Characterization of alpha-gliadin genes from diploid wheats and the comparative analysis with those from polyploid wheats

To carry out the comparative analysis of alpha-gliadin genes on A genomes of diploid and polyploid wheats, 8 full-length alpha-gliadin genes, including 3 functional genes and 5 pseudogenes, were obtained from diploid wheats, among which 2, 2 and 4 alpha-gliadin genes were isolated from T. urartu, T. monococcum and T. boeoticum, respectively. The results indicated that higher number of alpha-gliadin pseudogenes have been present in diploid wheats before the formation of polyploid wheats. Amino acid sequence comparative analysis among 26 alpha-gliadin genes, including 16 functional genes and 10 pseudogenes, from diploid and polyploid wheats was conducted. The results indicated that all alpha-gliadins contained four coeliac toxic peptide sequences (i.e., PSQQ, QQQP, QQPY and QPYP). The polyglutamine domains are highly variable, and the second polyglutamine stretch is usually disrupted by the lysine or arginine residue at the fourth position. The unique domain I is the most conserved domain. There are 4 and 2 conserved cysteine residues in the unique domains I and II, respectively. Comparative analysis indicated that the functional alpha-gliadin genes from A genome are highly conserved, whereas the identity of pseudogenes in diploid wheats are higher than those in hexaploid wheats. Phylogenetic analysis indicated that all the analyzed functional alpha-gliadin genes could be clustered into two major groups, among which one group could be further divided into 5 subgroups. The origin of alpha-gliadin pseudogene and functional genes were also discussed.     

Diversity and rearrangement of the human T cell rearranging gamma genes: nine germ-line variable genes belonging to two subgroups

We describe nine T cell gamma variable (V) gene segments isolated from human DNA. These genes, which fall into two subgroups, are mapped in two DNA regions covering 54 kb and probably represent the majority of human V gamma genes. One subgroup (V gamma I) contains eight genes, consisting of four active genes and four pseudogenes. The single V gamma II gene is potentially active. Sequence analysis of the V gamma I genes shows variation clustered in hypervariable regions, but somatic variability is restricted to N-region diversity. Studies on rearrangement in T cell lines and in thymic DNA show that major rearrangements can be observed that are attributable to the five active V gamma genes. In addition, human cells with the phenotype of helper T cells can undergo productive V gamma-J gamma joining.     

Organization and evolution of variable region genes of the human immunoglobulin heavy chain

We have isolated 23 different cosmid clones of the heavy-chain variable region genes (VH) of human immunoglobulin. These clones encompass about 1000 X 10(3) base-pairs of DNA containing 61 VH genes. Characterization of the 23 clones by Southern blot hybridization showed that VH genes belonging to different families were physically linked in many regions. Cluster 71, which was analyzed in detail, comprised seven VH segments arranged in the same orientation with different intervals. This clone contained internal homology regions, each carrying two VH segments of different families. Comparison of the nucleotide sequences of VH segments within each family showed that profiles of accumulation of mutations in framework (FR) and complementarity-determining (CDR) regions were different. CDR had more mutations at amino-acid-substituting positions than at silent positions, whereas FR had the reverse distribution of mutations. Five out of seven VH segments of this cluster were pseudogenes containing various mutations. VH pseudogenes were classified into two distinct groups; one with a few replacement mutations (conserved pseudogenes), and the other with rather extensive mutations (diverged pseudogenes). The possibility that conserved pseudogenes serve as a reservoir of VH segments is discussed.     

Murine ferritin heavy chain: isolation and characterization of a functional gene

A mouse liver genomic library screened with a full-length cDNA encoding murine ferritin heavy chain (mFHC) [Torti et al., J. Biol. Chem. 263 (1988) 12638-12644] yielded a functional genomic clone mFHC. The genomic clone isolated included a region of approximately 3 kb containing four exons and three introns. Sequence comparisons of the mouse genomic clone with other genomic clones from rat, human and chicken showed a high degree of similarity among species in the coding regions. Introns and flanking sequences were less conserved. However, comparison of mFHC promoter elements with FHC genes from other species revealed common elements. Analysis of the genomic structure of FHC suggested the presence of pseudogenes. S1 nuclease analysis, however, confirmed that this mouse clone, when transfected into human MRC-5 fibroblasts, was transcribed, indicating that this clone contains an FHC functional gene.     

A genome-wide survey of human thioredoxin and glutaredoxin family pseudogenes

The thioredoxin/glutaredoxin family consists of small heat-stable proteins that have a highly conserved CXXC active site and that participate in the regulation of many redox reactions. We have searched the human genome sequence to find putative pseudogenes (non-functional copies of protein-coding genes) for all known members of this family. This survey has resulted in the identification of seven processed pseudogenes for human Trx1 and two more for human Grx1. No evidence for the presence of processed pseudogenes has been found for the remaining members of this family. In addition, we have been unable to detect any non-processed pseudogenes derived from any member of the family in the human genome. The seven thioredoxin pseudogenes can be divided into two groups: Trx1-psi2, -psi3, -psi4, -psi5 and -psi6 arose from the functional ancestor, whereas Trx1-psi1 and -psi7 originated from Trx1-psi2 and -psi6, respectively. In all cases, the pseudogenes originated after the human/rodent radiation as shown by phylogenetic analysis. This is also the case for Grx1-psi1 and Grx1-psi2, which are placed between rodent and human sequences in the phylogenetic tree. Our study provides a molecular record of the recent evolution of these two genes in the hominid lineage.     

The genes of the lysosomal cysteine proteinases cathepsin B, H, L, and S map to different mouse chromosomes

The mouse genes for the lysosomal cysteine proteinases cathepsin B, H, L, and S were mapped to Chromosomes (Chrs) 14, 9, 13, and 3, respectively. Two of the DNA probes used in this study detected an additional, independently segregating locus. The cathepsin B-specific probe hybridized to a locus on Chr 2, and the cathepsin H probe to a locus on the X Chr. These loci either correspond to pseudogenes or to cathepsin B- and cathepsin H-related genes. The four cysteine proteinases mapped in this study lie within known regions of conserved synteny between mouse and human chromosomes, when compared with the corresponding positions of their human homologs. Assuming that the genes of the cysteine proteinase gene family arose from a common ancestral gene, our results suggest that these four cysteine proteinases had been dispersed over different chromosomes before separation of mouse and human in evolution. Received: 22 August 1996 / Accepted: 20 November 1996     

Human 28S ribosomal RNA sequence heterogeneity.

DNA sequencing of several cloned human 28S ribosomal RNA gene fragments has revealed sequence heterogeneity (1) but it was not clear whether these are inactive pseudogenes or are active genes that are transcribed and represented in ribosomes. S1 nuclease analysis allowed us to examine the population of ribosomal RNA molecules of a cell, and we found that 28S rRNA is a heterogeneous assortment of molecules in both mono- and polysomal preparations. Sequence variation, although largely concentrated in variable regions of the molecule, apparently also occurs in the conserved regions.     

Three parallel linkage groups of human acidic keratin genes.

Two regions of human genomic DNA, each containing several keratin genes, were isolated and partially sequenced. The keratin genes are inactive, having suffered deleterious mutations. Both regions contain at least four keratin genes arranged in a head-to-tail orientation including a pseudogene for keratin K#16. Within each segment there are two keratin genes in close linkage with only 1.5 kb of DNA between them. Sequence comparison of the two regions showed 98.9% identity in both the coding and the intronic segments of the pseudogenes. The pseudogenes show 94% identity to their functional counterparts. Southern hybridization analysis showed that the segments are paralogous, not allelic. The regions are products of two independent, recent duplication events. The first occurred approximately 24 million years ago, after the separation of primates from the rhesus/baboon line. The second is specific for the human lineage, having occurred approximately 3.8 million years ago. Analysis of the genomic DNAs of primates showed the presence of only one of the regions in the DNAs of gibbon and gorilla, while rhesus monkey and baboon were missing both copies. We conclude that the human keratin genes are still actively evolving, with new duplications having occurred as recently as after the separation of human and gorilla ancestors.     

Characterization of α-gliadin genes from diploid wheats and the comparative analysis with those from polyploid wheats

To carry out comparative analysis of the α-gliadin genes on A genomes of diploid and polyploid wheats, 8 full-length α-gliadin genes, including 3 functional genes and 5 pseudogenes, were obtained from diploid wheats, among which 2, 2 and 4 α-gliadin genes were isolated from T. urartu, T. monococcum, and T. boeoticum, respectively. The results indicated that higher number of α-gliadin pseudogenes have been present in diploid wheats before the formation of polyploid wheats. Amino acid sequence comparative analysis among 26 α-gliadin genes, including 16 functional genes and 10 pseudogenes, from diploid and polyploid wheats was conducted. The results indicated that all α-gliadins contained four coeliac toxic peptide sequences (i.e., PSQQ, QQQP, QQPY, and QPYP). The polyglutamine domains are highly variable, and the second polyglutamine stretch is usually disrupted by the lysine or arginine residue at the fourth position. The unique domain I is the most conserved domain. There are four and two conserved cysteine residues in the unique domains I and II, respectively. Comparative analysis indicated that the functional α-gliadin genes from A genome are highly conserved, whereas the identity of pseudogenes in diploid wheats are higher than those in hexaploid wheats. Phylogenetic analysis indicated that all the analyzed functional α-gliadin genes could be clustered into two major groups, among which one group could be further divided into 5 subgroups. The origin of α-gliadin pseudogene and functional genes were also discussed. The text was submitted by the authors in English.     

Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus laevis oocytes. Sequence variations of the conserved cis-acting 5' and 3' elements of U RNA genes may partly explain the low abundance of U7 RNA.     

Structural similarity of the HLA-DQ region in DQ3 and DQ4 haplotypes and structural diversity of the HLA-DQ region in HLA-DR7 haplotypes.

Genomic DNA obtained from a B lymphoblastoid cell line was digested with appropriate restriction endonuclease and hybridized with several probes specific for genes encoding HLA-DQ. Southern hybridization with a DQA1 3'untranslated (UT) region probe showed DQ2-type hybridization pattern in DR7DQ3 haplotype. On the contrary, DQB1 3'UT probe showed DQ3-type pattern in the same haplotype. Gene cloning and DNA sequencing analysis revealed a repetitive sequence, (TG)19, between DQA1 and DQB1 gene in the DR7DQ3 haplotype. These results suggest that a recombination event has occurred near this potential Z-DNA structure in the haplotype, DR7DQ3. The 3'UT region probes of DQA1 and DQB1 genes failed to detect restriction fragment length polymorphism (RFLP) differences between DR4DQ3 and DR4DQ4 haplotypes in this experiment, suggesting that the gene structure between DQA1 and DQB1 is conserved in these haplotypes.     

Evolutionary relationships of class II major-histocompatibility-complex genes in mammals

The major histocompatibility complex (MHC) class II molecule consists of noncovalently associated alpha and beta chains. In mammals studied so far, the class II MHC can be divided into a number of regions, each containing one or more alpha-chain genes (A genes) and beta-chain genes (B genes), and it has been known for some time that orthologous relationships exist between genes in corresponding regions from different mammalian species. A phylogenetic analysis of DNA sequences of class II A and B genes confirmed these relationships; but no such orthologous relationship was observed between the B genes of mammals and those of birds. Thus, the class II regions have diverged since the separation of birds and mammals (approximately 300 Mya) but before the radiation of the placental mammalian orders (60-80 Mya). Comparison of the phylogenetic trees for A and B genes revealed an unexpected characteristic of DP-region genes: DPB genes are most closely related to DQB genes, whereas DPA chain genes are most closely related to DRA-chain genes. Thus, the DP region seems to have originated through a recombinational event which brought together a DQB gene and a DRA gene (perhaps approximately 120 Mya). The 5' untranslated region of all class II genes includes sequences which are believed to be important in regulating class II gene expression but which are not conserved in known pseudogenes. These sequences are conserved to an extraordinary degree in the human DQB1 gene and its mouse homologue A beta 1, suggesting that regulation of expression of this locus may play a key role in expression of the entire class II MHC.     

Amplification of multiple copies of mitochondrial Cytochrome b gene fragments in the Australian freshwater crayfish,Cherax destructor Clark (Parastacidae: Decapoda)

Non-coding copies of fragments of the mitochondrial genome translocated to the nucleus or pseudogenes are being found with increasing frequency in a diversity of organisms. As part of a study to evaluate the utility of a range of mitochondrial gene regions for population genetic and systematic studies of the Australian freshwater crayfish, Cherax destructor (the yabby), we report the first detection of Cytochrome b (Cyt b) pseudogenes in crustaceans. We amplified and sequenced fragments of the mitochondrial Cyt b gene from 14 individuals of C. destructor using polymerase chain reaction (PCR) with primers designed from conserved regions of Penaeus monodon and Drosophila melanogaster mitochondrial genomes. The phylogenetic tree produced from the amplified fragments using these primers showed a very different topology to the trees obtained from sequences from three other mitochondrial genes, suggesting one or more nuclear pseudogenes have been amplified. Supporting this conclusion, two highly divergent sequences were isolated from each of two single individuals, and a 2 base pair (bp) deletion in one sequence was observed. There was no evidence to support inadvertent amplification of parasite DNA or contamination of samples from other sources. These results add to other recent observations of pseudogenes suggesting the frequent transfer of mitochondrial DNA (mtDNA) genes to the nucleus and reinforces the necessity of great care in interpreting PCR-generated Cyt b sequences used in population or evolutionary studies in freshwater crayfish and crustaceans more generally.     

DNA methylation profiling of pseudogene-parental gene pairs and two gene families

A substantial proportion of human genes contain tissue-specifically DNA-methylated regions (TDMRs). However, little is known about the evolutionary conservation of differentially methylated loci, how they evolve, and the signals that regulate them. We have studied TDMR conservation in the PLG and TBX gene families and in 32 pseudogene–parental gene pairs. Among the members of the recently evolved PLG gene family, 5′-UTR methylation is conserved and inversely correlated with the cognate gene expression, indicating as well a conserved regulatory role of DNA methylation. Conversely, many genes of the much older TBX family display complementary tissue-specific methylation, suggesting an epigenetic complementation in the evolution of this gene family. Similar to gene families, unprocessed pseudogenes arose from gene duplications and we found TDMR conservation in some pseudogene–parental gene pairs displaying short evolutionary distances. However, for the majority of unprocessed pseudogenes and for all processed pseudogenes examined, we found that tissue-specific methylation arose de novo after gene duplication.     

Examination of four HLA class I pseudogenes. Common events in the evolution of HLA genes and pseudogenes.

The HLA class I gene family in lymphoblastoid cell line 721 has been studied in detail and a number of sequences in addition to the classical genes have been identified. The cloning, characterization, and nucleotide sequences of four sequences, all full length HLA class I pseudogenes, are described in this report. These pseudogenes, contained within 5.4-, 5.9-, 7.0-, and 9.2-kb HindIII fragments, each have the class I exon-intron structure as well as class I homology in their 5' and 3' flanking regions. However, all four sequences have one or more substitutions that perturb the coding region, leaving little doubt that they are in fact pseudogenes. Comparisons among these sequences and the HLA class I genes revealed that their homology with the class I genes is patchwork. Thus, although some regions have diverged, other contiguous intron-exon sequences are highly conserved. Comparisons in the 5' regions indicate that the pseudogene promoters more closely resemble the classical HLA promoters than the nonclassical promoters as none of the unique structural features found in the HLA-E, -F, or -G regulatory regions are present in any of the pseudogene promoters. Further comparisons revealed that at least two putative gene conversion events, similar to those hypothesized to have occurred in the evolution of some HLA genes, may have occurred in the evolution of some of the pseudogenes. These and other hypothetical events in the evolution of the class I gene family are discussed.