Pseudogene Sequences Research Articles

The novel hemagglutinin-esterase genes of human torovirus and Breda virus

Human torovirus (HTV) and Breda virus (BRV), members of the genus torovirus in the family Coronaviridae, are established infectious agents of humans and cattle, respectively. The hemagglutinin-esterase (HE) gene of Breda virus serotype 2 (BRV-2) has been identified and the nucleotide sequence for BRV serotype 1 (BRV-1) genome which contains the open reading frames for the viral structural proteins has been reported revealing the presence of a 1.25 kb gene whose nucleotide sequence is identical to that of the BRV-2 HE gene. In this study, we amplified the 1.2kb HE gene from the HTV genome using long RT-PCR and sequenced the amplicon directly. At the nucleotide level, the HTV HE gene manifests 85% sequence identity to the HE genes of BRV-1 and BRV-2 and 89% identity with the X pseudogene sequence of BEV. The 1.25 kb amplicons which contained the HE genes of BRV-1 and HTV were cloned and expressed in a baculovirus system and the proteins purified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Hyperimmune sera prepared in guinea pigs against these proteins were reactive with both bovine torovirus (BTV) and human torovirus (HTV) antigens. By immunoblot, they reacted specifically with a 65 kDa protein corresponding in size to the torovirus HE protein. Furthermore, the hyperimmune sera but not the preimmune sera reacted with a series of BTV-positive and HTV-positive fecal specimens by immunoblot and dot blot analysis. By immunoelectron microscopy (IEM) torovirus particles from BTV-positive specimens from calves with diarrhea and HTV-positive specimens from patients were aggregated by the hyperimmune sera. Human convalescent sera and gnotobiotic calf post-infection sera reacted by immunoblot with the expressed 65 kDa protein. The expressed HE protein of HTV has important diagnostic potential.

The critical region of overlap defining theAZFa male infertility interval of proximal Yq contains three transcribed sequences

The position of deletion breakpoints in a series of fourAZFa male infertility patients has been refined using new markers derived from BAC clone DNA sequence covering the AZFa male infertility...

Phylogenetic relationships among Brazilian howler monkeys, genus Alouatta (Platyrrhini, Atelidae), based on <FONT FACE=Symbol>g</FONT>1-globin pseudogene sequences

The genus Alouatta (howler monkeys) is the most widely distributed of New World primates, and has been arranged in three species groups: the Central American Alouatta palliata group and the South American Alouatta seniculus and Alouatta caraya groups. While the latter is monotypic, the A. seniculus group encompasses at least three species (A. seniculus, A. belzebul and A. fusca). In the present study, approximately 600 base pairs of the g1-globin pseudogene were sequenced in the four Brazilian species (A. seniculus, A. belzebul, A. fusca and A. caraya). Maximum parsimony and maximum likelihood methods yielded phylogenetic trees with the same arrangement: {A. caraya [A. seniculus (A. fusca, A. belzebul)]}. The most parsimonious tree had bootstrap values greater than 82% for all groupings, and strength of grouping values of at least 2, supporting the sister clade of A. fusca and A. belzebul. The study also confirmed the presence of a 150-base pair Alu insertion element and a 1.8-kb deletion in the g1-globin pseudogene in A. fusca, features found previously in the remaining three species. The cladistic classification based on molecular data agrees with those of morphological studies, with the monospecific A. caraya group being clearly differentiated from the A. seniculus group.

A Novel Mitochondrial DNA-like Sequence in the Human Nuclear Genome

We describe here a nuclear mitochondrial DNA-like sequence (numtDNA) that is nearly identical in sequence to a continuous 5842 bp segment of human mitochondrial DNA (mtDNA) that spans nucleotide positions 3914 to 9755. On the basis of evolutionary divergence among modern primates, this numtDNA molecule appears to represent mtDNA from a hominid ancestor that has been translocated to the nuclear genome during the recent evolution of humans. This numtDNA sequence harbors synonymous and nonsynonymous nucleotide substitutions relative to the authentic human mtDNA sequence, including an array of substitutions that was previously found in the cytochrome c oxidase subunit 1 and 2 genes. These substitutions were previously reported to occur in human mtDNA, but subsequently contended to be present in a nuclear pseudogene sequence. We now demonstrate their exclusive association with this 5842-bp numtDNA, which we have characterized in its entirety. This numtDNA does not appear to be expressed as a mtDNA-encoded mRNA. It is present in nuclear DNA from human blood donors, in human SH-SY5Y and A431 cell lines, and in ρ0 SH-SY5Y and ρ0 A431 cell lines that were depleted of mtDNA. The existence of human numtDNA sequences with great similarities to human mtDNA renders the amplification of pure mtDNA from cellular DNA very difficult, thereby creating the potential for confounding studies of mitochondrial diseases and population genetics.

Identification of a sex-linked GH pseudogene in one of two species of Japanese salmon ( Oncorhynchus masou and O. rhodurus)

The organization of growth hormone (GH) genes has been examined in two closely related species of salmonids from Japan, masu salmon ( Oncorhynchus masou) and amago salmon ( O. rhodurus). In two other species of salmonid, coho ( O. kisutch) and chinook ( O. tshawytscha), a type-2 GH pseudogene sequence has been identified on the Y chromosome, and this sequence also appears to be located on the Y chromosome of O. masou. Interestingly, despite the close relationship between amago and masu salmon based on mtDNA sequence data, a Y-linked GH sequence was not observed in O. rhodurus and thus provides a molecular marker that can distinguish males of these two species. In addition, the Y-linked GH sequence in masu salmon provides a useful tool for the production of monosex all-female populations for aquaculture.

Phylogenetic Studies of Marsupials Based on Phosphoglycerate Kinase DNA Sequences

Phosphoglycerate kinase sequences were obtained for 313 aligned bases of 41 individuals from 39 marsupial species. In contrast to previous molecular analyses, the relationships suggested by these data show a high level of congruence with morphologically defined orders and families. Four main monophyletic lineages are recognizable. These are the monogeneric orders Microbiotheria (Dromiciops australis) and Notoryctemorphia (Notoryctes typhlops), a grouping of the American orders Didelphimorphia and Paucituberculata, and the Australasian species other thanN. typhlops.Within the Australasian lineage, there are again four main monophyletic groups; the Dasyuridae, two peramelemorph (bandicoot) lineages (one comprised of pseudogene sequences) and the Diprotodontia. This topology is not greatly affected by the exclusion of pseudogenes except that a clade of syndactylous species (Peramelemorphia plus Diprotodontia) is recovered. Two other peramelemorph pseudogenes have inserts of about 1 kb with high levels of similarity to LINE 1 elements. The Diprotodontia is notable for its relative lack of intersequence variation in comparison to the Dasyuromorphia.

Alpha-Gal epitopes on viral glycoproteins.

The α-gal epitope (Galα1,3Galβl,4GlcNAc-R) is a terminal glycosidic structure that is expressed on the surface of cells from most mammalian species other than humans, apes and Old World monkeys (Galili et al., 1988a; Galili et al., 1987). The terminal α-galactosyl unit of this epitope is added to nascent glycolipids and glycoproteins in the Golgi apparatus by α1,3galactosyltransferase (α1,3GT). In primates lacking the α-gal epitope, the α1,3GT gene is not transcribed, and nonsense mutations are present within the coding region of some species (Galili and Swanson, 1991; Larsen et al., 1990a; Joziasse et al., 1989). The presence of a functional α1,3GT in New World monkeys suggests that this gene was inactivated in ancestral Old World monkeys and apes after their divergence from New World monkeys (Galili et al., 1988a). A detailed comparison of the α1,3GT pseudogene sequences in Old World monkeys and apes further suggests that this gene was inactivated after these two groups diverged from each other (Galili and Andrews, 1995; Galili and Swanson, 1991).

Structural organization of the human short-chain L-3-hydroxyacyl-CoA dehydrogenase gene.

The third step in the mitochondrial beta-oxidation spiral of short-chain fatty acids is catalyzed by short-chain L-3-hydroxyacyl-CoA dehydrogenase (HADHSC; EC 1.1.1.35). We have determined the structural organization of the human HADHSC gene by sequencing of cloned genomic amplification products, obtained using HADHSC-specific cDNA-based primers, as well as by direct sequencing of an isolated PAC clone containing the HADHSC gene. Upon comparison with the HADHSC cDNA sequence, HADHSC was shown to encompass at least eight exons, ranging in size from 73 to 158 bp, and 7 introns. The total HADHSC gene spans approximately 49 kb. The HADHSC 5'-flanking region was characterized with an AluI plasmid library constructed from a partially AluI-digested PAC clone containing the human HADHSC gene. Several typical promoter elements such as a CAAT-box, Sp1, AP1, and AP2 sites were found, while a TATA-box was apparently absent. Among other putative regulatory elements, a NRRE-1 site was identified. By radiation hybrid panel, assisted fine-mapping HADHSC was linked to marker AFM070TH5, corresponding to Chromosome (Chr) 4q22-26, and a putative HADHSC pseudogene was linked to marker D15S1324, located at Chr 15q17-21. Knowledge of the genomic organization and 5'-flanking region of HADHSC will enable genomic mutation analysis of patients suspected of HADHSC deficiency, as well as facilitate the investigation into the transcriptional regulation of short-chain fatty acid oxidizing gene products in general and HADHSC expression in particular.

The angiogenins.

The angiogenic and other biological functions of the angiogenins, members of the pancreatic RNase superfamily of proteins, are reviewed in the context of their primary and tertiary structures. The ribonucleolytic activity and interactions with the placental ribonuclease inhibitor have seen much study in the last few years. The mechanism of the angiogenic activity of angiogenin has recently been postulated as involving multiple interactions with other proteins through specific regions on the molecular surface of angiogenin. These molecular partners include heparin, plasminogen, elastase, angiostatin, actin and most importantly a 170-kilodalton receptor on subconfluent endothelial cells. The existence of the latter receptor was established in conjunction with a mitogenic activity of angiogenin on subconfluent cells. The levels of angiogenin in various physiological and disease states are summarized, including various studies on pregnancy and angiogenin. Correlations are seen between states of enhanced angiogenesis and angiogenin levels. An overview of the relationship of angiogenin and the other RNases of the superfamily showed that their genes all are in relative close proximity on human chromosome 14. Examination of the many expressed sequence tags published in the public databanks, for angiogenin and the other RNases, revealed that angiogenin and RNase-4 (the most evolutionarily conserved RNase), share various identical 5'-untranslated regions on their sets of messenger RNAs, suggesting that their genes are in very close proximity on chromosome 14 and that they are products of differential splicing. This in turn suggests that, in both humans and mice, expression of these two proteins is under identical control, with obvious implications for their biological activities. The evolutionary history of the angiogenins is examined briefly on the basis of the protein sequences of the human, rabbit, pig, two bovine and four mouse angiogenins, and two mouse angiogenin pseudogene sequences. The discrepancy between the conventional requirement for conservatism in structure to allow multimolecule interactions, and the actual fast-changing sequence of the angiogenins, in concert with the wide-ranging activity even in birds, of human angiogenin, is discussed.

The maize regulatory gene B-Peru contains a DNA rearrangement that specifies tissue-specific expression through both positive and negative promoter elements.

The B-Peru allele of the maize b regulatory gene is unusual relative to most b alleles in that it is expressed in the aleurone layer of the seed. It is also expressed in a subset of plant vegetative tissues. Transgenic maize plants containing the B-Peru gene with the first 710 bases of upstream sequence conferred the same levels of aleurone expression as nontransgenic B-Peru plants, but no pigment was made in vegetative tissues. Transient transformation assays in aleurone tissue localized the aleurone-specific promoter to the first 176 bases of the B-Peru upstream region and identified two critically important regions within this fragment. Mutation of either region alone reduced expression greater than fivefold. Surprisingly, the double mutation actually increased expression to twice the native promoter level. Our results suggest that these two critical sequences, which lie close together in the promoter, may form a negative regulatory element. Several lines of evidence suggest that the B-Peru promoter arose through the translocation of an existing aleurone-specific promoter to the b locus. Immediately upstream of the aleurone-specific promoter elements and in the opposite orientation to the b coding sequence is a pseudogene sequence with strong similarity to a known class of proteins. Our findings that novel aleurone-specific promoter sequences of the B-Peru transcription factor are found adjacent to part of another gene in a small insertion are quite unexpected and have interesting evolutionary implications.

Genomics and polymorphism of Agph-DAB1, an Mhc class II B gene in red-winged blackbirds (Agelaius phoeniceus).

To further our understanding of the evolution of avian Mhc genes at the genomic level, we screened a cosmid library made from a red-winged blackbird (Agelaius phoeniceus) with a blackbird cDNA probe and subcloned from one of the Mhc-containing cosmids a gene which we designate Agph-DAB1. The structure of the gene is similar to that found for chicken class II B genes, except that the introns are surprisingly large, ranging from 98 to over 600 bp, making this the longest avian class II B gene to date. Using primers targeted toward the introns flanking the peptide-binding region (PBR), we amplified the entirety of the second exon and determined nucleotide sequences of 41 PCR products from eight individual blackbirds. The 10 sequence types found, among which were two probable pseudogene sequences, exhibit the classic hallmarks for evolution of PBRs, namely, an excess of nonsynonymous over synonymous substitutions and evidence of gene conversion events in polymorphic subdomains. Despite these patterns and our use of intron primers, the distribution of sequences among individuals suggests that more than one locus was amplified in most individuals, and the bushlike tree of sequences provides little information as to locus-specific clusters. These results imply a complex history of gene conversion, recent duplication, or possibly, concerted evolution among multiple loci, although Agph-DAB1, the first genomic Mhc sequence from a bird other than chicken, provides important clues in the quest for locus-specific Mhc primers in birds.

Infection characteristics of rabies virus variants with deletion or insertion in the pseudogene sequence.

We investigated the infection characteristics of recombinant rabies virus variants modified in the pseudogene sequence. Infection of neuronal cell lines by the SAD W9 and SAD V* variants (respectively with deletion or insertion in this sequence) showed no significant differences as compared to the parental strain, the attenuated strain SAD B19, in infection characteristics such as number of infected cells or viral yield. The inoculation of mice by these variants resulted in similar infection patterns and pathogenicity. Stereotaxic inoculation of the different variants into the rat striatum showed that deletion or insertion did not affect the axonal virus spread, nor did insertion of a complete additional transcription unit, that could be expressed in the areas connected to the inoculation site. These results show that the pseudogene sequence is not involved in viral spread and pathogenicity and confirm the availability of this domain for targeting and expression of foreign genes into neurons.

A human serotonin-7 receptor pseudogene

Although the serotonin-7 receptor was cloned several years ago, its localization in brain tissues remains confusing because of the existence of a related expressed pseudogene, the sequence of which has not hitherto been reported. During the course of searching for related receptor genes, we also searched for this pseudogene to determine its sequence. Human genomic DNA was screened for dopamine and serotonin receptor-like genes, using the polymerase chain reaction method and degenerate oligonucleotide primers based on the similar sequences in the transmembrane-6 and -7 regions of the serotonin-5A, the serotonin-7, and the dopamine D2, D3 and D4 receptors. This resulted in one of the clones having a 115 bp fragment, of which 89% of the bases were identical to the transmembrane-6 and -7 regions of the serotonin-7 receptor sequence. The fragment was radiolabelled and used to screen a human fetal brain cDNA library. A novel cDNA clone of 1326 bp was isolated. Based on the nucleotide sequence, 88% of the bases in this sequence of the pseudogene are identical to the human serotonin-7 receptor coding sequence. However, compared to the serotonin-7 receptor DNA sequence, the pseudogene sequence has nucleotide deletions and insertions, resulting in frame-shifts and stop codons. It was concluded that this sequence represented a pseudogene related to the serotonin-7 receptor gene.

Molecular evolution of cdc2 pseudogenes in spruce (Picea).

The p34cdc2 protein and other cyclin-dependent protein kinases (CDK) are important regulators of eukaryotic cell cycle progression. We have previously cloned a functional cdc2 gene from Picea abies and found it to be part of a family of related sequences, largely consisting of pseudogenes. We now report on the isolation of partial cdc2 pseudogenes from Picea engelmannii and Picea sitchensis, as well as partial functional cdc2 sequences from P. engelmannii, P. sitchensis and Pinus contorta. A high level of conservation between species was detected for these sequences. Phylogenetic analyses of pseudogene and functional cdc2 sequences, as well as the presence of shared insertions or deletions, support the division of most of the cdc2 pseudogenes into two subfamilies. New cdc2 pseudogenes appear to have been formed in Picea at a much higher rate than they have been obliterated by neutral mutations. The pattern of nucleotide changes in the cdc2 pseudogenes, as compared to a presumed ancestral functional cdc2 gene, was similar to that previously found in mammalian pseudogenes, with a strong bias for the transitions C to T and G to A, and the transversions C to A and G to T.

Sensitive and specific cytokeratin 18 reverse transcription-polymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNA

Processed pseudogenes of residual contaminating genomic DNA interfere with a sensitive detection of cytokeratin 18 (CK18) mRNA by reverse transcription and polymerase chain reaction (RT-PCR). This may cause false-positive results when CK18 mRNA is used as a marker for ectopic tumor cells in specimens from cancer patients. To establish a sensitive CK18 RT-PCR by excluding the amplification of processed pseudogenes, the following strategy was chosen: (a) CK18 pseudogene sequences were cloned from genomic DNA by PCR; (b) cDNA-specific primers were designed on the basis of mismatches between pseudogenes and cDNA; (c) PCR conditions were adjusted to reach maximum sensitivity and specificity. Epithelial cells (1-10) could be detected in 1 mL of blood. Among the numerous CK18 genes homologous to the transcribed gene, at least two different processed pseudogenes exist that are highly homologous to each other and to the exons of the transcribed CK18 gene.

Tail-to-head arrangement of a partial chicken glyceraldehyde-3-phosphate dehydrogenase processed pseudogene.

A chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) processed pseudogene was identified by inverse PCR using oligonucleotide primers specific for the 5' region of the GAPDH mRNA. Molecular cloning and sequence analysis of this genomic sequence shows that the processed pseudogene is incomplete and arranged in a permuted tail-to-head order. We propose that the tail-to-head organization is the result of circularization and breakage of a GAPDH retrogene prior to chromosomal integration. PCR analysis of DNAs from quail, pheasant, and various jungle fowl, shows that the processed pseudogene was formed after the three genera diverged but prior to Gallus speciation. This is the first report of a chicken GAPDH processed pseudogene sequence. This is also the first published report of a processed pseudogene with a tail-to-head organization.

Identification of de novo deletions at the NF1 gene: no preferential paternal origin and phenotypic analysis of patients.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. To date, a relatively small number of NF1 mutations have been characterized, thus precluding genotype-phenotype correlations. By genotyping 75 NF1 families, we have detected six hemizygous patients (two of whom are members of the same family). The five presumed deletions were confirmed by two quantitative methods of analysis of NF1 copy number: Southern hybridization with cDNA probes and a single-strand conformation polymorphism analysis that discriminates between the NF1 gene and the pseudogene sequences. The five deletions remove most of the NF1 gene, at least 225 kb, from exon 9 to the 3' end of the coding sequence. The origin of de novo mutations in the NF1 gene has been reported to be mainly paternal but we have determined that four of the de novo deletions involved the maternal chromosome and one the paternal chromosome. The six patients with deletions exhibited precocious, multiple clinical features of the disease. The incidence of tumor complications, particularly plexiform neurofibromas and intracranial tumors, among this group of patients is higher than the observed incidence in our NF1 population, suggesting that NF1 haploinsufficiency may cause a more severe phenotype with regard to tumor development. In contrast to other reports that associated large deletions with mildly dysmorphic facies, mental retardation and a large number of cutaneous neurofibromas, only one out of our six patients presented this phenotype.

A DP1 pseudogene derived from an aberrantly processed RNA

A fragment of mouse genomic DNA containing a pseudogene corresponding to the processed transcript of the DP1 locus was isolated and analysed. The pseudogene sequence, on comparison with the genomic locus and the corresponding mRNA indicated the presence of several small deletions and point mutations. In addition, the pseudogene showed a deletion for the second exon of the DP1 gene indicating the occurrence of an exon slippage event during its formation. We also describe the chromosomal mapping of the pseudogene to chromosome 1 by fluorescence in situ hybridisation and distinguish it from the localisation of the actual murine DP1 genomic locus on mouse chromosome 8.

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche.

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56-69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O. ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P < 0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.

Cloning and Expression of a cDNA Encoding Uridine Kinase from Mouse Brain

Uridine kinase is the rate-limiting enzyme in the pyrimidine salvage pathway of all mammalian cells. A cDNA for uridine kinase from mouse brain has been isolated, sequenced, and characterized. This is the first report of a complete nucleotide sequence for mammalian uridine kinase. The isolated cDNA is only 95% complete, missing the first 17 codons. The correct 5′-terminus sequence was obtained from high-stringency screening of a mouse liver genomic DNA library. The translated cDNA sequence encodes a protein of 277 amino acids (Mr31,068). A truncated form of the cDNA was expressed inEscherichia coli.The expressed protein displayed uridine kinase activity and readily formed a tetramer, the most active form of the wild-type enzyme. Analysis of the amino acid sequence identified the three ATP-binding site consensus motifs. The predicted secondary structure for uridine kinase and the sequence comparison with three kinases having known crystal structures are consistent with uridine kinase having an α/β core structure of the nucleotide-binding fold found in many kinases. We have also isolated and cloned a nonfunctional, processed pseudogene from mouse genomic DNA. This pseudogene sequence is 94% identical with the coding DNA.