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PubMed1995

The presence of tRNA pseudogenes in mammalia and plants and their absence in yeast may account for different specificities of pre-tRNA processing enzymes.

Six of 13 cloned members of the human tRNA(Val) gene family code for tRNA(Val) pseudogenes, of which all but one are transcribed efficiently in HeLa cell extracts. Due to single or multiple mismatches in stem regions, the corresponding pre-tRNAs are resistant against the action of human 5'- and 3'-processing enzymes and are thus prevented from being converted to mature tRNAs. Surprisingly, all of them are accurately and efficiently processed to mature-sized tRNA in yeast nuclear extract. This is in agreement with corresponding studies of plant pre-tRNAs which are not processed in wheat germ extract but are rapidly processed in yeast extract. These observations imply that the yeast pre-tRNA 5'- and 3'-maturases do not monitor the three-dimensional structure of their substrates as stringently as mammalian and plant enzymes, possibly because tRNA pseudogenes do not occur in yeast.

Cell Extracts↗

PubMed1996

Olfactory receptor-encoding genes and pseudogenes are expressed in humans.

A putative olfactory receptor-encoding gene was cloned from human genomic DNA and shown to be expressed by isolation of a full-length cDNA from olfactory tissue. A second cDNA clone was found to encode an olfactory receptor pseudogene. The expression of a pseudogene from the olfactory gene repertoire, in neurons which express only a single receptor type, implies that many neurons will be non-functional.

Amino Acid Sequence↗

PubMed1989

The human glucocerebrosidase gene and pseudogene: structure and evolution.

We report the sequence of the entire human gene encoding beta-glucocerebrosidase and that of the associated pseudogene. The gene contains 11 exons extending from base pair 355 to base pair 7232 in the overall sequence. The gene promoter contains TATA- and CAT-like boxes upstream of the major 5' end of the glucocerebrosidase RNA. The two TATA boxes lie between nucleotides (-23)-(-27) and (-33)-(-39) and the two possible CAT boxes reside between nucleotides (-90)-(-94) and (-96)-(-99) in relation to the major 5' end of the mRNA. The functionality of the promoter region was monitored by coupling it to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) and assaying the expression of the enzyme in cells transfected with this vector. The glucocerebrosidase promoter not only directs synthesis of the bacterial enzyme but also exhibits the same pattern of tissue-specific expression as that of the endogenous gene. An apparently tightly linked pseudogene is approximately 96% homologous to the functional gene. However, introns 2, 4, 6, and 7 have large "deletions" consisting of Alu sequences 313, 626, 320, and 277 bp in length, respectively. It is entirely possible that the ancestral gene lacks these sequences and that they have been inserted into the introns of the functioning gene. There is also a 55-bp deletion from a part of exon 9 flanked by a short inverted repeat. The sequence data should facilitate development of methods for diagnosis of Gaucher disease at the molecular level.

Base Sequence↗

PubMed1990

Isolation and characterization of a rat delta-aminolevulinate dehydratase processed pseudogene.

Southern blot analysis of genomic DNA from different strains of rat indicated that there were multiple copies of the gene encoding the second enzyme of the heme biosynthetic pathway, delta-aminolevulinate dehydratase (ALA-D). Two types of genomic clones were isolated from a Sprague-Dawley rat library. One appears to be the expressed gene, whereas the nucleotide sequence of the other suggests that it contains an ALA-D processed pseudogene because (1) there are no introns, (2) there are multiple mutations that alter the predicted amino acid sequence of ALA-D and cause premature termination, (3) there is a 3' polyadenylated tract, and (4) there is an 8-bp direct repeat flanking the gene. The rat genome is unusual in this respect since ALA-D pseudogenes have not been detected in Southern blot analyses of other mammals, including human, gorilla, chimpanzee, orangutan, rabbit, mouse, and Chinese hamster.

Animals↗

PubMed1991

Assignment of human prochymosin pseudogene to chromosome 1.

Chymosin is an extremely specific aspartatic protease responsible for milk coagulation. Chymosin is expressed in a number of mammalian offspring, yet its presence in the gastric tissue of human infants remains a matter of controversy. In any event, the human genome contains chymosin-related sequences that probably represent a pseudogene. Using DNA obtained from human x hamster somatic cell hybrids as the template and polymerase chain reaction, we have mapped the human prochymosin pseudogene to chromosome 1.

Animals↗

PubMed1992

Genetic variability of the murine creatine kinase B gene locus and related pseudogenes in different inbred strains of mice.

The role of genetic variation in isoenzyme gene families is often poorly appreciated. We report here on the determination of DNA sequences and typing of genetic variability in four creatine kinase B (CKB) gene loci in different inbred strains of mice. The unique functional murine CKB gene was found to be nearly identical to the previously characterised rat and human sequences in both size and exon-intron structure. In this gene, approximately 0.5% allelic nucleotide positions as well as the lengths of simple A-rich and [TG]n repetitive elements located at the 5' and 3' sides of the transcribed segment, differed between inbred strains of mice. Preliminary experiments suggest that this sequence divergence is of importance for design of gene targeting strategies involving homologous DNA recombination. The three additional CKB-like gene loci in mice all had the characteristics of processed pseudogenes. By Southern blot analysis we could demonstrate that both the type and number of pseudogenes differed between inbred strains. Analysis of the CKB gene sequences enabled us to speculate about the evolutionary history of this highly polymorphic subfamily of genes.

Amino Acid Sequence↗

PubMed1995

The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) rev protein, eIF-5A, maps to chromosome 17p12-p13: three eIF-5A pseudogenes map to 10q23.3, 17q25, and 19q13.2.

The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 transactivator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent-human cell hybrids in combination with fluorescence in situ hybridization analysis, we show that the four genes map to three different chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12-p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein.

Base Sequence↗

PubMed1995

Molecular cloning and characterization of the active human mitochondrial NADH:ubiquinone oxidoreductase 24-kDa gene (NDUFV2) and its pseudogene.

Two distinct loci for the 24-kDa subunit of the mitochondrial NADH:ubiquinone oxidoreductase (complex I of the respiratory chain) were detected in the human genome: a transcribed gene from chromosome 18 and an inactive locus on chromosome 19. Cosmid clones containing the functional gene (NDUFV2) and the pseudogene (NDUFV2P1) were isolated. The NDUFV2 gene spans approximately 20 kb and contains 8 exons. Refined mapping of both NDUFV2 genes by FISH resulted in an assignment of the NDUFV2 gene to 18p11.2-p11.31 and of the NDUFV2P1 gene to 19q13.3-qter. The nucleotide sequence of the NDUFV2P1 pseudogene differs from the cDNA sequence by the lack of the methionine initiator codon, an additional 165 bp of the first intron sequence, and a 1-nucleotide deletion.

Base Sequence↗

PubMed2003

Multiple single-nucleotide polymorphisms in the methylenetetrahydrofolate reductase and its truncated pseudogene of 23 inbred strains of mice.

Reduced 5,10-methylenetetrahydrofolate reductase (MTHFR) results in a number of human diseases. To find a model mouse sensitive to these diseases, we analyzed single-nucleotide polymorphisms (SNPs) of the mouse Mthfr using 23 phylogenetically distant strains of mouse. We found five SNPs: two nonsynonymous and three synonymous. The CAST/Ei strain has the nonsynonymous SNP L350V and five strains (NMRI, KJR, SWN2, MSM, and JF1) have the nonsynonymous SNP S22G. The MTHFR activity of CAST/Ei and MSM showed no significant difference in activity or thermostability compared with that of C57BL/6J. We also found a pseudogene segment of the mouse Mthfr that was not present in human and was more frequently variable than the functional gene. These results suggest a possibility that the truncated pseudogene may buffer variations of the mouse Mthfr functional gene, and the mouse has evolved fewer variations of the gene than human.

Amino Acid Sequence↗

PubMed2005

Identification of a transcriptionally active hVH-5 pseudogene on 10q22.2.

Mitogen-activated protein kinases (MAPKs) are important regulators of a vast number of biological functions that affect life and death of eukaryotic cells and are tightly regulated by the concerted action of several phosphatases. Among these is the human homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH-5) product, which dephosphorylates and thus inhibits members of the MAPK family. Here, we analyzed hVH-5 transcripts in mammary carcinoma cell lines and discovered a sequence with 88% similarity to hVH-5 transcripts. Because this variant of hVH-5 lacked intronic sequences in its genomic structure, we suggest it might be a processed pseudogene of hVH-5. psihVH-5 transcripts were detected in human peripheral tissues as well as in 11 of 14 breast cancer cell lines. In respect to the normal hVH-5 gene, the pseudogene contains several point mutations and a frame shift due to the deletion of 2 bases that would lead to the truncation of the putative psihVH-5 product.

Amino Acid Sequence↗

PubMed2005

Sequence-based typing of the HLA-A10/A19 group and confirmation of a pseudogene coamplified with A*3401.

The strategy for sequencing human leukocyte antigen (HLA)-A was based on separate amplification of exons 2 and 3, followed by forward and reverse heterozygous sequencing of the alleles. Validation of the method was obtained by sequencing 11 individuals carrying alleles from all different HLA-A allele groups, except *43. All alleles could be correctly identified except A*3401. Unexpected polymorphic positions were identified in exon 3, even in individuals homozygous for A*3401. In addition, the pseudogene HLA-COQ or HLA-DEL linked to A*3401 was coamplified and sequenced. The problem was solved by using different amplification primers for exon 3 with mismatches for the two pseudogenes. A total of 252 unrelated individuals with at least one allele belonging to the A10 or A19 group were typed for HLA-A by this strategy. Ten different alleles were identified in the A10 group and 14 in the A19 group. As second allele a further 30 different subtypes from all different groups were sequenced. In 21 individuals, sequencing exon 1 was necessary to distinguish A*7401 from A*7402. The sequencing strategy, with separate amplification of the exons, has proven to be a robust method, resulting in reliable and efficient high-resolution HLA-A typing.

Alleles↗

PubMed2005

Mitochondrial DNA depletion analysis by pseudogene ratioing.

The mitochondrial DNA (mtDNA) depletion status of rho(0) cell lines is typically assessed by hybridization or polymerase chain reaction (PCR) experiments, in which the failure to hybridize mtDNA or amplify mtDNA using mtDNA-directed primers suggests thorough mitochondrial genome removal. Here, we report the use of an mtDNA pseudogene ratioing technique for the additional confirmation of rho0 status. Total genomic DNA from a U251 human glioma cell line treated with ethidium bromide was amplified using primers designed to anneal either mtDNA or a previously described nuclear DNA-embedded mtDNA pseudogene (mtDNApsi). The resultant PCR product was used to generate plasmid clones. Sixty-two plasmid clones were genotyped, and all arose from mtDNApsi template. These data allowed us to determine with 95% confidence that the resultant mtDNA-depleted cell line contains less than one copy of mtDNA per 10 cells. Unlike previous hybridization or PCR-based analyses of mtDNA depletion, this mtDNApsi ratioing technique does not rely on interpretation of a negative result, and may prove useful as an adjunct for the determination of rho0 status or mtDNA copy number.

Cell Line, Tumor↗

PubMed2006

Upregulation of dihydropyrimidinase-related protein 2, spectrin alpha II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats--a proteomics approach.

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18-22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3-46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin alpha II chain (rat fragment, also known as alpha-fodrin or non-erythroid alpha chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24h after the induction of cerebral ischemia.

Animals↗

PubMed2005

Retroposition of processed pseudogenes: the impact of RNA stability and translational control.

Human processed pseudogenes are copies of cellular RNAs reverse transcribed and inserted into the nuclear genome by the enzymatic machinery of L1 (LINE1) non-LTR retrotransposons. Although it is generally accepted that germline expression is crucial for the heritable retroposition of cellular mRNAs, little is known about the influences of RNA stability, mRNA quality control and compartmentalization of translation on the retroposition of processed pseudogenes. We found that frequently retroposed human mRNAs are derived from stable transcripts with translation-competent functional reading frames that are resistant to nonsense-mediated RNA decay. They are preferentially translated on free cytoplasmic ribosomes and encode soluble proteins. Our results indicate that interactions between mRNAs and L1 proteins seem to occur at free cytoplasmic ribosomes.

Animals↗

PubMed2006

Fugu genome does not contain mitochondrial pseudogenes.

Contrary to previous observations that fish genomes are devoid of nuclear mitochondrial pseudogenes, a genome-wide survey identified a large number of "recent" and "ancient" nuclear mitochondrial DNA fragments (Numts) in the whole-genome sequences of the fugu (Takifugu rubripes), Tetraodon nigroviridis, and zebrafish (Danio rerio). We have analyzed the latest assembly (v4.0) of the fugu genome and show that, like the Anopheles genome, the fugu nuclear genome does not contain mitochondrial pseudogenes. Fugu assembly v4.0 contains a single scaffold representing the near complete sequence of the fugu mitochondria. The "recent" Numts identified by the previous study in fugu assembly v2.0 are in fact shotgun sequences of mitochondrial DNA that were misassembled with the nuclear sequences, whereas the "ancient" Numts appear to be the result of spurious matches. It is likely that the Numts identified in the genomes of Tetraodon and zebrafish are also similar artifacts. Shotgun sequences of whole genomes often include some mitochondrial sequences. Therefore, any Numts identified in shotgun-sequence assemblies should be verified by Southern hybridization or PCR amplification.

Animals↗

PubMed1988

Cloning, nucleotide sequence and molecular evolution of a rabbit processed metallothionein MT-2 pseudogene.

A rabbit metallothionein-2 pseudogene (MT-2 psi) has been isolated from a partial rabbit genomic library. Its unusual sequence shows evidence of complex rearrangements involving recombination and deletion events. There are no intervening sequences, 3' poly A tract or 5' regulatory DNA sequences. The pseudogene is flanked by two sets of direct repeats (CT)3 GT(CT)4 and CTGG(G)CTC. They are most probably the sites of insertion of MT-2 psi in the rabbit genome. In addition, a number of repetitive DNA sequences are observed flanking the MT-2 psi gene. These are features of a processed retrogene.

Amino Acids↗

PubMed1995

Rabies virus glycoprotein gene contains a long 3' noncoding region which lacks pseudogene properties.

Analysis of a limited number of laboratory strains of rabies virus had demonstrated the presence of a genome region bounded by two transcription termination and polyadenylation-like (TTP) signals (approximately 400 to 450 nucleotides apart) which was located between the end of the glycoprotein (G) coding sequence and the beginning of the L polymerase coding sequence. Although this region had been suggested to represent a remnant or pseudogene (psi), no detailed analysis had been carried out to examine this possibility. Here we present the nucleotide sequence analysis of this genome region for several laboratory rabies virus strains and a large number of diverse rabies viruses detected directly in brain tissue of naturally infected animals. Only one distinct lineage of the laboratory strains and none of the wild-type rabies viruses contained the upstream TTP-like signal, indicating that only the downstream TTP motif is the authentic G mRNA transcription termination and polyadenylation and signal. Phylogenetic analysis of sequence differences provided no evidence of laboratory strains containing the two TTP-like signals being ancestral to any of the viruses possessing only the downstream TTP sequence motif. These data indicate that this region of the rabies virus genome encodes a G mRNA with a long 3' noncoding region with no evidence of a pseudogene.

Animals↗

PubMed2001

Genomic organization of a human 5beta-reductase and its pseudogene and substrate selectivity of the expressed enzyme.

The enzyme 5beta-reductase catalyzes the reduction of the 4-ene of 3-ketosteroids, converting them into 5beta-dihydro-3-ketosteroids and, thus, could be involved in the metabolism of 4-cholestene-3-one, progesterone, 17~-hydroxyprogesterone, aldosterone, corticosterone, cortisol, 4-androstenedione, and testosterone. In this study, we report the genomic structure of a human 5beta-reductase gene, its tissue distribution, the characterization of an intronless pseudogene and the substrate selectivity of the enzyme. The gene coding for the active 5beta-reductase contains nine exons like most members of the aldo-keto reductase family, but the sequence covered by the gene, more than 42 kb, is much longer than the sequence of other members of this family. There are many large introns, especially introns 3, 4 and 7 that span approx. 7 kb, and intron 1 that contains more than 10 kb. Northern blot analysis showed three band sizes of 1.3, 2.2 and 2.7 kb. The 1.3 and 2.7 kb bands are highly expressed in the liver while weaker 2.2 and 1.3 kb bands have been observed in the testis and colon, respectively. We also identified an intronless gene having 86% homology with the 5beta-reductase cDNA sequence. Since its sequence contains many stop codons, this gene is most probably a pseudogene. To determine more precisely the substrate selectivity of the enzyme, we established a stable cell line expressing human 5beta-reductase in transformed embryonic kidney (HEK-293) cells. The transfected cells efficiently catalyze the transformation of progesterone, androstenedione, 17alpha-hydroxyprogesterone and testosterone. However, they catalyze much less efficiently the transformation of compounds containing an 11beta-hydroxy group, such as aldosterone, corticosterone and cortisol. In addition to its role in cholesterol catabolism, it is well recognized that 5beta-reductase inactivates active androgens. Indeed, 5beta-dihydrotestosterone (5beta-DHT), the product of the reduction of testosterone by 5beta-reductase, is not active while its 5~-isomer (DHT) is the most potent natural androgen. Recent findings show that 5beta-pregnanes are active ligands in the induction of CYP3A through the orphan receptor hPAR. Our results thus open an opportunity for studying the new role of 5beta-reductase in the formation of a new type of active steroids.

Base Sequence↗