presence of functional RT (from human L1, yeast Tyl or Crithidia CRE1), and in the absence of homologous recombination, an HO endonuclease-induced DSB at the mating type (MAT) locus is the primary site at which a marked cDNA is observed among surviving cells. The structure and junctional sequences of these insertions suggest that repair occurs primarily by non-homologous recombination. Our data support a role for endogenous retroelements in the repair of chromosomal breaks.

Duplication of a Gene-Rich Cluster between 16p11.1 and Xq28: A Novel Pericentromeric-Directed Mechanism for Paralogous Genome Evolution

Abstract: We have identified a 26.5 kb gene-rich duplication shared by human Xq28 and 16p11.1. Complete comparative sequence analysis of cosmids from both loci has revealed identical Xq28 and 16p11.1 genomic structures for both the human creatine transporter gene (SLC6A8) and five exons of the CDM gene (DXS1357E). Overall nucleotide similarity within the duplication was found to be 94.6%, suggesting that this interchromosomal duplication occurred within recent evolutionary time (7-10 mya). Based on comparisons between genomic and cDNA sequence, both the Xq28 creatine transporter and DXS1357E genes are transcriptionally active. Predicted translation of exons and RT-PCR analysis reveal that chromosome 16 paralogs likely represent pseudogenes. Comparative fluorescent in situ hybridization (FISH) analyses of chromosomes from various primates indicate that this gene-rich segment has undergone several duplications. In gorilla and chimpanzee, multiple pericentromeric localizations on a variety of chromosomes were found using probes from the duplicated region. In other species, such as the orangutan and gibbon, FISH signals were only identified at the distal end of the X chromosome, suggesting that the Xq28 locus represents the ancestral copy. Sequencing of the 16p 11.1/Xq28 duplication breakpoints has revealed the presence of repetitive immunoglobulin-like CAGGG pentamer sequences at or near the paralogy boundaries. The mobilization and dispersal of this gene-rich 27 kb element to the pericentromeric regions of primate chromosomes defines an unprecedented form of recent genome evolution and a novel mechanism for the generation of genetic diversity among closely related species.

Gene conversion contributes to Ig light chain diversity in cattle.

Abstract: In humans and mice, extensive gene rearrangement is the major mechanism of diversification of the primary Ig repertoire This study shows that cattle depart from this pattern because rearrangement in the light chain locus is sharply limited Furthermore, in cattle, gene conversion contributes to the diversification of the primary light chain repertoire Sequencing of germ-line and expressed Vlambda genes revealed three important features First, the germ line contained a number of Vlambda pseudogenes In fact, 14 (70%) of the 20 germ-line genes identified and sequenced were pseudogenes, because they had one or more of the following defects: lack of recombination signal sequences at the 3' end, stop codons within the reading frame or truncations, and/or insertions or deletions that resulted in loss of reading frame Second, Vlambda cDNA from ileal Peyer's patch B cells demonstrated that the light chain repertoire arises from only a small number of V(J) rearrangements Even though two J genes were identified in the germ line, all of the expressed Vlambda genes examined contained the same J segment, indicating that only a single J gene participates in rearrangement at the lambda locus Third, a significant number of departures from the germ-line sequences of rearranged Vlambda can be traced to donor sequences of one or more Vlambda pseudogenes We conclude that a limited number of rearrangements and gene conversion play a role in contributing to the diversification of the primary lambda repertoire Furthermore, while clear indications of a role for somatic mutation in lambda diversification was seen, V gene rearrangement was not a major factor

Molecular basis for secretor type alpha(1,2)-fucosyltransferase gene deficiency in a Japanese population: a fusion gene generated by unequal crossover responsible for the enzyme deficiency.

Abstract: About 20%-25% of Caucasian individuals are nonsecretors who fail to express soluble A, B, H, and Lewis b histo-blood group antigens in secretory organs and secretory fluids because of the absence of the Secretor gene (FUT2)-encoded alpha(1,2)-fucosyltransferase (Se enzyme) activity. Recently, the FUT2 and a pseudogene have been isolated, and an Se enzyme-deficient allele (se) caused by a nonsense mutation (G428A, se1) in Caucasians has also been reported. Although we were unable to find the se1 allele, we have found a missense mutation (A385T, se2) and two nonsense mutations (C571T, se3 and C628T, se4) in the Japanese Se enzyme-deficient alleles. In addition, we have found a fusion gene, which consisted of the 5'-region of the pseudogene and the 3'-region of the functional FUT2, as a Se enzyme-deficient allele (se5). The DNA sequence analysis of the fusion gene indicated that the crossover region corresponded to regions between bases 253 and 313 of the pseudogene and between bases 211 and 271 of the FUT2. This finding suggested that the fusion gene was generated by homologous but unequal crossover. A population study on 141 randomly selected Japanese has indicated that the se2 is a common Se enzyme-deficient allele in the Japanese population. The results suggest that Se enzyme-deficient alleles are race specific.

Zea ribosomal repeat evolution and substitution patterns.

Abstract: Zea and Tripsacum nuclear ribosomal internal transcribed spacer (ITS) sequences were used to evaluate patterns of concerted evolution, rates of substitutions, patterns of methylation-induced deamination, and structural constraints of the ITS. ITS pseudogenes were identified by their phylogenetic position, differences in nucleotide composition, extensive deamination at ancestral methylation sites, and substitutions resulting in low-stability secondary RNA structures. Selection was important in shaping the kinds of polymorphisms and substitutions observed in the ITS. ITS substitution rates were significantly different among the Zea taxa. Deamination of cytosines at methylation sites was a potent mutation source, but selection appeared to maintain high methylation site density throughout the ribosomal repeat except for the gene promoter. Nucleotide divergence statistics identified selectively constrained regions at the 5' ends of the ITS1 and ITS2.

Immunoglobulin gene hyperconversion ongoing in chicken splenic germinal centers.

Abstract: It has been believed that the peripheral lymphocytes in chickens proliferate by self-renewing amplification of the preimmune repertoire generated in bursa. We amplified rearranged immunoglobulin variable (V) region genes from the single germinal centers induced by immunization. The sequence analysis of these genes revealed that most were derived from distinct B-cell clones which expanded locally, generating somatic antibody mutants at a high rate. Somatic hypermutations included unlinked base changes and the linked base modifications interpreted as unidirectional transfer of sequences from V region pseudogenes. This finding demonstrates the ongoing post-bursal diversification of B-cells in splenic germinal centers by templated gene conversion as well as untemplated point mutations.

Molecular cloning and characterization of a gibberellin-inducible, putative alpha-glucosidase gene from barley.

Abstract: A putative α-glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A3 (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal α-glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI α-amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this α-glucosidase gene and two additional sequences that may be related genes or pseudogenes.

Sterol 14-Demethylase P450 (P45014DM*) Is One of the Most Ancient and Conserved P450 Species

Abstract: To determine the orthology of sterol 14-demethylase (P45014DM), the only known P450 enzyme distributed widely in eukaryotes with a conserved metabolic role, the full-length amino acid sequences of rat and human P45014DMs were determined from the cloned cDNA sequences, and compared with those of the corresponding fungal proteins (CYP51). The amino acid identity value between given pairs of P45014DMs ranged from 93% (human/rat) to 39% (human or rat/Saccharomyces cerevisiae). All the P45014DMs formed a single cluster in a phylogenetic tree constructed from representative P450 protein sequences currently available. The nearest neighbors to the P45014DM cluster in the phylogenetic tree were CYP7 (cholesterol 7 alpha-hydroxylase) and CYP8 (prostacyclin synthase), and the divergence point of fungal and mammalian P45014DMs was clearly more recent than that of P45014DM and CYP7/CYP8. These lines of evidence show that fungal and mammalian P45014DMs are really orthologous. This is the first example of orthologous P450s occurring in distinct kingdoms. P45014DM may be an ancient P450 which arose before the divergence of major eukaryotic branches and has been conserved throughout evolution. The amino acid identity value (93%) between human and rat P45014DMs was comparable to those observed for some housekeeping enzymes. In addition, a processed pseudogene of P45014DM was found in a rat genomic DNA library, suggesting the expression of P45014DM in germ line cells. These facts suggest that P45014DM may be a housekeeping enzyme essential for the viability of mammals.

Direct molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia.

Abstract: The majority of congenital adrenal hyperplasia (CAH) cases arise from mutations in the steroid 21-hydroxylase (CYP21) gene. Without reliance on HLA gene linkage analysis, we have developed primers for differential polymerase chain reaction (PCR) amplification of the CYP21 gene and the non-functional CYP21P gene. Using the amplification created restriction site (ACRS) approach for direct mutational detection, a secondary PCR was then performed using a panel of primers specific for each of the 11 known mutations associated with CAH. Subsequent restriction analysis allowed not only the detection but also the determination of the zygosity of the mutations analysed. Existing deletion of the CYP21 gene could also be detected. In the analysis of 20 independent chromosomes in 11 families of CAH patients in Taiwan, four CYP21 mutation types, besides deletion, were detected. Interestingly, in five different alleles, the CYP21P pseudogene contained some polymorphisms generally associated with the CYP21 gene. These results suggest gene conversion events that are occurring in both CYP21P and CYP21 genes. Our combined differential PCR-ACRS protocol is simple and direct and is applicable for prenatal diagnosis of CAH using chorionic villi or amniotic cells.

Point mutations in Italian patients with classic, non-classic, and cryptic forms of steroid 21-hydroxylase deficiency.

Abstract: Seventy-three Italian patients affected by steroid 21-hydroxylase deficiency were studied by a PCR –allele-specific oligonucleotide protocol in order to evaluate the presence of eight known point mutations. The majority of chromosomes were found to carry point gene conversions normally present in the pseudogene. Within the classic form, the most common mutations were the splicing mutation A/C-655 to G in intron 2 (34.2%), the nonsense mutation C-1993 to T in exon 8 (10.8%), and the missense mutation T-999 to A in exon 4 (10%). Within the non-classic form, the missense mutation G-1683 to T was the most common (57.7%). Other mutations were either absent, such as the three clustered missense mutations T-1380, T-1383, T-1389 to A in exon 6, or very rare, like the 1761 + T in exon 7 and the C-2108 to T in exon 8. Family genotyping revealed the presence of ten asymptomatic parents carrying mutations in both chromosomes, thus identifying the gene defect in cryptic subjects. Interestingly, the same mutations were found in both symptomatic and asymptomatic forms.

The 5' end of the BRCA1 gene lies within a duplicated region of human chromosome 17q21

Abstract: To begin to address the hypothesis that abnormal regulation of the breast/ovarian cancer susceptibility gene BRCA1 is a critical step in sporadic breast/ovarian tumorigenesis, we have determined the detailed structure of the BRCA1 genomic region, We show that this region of the genome contains a tandem duplication of approximately 30 kilobases, which results in two copies of BRCA1 exons 1 and 2, of exons 1 and 3 of the adjacent 1A1-3B gene and of the previously reported 295 base pair intergenic region. Sequence analysis of the duplicated exons of BRCA1 and 1A1-3B and flanking genomic DNA reveals maintenance of the intron-exon structure and a high degree of nucleotide sequence identity, suggesting that these are non-processed pseudogenes and that the duplication is a recent event in evolutionary terms, We also show that a processed pseudogene of the acidic ribosomal phosphoprotein P1 (ARPP1) is inserted directly upstream of pseudo-BRCA1 exon 1A, We believe that these findings could not only confound BRCA1 mutation analysis, but could have implications for the normal and abnormal regulation of BRCA1 transcription, translation and function.

Identification of the rhesus monkey HLA-G ortholog. Mamu-G is a pseudogene.

Abstract: HLA-G is a nonclassical MHC class I molecule that is primarily expressed in the placenta. To investigate whether rhesus monkeys possess an HLA-G ortholog, we cloned and sequenced MHC class I cDNAs from the rhesus placenta. We identified two rhesus MHC class I cDNAs with sequence similarity to HLA-G. Each cDNA contained premature stop codons and frameshift mutations, suggesting that it was derived from an MHC class I pseudogene. Gene trees constructed using MHC class I alleles from human and nonhuman primates revealed that the rhesus placental pseudogene alleles clustered with HLA-G orthologs from the human, chimpanzee, and gorilla. These data suggested that this rhesus MHC class I pseudogene is an HLA-G ortholog. This locus was, therefore, designated Mamu (Macaca mulatta)-G. PCR amplification of a portion of Mamu-G from the genomic DNA of five rhesus monkeys resulted in the identification of five additional Mamu-G alleles and revealed the presence of four Mamu-G alleles in one rhesus monkey, suggesting that Mamu-G had been duplicated in this individual. Furthermore, the analysis of 81 MHC class I clones isolated from a rhesus placenta cDNA library did not result in the isolation of Mamu-G cDNAs, nor the isolation of any additional HLA-G homologs, suggesting that Mamu-G was transcribed at negligible levels. Given the similarity of rhesus monkey and human placenta structure and function, these data raise interesting questions regarding the role of HLA-G in pregnancy.

Characterization of a Cluster of Sulfatase Genes on Xp22.3 Suggests Gene Duplications in an Ancestral Pseudoautosomal Region

Abstract: An obligatory crossing-over event between the X and Y chromosomes in mammals occurs at each male meiosis within the 2.6 Mb of DNA defining the pseudoautosomal region (PAR). Genes located within or near the human PAR have homologous copies on the X and Y chromosomes, escape X inactivation and appear to be highly divergent throughout evolution. We have characterized the genomic structure of two genes from a recently identified cluster of sulfatase genes (ARSD and ARSE) located in the Xp22.3 region, and of their homologs on the Y chromosome. Our results indicate that the ARSD and ARSE genes from within this cluster have a conserved genomic organization, shared also by another Xp22.3 gene, STS, but completely different from that of all the other sulfatase genes. Sequence analysis of the Y-linked homologs indicate that they represent truncated pseudogenes. Sequence identity values between the X and Y copies of each gene is on average 91%, significantly higher than the values obtained by comparing different members of the family. FISH mapping experiments performed in several primate species revealed an identical localization of the X-linked copies to that in man, but different localizations of the Y homologs. Together, our data indicate that the cluster of sulfatase genes on human Xp22.3 was created through duplication events which probably occurred in an ancestral PAR, and support the view that the PAR has undergone multiple changes during recent mammalian evolution.

Amylase gene structures in primates: retroposon insertions and promoter evolution.

Abstract: Amylase transcription in the human salivary gland results from the evolutionary juxtaposition of two inserted elements, a gamma-actin pseudogene and an endogenous retrovirus, to create an unusual salivary-specific promoter. We utilized these structures as molecular tags to characterize the amylase genes in extant primates by polymerase chain reaction amplification of promoter fragments from genomic DNA. Six distinct amylase promoter structures were identified, which allowed us to infer the structures of common ancestors and trace the evolution of the modern human amylase promoters. Our data show that integration of the pseudogene and retrovirus were evolutionarily recent events. The gamma-actin pseudogene integrated after the divergence of the New World monkeys from the primate ancestral tree, and the retrovirus integrated later, after the divergence of the Old World monkeys. The New World monkey amylase promoter represents the mammalian amylase precursor structure before integration of the two retroposons. Two distinct amylase genes were identified in the Old World monkeys, one with a complete gamma-actin pseudogene insert and another novel structure with a truncation of the gamma-actin sequences. We demonstrated abundant amylase expression in the saliva of an Old World monkey, indicating that the endogenous retrovirus is not required for amylase transcription in the primate salivary gland.