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Cloning of a mouse protein kinase A catalytic subunit pseudogene and chromosomal mapping of C subunit isoforms.

Two isoforms of the protein kinase A catalytic subunit, C alpha and C beta, have previously been described in the mouse. We now report the cloning and characterization of a novel C-related sequence, Cx, from a murine genomic library. Cx is 89.8% identical to part of the C alpha coding region, but lacks all of the introns present in this gene, suggesting that it arose via retroposition. The existence of several frameshift mutations, premature termination codons, and missense mutations at critical sites confirms that it is a pseudogene. Furthermore, we are unable to detect any expression. Homology with functional protein kinase genes commences exactly at the first intron splice junction in C alpha, downstream of the expected translational start codon. Cx is also truncated at its 3' end by the interposition of two distinct, contiguous LINE-1 elements. By fluorescence in situ hybridization, we demonstrate that Cx is located on the X Chromosome (Chr), at band F3. This is displaced from its functional homologs, C alpha and C beta, which we map to mouse Chrs 8 (band C3) and 3 (band H3), respectively.

Animals↗

Presence of a 16S rRNA pseudogene in the bi-molecular plastid genome of the primitive brown alga Pylaiella littoralis. Evolutionary implications.

The plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm. is composed of two different circular DNA molecules: the largest carries two rrn operons, and the smallest, only one copy of both 16S and 23S rDNAs. 16S rDNA copies located on both molecules have been cloned and their nucleotide sequences determined: they are 65% homologous to one another. The expression of these genes was assayed by hybridizing in vivo labelled P. littoralis rRNAs to both clones, and specific oligonucleotides to total RNA from P. littoralis. Results indicate that the 16S rDNA copy located on the small molecule is a pseudogene. Comparisons of the functional gene with other 16S rRNA genes shows that chloroplasts from green plants emerged earlier from the cyanobacterial lineage than Euglena gracilis and Pylaiella littoralis plastids.

Base Sequence↗

The human T-cell receptor gamma variable pseudogene V10 is a distinctive marker of human speciation.

The V10 variable gene of the human T-cell receptor gamma locus (TCRG-V10), the only member of the subgroup III, has a structural defect which inhibits the splicing of the leader intron. We show that there is a single point mutation in the V10 leader donor splice site responsible for this situation and that this mutation is found in the different populations tested, indicating that V10 corresponds to a pseudogene in humans. We restored the splice site by mutagenesis and obtained correct splicing in vitro. Analysis of the V10 germline gene in different primates reveals functional splice sites in the closest human apes, the chimpanzee and the gorilla. The splice competence of TCRG-V10 in higher primates was addressed in peripheral blood lymphocytes from chimpanzee by specific cDNA amplification, and correct splicing of the TCRG-V10 leader intron was found as well as a majority of in frame rearrangements involving only the TCRG-J1 or J2 segments. These results suggest that V10(+)gamma /delta T cells may represent an important subset in the non-human higher primates, contrary to the situation observed in the human.

Amino Acid Sequence↗

New chromosomal mapping assignments for argininosuccinate synthetase pseudogene 1, interferon-beta 3 gene, and the diazepam binding inhibitor gene.

Argininosuccinate synthetase pseudogene 1 (ASSP1), interferon-beta 3 (IFNB3) gene, and diazepam binding inhibitor (DBI) gene have previously been mapped to human chromosome 2. Their nucleotide sequences, recorded in the GENBANK data base, were used to generate DNA primers to amplify specific sequences using the polymerase chain reaction (PCR). These primers failed to amplify DNA sequences when used to analyze microcell hybrid clones containing human chromosome 2. In order to map these genes, a panel of somatic cell hybrids was analyzed by PCR with these primer sets. The results of these experiments place ASSP1 sequences on human chromosome 6, IFNB3 on human chromosome 8, and DBI on human chromosome 6.

Argininosuccinate Synthase↗

Parallel origins of duplications and the formation of pseudogenes in mitochondrial DNA from parthenogenetic lizards (Heteronotia binoei; Gekkonidae).

Analysis of mitochondrial DNAs (mtDNAs) from parthenogenetic lizards of the Heteronotia binoei complex with restriction enzymes revealed an approximately 5-kb addition present in all 77 individuals. Cleavage site mapping suggested the presence of a direct tandem duplication spanning the 16S and 12S rRNA genes, the control region and most, if not all, of the gene for the subunit 1 of NADH dehydrogenase (ND1). The location of the duplication was confirmed by Southern hybridization. A restriction enzyme survey provided evidence for modifications to each copy of the duplicated sequence, including four large deletions. Each gene affected by a deletion was complemented by an intact version in the other copy of the sequence, although for one gene the functional copy was heteroplasmic for another deletion. Sequencing of a fragment from one copy of the duplication which encompassed the tRNA(leu)(UUR) and parts of the 16S rRNA and ND1 genes, revealed mutations expected to disrupt function. Thus, evolution subsequent to the duplication event has resulted in mitochondrial pseudogenes. The presence of duplications in all of these parthenogens, but not among representatives of their maternal sexual ancestors, suggests that the duplications arose in the parthenogenetic form. This provides the second instance in H. binoei of mtDNA duplication associated with the transition from sexual to parthenogenetic reproduction. The increased incidence of duplications in parthenogenetic lizards may be caused by errors in mtDNA replication due to either polyploidy or hybridity of their nuclear genomes.

Amino Acid Sequence↗

A human metallothionein pseudogene containing AG/CT repetitive elements.

A lambda phage recombinant clone, 25 S, which contains a 15.5-kb EcoRI human genomic DNA fragment, has been characterized. Restriction mapping and Southern blot hybridization indicated a 3.0-kb HindIII fragment containing metallothionein (MT)-like sequences. Several interesting features were found upon comparison of this nucleotide sequence with that of other human MT genes: (1) sequences representing the 5' regulatory region, the 5' untranslated region, and the first exon are not contained in the 3.0-kb HindIII fragment; (2) the coding sequence of the second exon (amino acids 10-31 encoding a portion of the beta-domain of the MT protein) has 11 amino acid changes out of a total of 21, whereas, the third exon (amino acids 32-61, representing the complete alpha-domain of the MT protein) has only 4 amino acid substitutions; however, all cysteine residues are conserved; (3) this MT-like gene retains intron sequences and processing signals; (4) Southern blot analysis of human genomic DNA indicated this MT-like gene is located on a 10.5-kb EcoRI genomic DNA fragment; and (5) unusual AG/CT-rich repetitive elements are located within the second intron and upstream of the second exon of this MT-like gene. This gene is not expressed in response to metal induction in two human cell lines, as shown by northern blot analyses. Based on these observations, this MT-like gene represents a unique nonprocessed pseudogene of the human MT multigene family.

Amino Acid Sequence↗

Structure and evolution of 5S rRNA genes and pseudogenes in the genus Aspergillus.

We have cloned and determined the nucleotide sequence of 18 DNA fragments hybridizing to 5S rRNA from two Aspergillus species--A. wentii and A. awamori. Four of the analyzed sequences were pseudogenes. The gene sequences of these two species were very similar and differed from Aspergillus nidulans at both constant and microheterogeneous sites.

Aspergillus nidulans↗

Recent evolutionary origin within the primate lineage of two pseudogenes with similarity to members of the transforming growth factor-beta superfamily.

Using a search engine called Motifer, we searched the public database of the human genome for genes matching a consensus pattern of cysteine residues derived from members of the transforming growth factor-beta (TGF-beta) superfamily. We identified two genes (named MDF451 and MDF628) that display sequence similarity to members of the TGF-beta superfamily in the arrangement of six conserved cysteine residues. Phylogenetic analyses revealed that MDF451 and MDF628 constitute a distinct subgroup within the TGF-beta superfamily, distantly related to the GDNF subfamily of ligands. Both genes could be identified in several primate species in addition to human, including chimpanzee, gorilla, guereza, and green and gray monkey, but not in rodents or other non-primate mammals, and appear not to be present in the genomes of mouse, rat or zebrafish. RNAs for MDF451 and MDF628 were expressed at low levels within distinct regions of the human central nervous system, including adult cerebellum, adult spinal cord and fetal brain. Despite expression at the RNA level, both genes presented a transcribed upstream stop codon that would prevent translation of the TGF-beta-like reading frame. The coding potential of alternative reading frames was not immediately apparent. The two genes may represent TGF-beta-like pseudogenes that have recently appeared in evolution in a common ancestor of the primate lineage by duplication from a GDNF/TGF-beta-like ancestral gene.

Amino Acid Sequence↗

A novel Q378X mutation exists in the transmembrane transporter protein ABCC6 and its pseudogene: implications for mutation analysis in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is an inherited disorder of the elastic tissue with characteristic progressive calcification of elastic fibers in skin, eye, and the cardiovascular system. Recently mutations in the ABCC6 gene, encoding a transmembrane transporter protein, were identified as cause of the disease. Surprisingly, sequence and RFLP analysis for exon 9 with primers corresponding to flanking intronic sequence in diseased and haplotype negative members from all of our families and in a control population revealed either a homozygous or heterozygous state for the Q378X (1132C-->T) nonsense mutation in all individuals. With the publication of the genomic structure of the PXE locus we had identified the starting point of a large genomic segmental duplication within the locus in the cytogenetic interval defined by the Cy19 and Cy185 somatic cell hybrid breakpoints on chromosome 16p13.1. By means of somatic cell hybrid mapping we located this starting point telomeric to exon 10 of ABCC6. The duplication, however, does not include exon 10, but exons 1-9. These findings suggest that one or several copies of an ABCC6 pseudogene (psiABCC6) lie within this large segmental duplication. At least one copy contains exons 1-9 and maps to the chromosomal interval defined by the Cy163 and Cy11 breakpoints. Either this copy and/or an additional copy of psiABCC6 within Cy19-Cy183 carries the Q378X mutation that masks the correct identification of this nonsense mutation as being causative in pseudoxanthoma elasticum. Long-range PCR of exon 9 starting from sequence outside the genomic replication circumvents interference from the psiABCC6 DNA sequences and demonstrates that the Q378X mutation in the ABCC6 gene is associated with PXE in some families. These findings lead us to propose that gene conversion mechanisms from psiABCC6 to ABCC6 play a functional role in mutations causing PXE.

Alleles↗

Heme uptake genes in human and fish isolates of Photobacterium damselae: existence of hutA pseudogenes.

The marine bacterium Photobacterium damselae includes strains classified into two distinct subspecies, namely subsp. damselae and subsp. piscicida, which have been reported to cause disease in a variety of marine animals and in humans. P. damselae strains utilize heme compounds as sole iron sources. In the present study, ten potential heme uptake and utilization genes are described in P. damselae subsp. damselae and subsp. piscicida. One gene cluster includes the genes coding for putative proteins HutZ, HutX and HutW; TonB, ExbB and ExbD, the three components of the TonB system; HutB, the putative periplasmic binding protein; HutC, the putative inner membrane permease; and HutD, the putative ABC-transporter ATP-ase. A gene coding for HutA, the outer membrane heme receptor, has also been identified, but it is not linked to the rest of the heme transport genes. RT-PCR analyses showed that heme uptake genes are arranged in three iron-regulated transcriptional units. A plasmid carrying the gene for the heme receptor HutA in combination with a plasmid carrying tonBexbBDhutBCD genes conferred to Escherichia coli 101ESD (ent) the ability to use heme and hemoglobin as iron sources. The hutA gene was present in strains isolated from humans and a variety of fish species, but it was shown to be interrupted in some subsp. piscicida strains, constituting a pseudogene. This is the first description of a heme-uptake system in a Photobacterium species, and shows some structural and functional similarities to heme-uptake systems reported in other gram-negative bacteria.

Amino Acid Sequence↗

Forty million years of independent evolution: a mitochondrial gene and its corresponding nuclear pseudogene in primates.

Sequences from nuclear mitochondrial pseudogenes (numts) that originated by transfer of genetic information from mitochondria to the nucleus offer a unique opportunity to compare different regimes of molecular evolution. Analyzing a 1621-nt-long numt of the rRNA specifying mitochondrial DNA residing on human chromosome 3 and its corresponding mitochondrial gene in 18 anthropoid primates, we were able to retrace about 40 MY of primate rDNA evolutionary history. The results illustrate strengths and weaknesses of mtDNA data sets in reconstructing and dating the phylogenetic history of primates. We were able to show the following. In contrast to numt-DNA, (1) the nucleotide composition of mtDNA changed dramatically in the different primate lineages. This is assumed to lead to significant misinterpretations of the mitochondrial evolutionary history. (2) Due to the nucleotide compositional plasticity of primate mtDNA, the phylogenetic reconstruction combining mitochondrial and nuclear sequences is unlikely to yield reliable information for either tree topologies or branch lengths. This is because a major part of the underlying sequence evolution model--the nucleotide composition--is undergoing dramatic change in different mitochondrial lineages. We propose that this problem is also expressed in the occasional unexpected long branches leading to the "common ancestor" of orthologous numt sequences of different primate taxa. (3) The heterogeneous and lineage-specific evolution of mitochondrial sequences in primates renders molecular dating based on primate mtDNA problematic, whereas the numt sequences provide a much more reliable base for dating.

Animals↗

Processed pseudogenes, processed genes, and spontaneous mutations in the Arabidopsis genome.

We identified 411 processed sequences in the Arabidopsis thaliana genome based on the fact that they have lost their intron(s) and have a length that is at least 95% of the length of the gene that gave rise to them. These sequences were generated by 230 different genes and clearly originated from retrotranspositons events because most of them (91%) have a poly(A)-tail. They are composed of 376 sequences with frame shifts and/or premature stop codons (processed pseudogenes) and 35 sequences without disablements (processed genes). Eleven of these processed genes are likely functional retrotransposed genes because they have low Ka/Ks ratios and high Ks values, and their sequences match numerous Arabidopsis ESTs. Processed sequences are mostly randomly distributed in the Arabidopsis genome and their rate of accumulation has steadily been decreasing since it peaked some 50 MYA. In contrast with the situation observed in mammals, the processed sequences found in the Arabidopsis genome originate from genes with high copy numbers and not from highly expressed genes. The patterns of spontaneous mutations in Arabidopsis are slightly different than those of mammals but are similar to those observed in Drosophila. This suggests that methylated cytosine deamination is less frequent in Arabidopsis than in mammals.

Arabidopsis↗

Identification of two new alleles, IGHV3-23*04 and IGHJ6*04, and the complete sequence of the IGHV3-h pseudogene in the human immunoglobulin locus and their prevalences in Danish Caucasians.

More than 100 variable (V), 27 diversity (D), and six joining (J) genes are encoded in the human heavy chain locus, and many of these genes exists in different allelic forms. The number of genes and the allelic differences help to create diversity in the immunoglobulin receptors, a key feature of the adaptive immune system. We here report the identification of two novel and seemingly functional alleles of human heavy chain genes. The variable IGHV3-23*04 allele is found with an allele frequency of 0.21 amongst Danish Caucasians, whereas the novel joining IGHJ6*04 allele is rare (allele frequency 0.02). We also report the full sequence of IGHV3-h. The gene exists in two allelic forms but is only found in 58% of the Danish Caucasians studied. The methionine translation initiation codon is mutated, ATG-->AAG, and we therefore propose that the gene is a pseudogene incapable of being translated.

Adult↗

Complex group-I introns in nuclear SSU rDNA of red and green algae: evidence of homing-endonuclease pseudogenes in the Bangiophyceae.

The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5' end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors a sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein on the complementary strand. A comparison between related group-I introns in the Bangiophyceae revealed homing-endonuclease-like pseudogenes due to frame-shifts and deletions in Porphyra and Bangia. The Scenedesmus and Porphyra introns provide new insights into the evolution and possible novel functions of nuclear group-I intron proteins.

Amino Acid Motifs↗

Mapping of the mouse Tdgf1 gene and Tdgf pseudogenes.

Teratocarcinoma-derived growth factor-1 (Tdgf1), a member of the "EGF family" of growth factors, is expressed during mouse gastrulation in the forming mesoderm and later in the truncus arteriosus of the developing heart. In humans, TDGF1 is highly expressed in germ cell tumors and in colon and mammary carcinomas. In mouse, one gene (Tdgf1) and two pseudogenes (Tdgf1-ps1 and Tdgf1-ps2) have been isolated and characterized. Tdgf1 corresponds to the gene expressed in F9 teratocarcinoma cells. Tdgf1-ps1 and Tdgf1-ps2 are two intronless sequences with all the characteristics of retroposons. In the present paper, we assign the chromosomal location for Tdgf1, Tdgf1-ps1, and Tdgf1-ps2 sequences to Chromosomes (Chrs) 9, 16, and 17, respectively. Two previously described mouse mutants, scant hair (sch) and fur deficient (fd), map near the Tdgf1 gene. Analysis of their DNA coding region provided no evidence that Tdgf1 could be the responsible gene for these phenotypes. Finally, analysis of the DNA from several Mus musculus strains and from Mus spretus mice revealed a highly variable restriction pattern and the absence of the Tdgf1-ps1 genomic sequence from the Mus spretus genome.

Animals↗

Sequence polymorphism at the tetranucleotide repeat of the human beta-actin related pseudogene H-beta-Ac-psi-2 (ACTBP2) locus.

The tetranucleotide repeat polymorphism in the human beta-actin related pseudogene H-beta-Ac-psi-2 (ACTBP2) locus has become a widely used short tandem repeat (STR) system in paternity testing and human identification. The discrimination index of this locus has been reported to be as high as 99.65%. To study the overall variability of this locus, 222 alleles were sequenced in our laboratory. Here, we report the sequences of the 102 different alleles observed. In addition to the length polymorphism, up to ten different sequence variants for single fragment lengths have been detected.

Actins↗

Human type I hair keratin pseudogene phihHaA has functional orthologs in the chimpanzee and gorilla: evidence for recent inactivation of the human gene after the Pan-Homo divergence.

In addition to nine functional genes, the human type I hair keratin gene cluster contains a pseudogene, phihHaA (KRTHAP1), which is thought to have been inactivated by a single base-pair substitution that introduced a premature TGA termination codon into exon 4. Large-scale genotyping of human, chimpanzee, and gorilla DNAs revealed the homozygous presence of the phihHaA nonsense mutation in humans of different ethnic backgrounds, but its absence in the functional orthologous chimpanzee (cHaA) and gorilla (gHaA) genes. Expression analyses of the encoded cHaA and gHaA hair keratins served to highlight dramatic differences between the hair keratin phenotypes of contemporary humans and the great apes. The relative numbers of synonymous and non-synonymous substitutions in the phihHaA and cHaA genes, as inferred by using the gHaA gene as an outgroup, suggest that the human hHaA gene was inactivated only recently, viz., less than 240,000 years ago. This implies that the hair keratin phenotype of hominids prior to this date, and after the Pan-Homo divergence some 5.5 million years ago, could have been identical to that of the great apes. In addition, the homozygous presence of the phihHaA exon 4 nonsense mutation in some of the earliest branching lineages among extant human populations lends strong support to the "single African origin" hypothesis of modern humans.

Amino Acid Sequence↗

The human CDC42 gene: genomic organization, evidence for the existence of a putative pseudogene and exclusion as a SJS1 candidate gene.

Schwartz-Jampel syndrome (SJS) is an autosomal recessive human disorder characterized by myotonia and osteoarticular deformities. Three types are distinguished based on age at onset: types 1A, 1B and 2. We have previously localized the SJS1 gene, responsible for types 1A and 1B, on human chromosome 1p35-p36.1 in a region frequently rearranged in human tumours. The CDC42 gene, for which divergent localizations have previously been described (chromosomes 4, 7 and 20), has been mapped within the SJS1 critical interval by radiation hybrid and yeast/P1 artificial-chromosome-based physical map analyses. The CDC42 gene product is a small GTPase protein of the Rho family mediating a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression and transformation. To search for mutations in SJS1 patients, we have determined the organization of the human CDC42 gene on chromosome 1p and found that it encodes for the placental and brain isoforms generated by alternative splicing. No mutations have been found in SJS1 patients, excluding CDC42 as the SJS1 gene. Interestingly, we have demonstrated that a CDC42-like transcript gene located on chromosome 4 does not contain introns and is similar to the placental isoform, suggesting that it is a processed pseudogene. The determination of the CDC42 gene structure described in this report should facilitate future studies of the potential role of CDC42 in human disorders.

Amino Acid Sequence↗