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ATP synthase from bovine mitochondria: complementary DNA sequence of the import precursor of a heart isoform of the alpha subunit.

The alpha-subunit of ATP synthase from mitochondria is a major component of the extrinsic membrane sector of the enzyme. It is encoded in nuclear DNA. A family of overlapping complementary DNA clones encoding its precursor has been isolated from a bovine library by using in the first instance a mixture of 128 synthetic oligonucleotides designed on the basis of the known protein sequence, and the sequence of the full-length cDNA has been determined. The deduced protein sequence shows that the alpha-subunit of ATP synthase has a presequence of 43 amino acids that is not present in the mature protein. Presumably it directs the protein into the mitochondrial matrix and is removed during the import process. The encoded protein sequence is also longer by one amino acid at its C-terminal end than the protein isolated from F1-ATPase, but this alanine residue may have been removed artifactually during release of the F1-ATPase particle from the inner mitochondrial membrane. With the exception of one uncertainty caused by an ambiguity at one position in the nucleotide sequence, the mature protein sequence encoded in the cDNA is exactly the same as the sequence determined previously by direct analysis of the protein isolated from bovine heart mitochondria [Walker et al. (1985) J. Mol. Biol. 184, 677-701]. The cDNA sequence differs in 158 nucleotides over a region of alignment of 1097 nucleotides from a partial cDNA for the alpha-subunit that has been isolated from a bovine cDNA derived from liver RNA [Breen (1988) Biochem. Biophys. Res. Commun. 152, 264-269].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal.

The genomic RNA of the coronavirus IBV contains an efficient ribosomal frameshift signal at the junction of the overlapping 1a and 1b open reading frames. The signal is comprised of two elements, a heptanucleotide "slip-site" and a downstream tertiary RNA structure in the form of an RNA pseudoknot. We have investigated the structure of the pseudoknot and its contribution to the frameshift process by analysing the frameshifting properties of a series of pseudoknot mutants. Our results show that the pseudoknot structure closely resembles that which can be predicted from current building rules, although base-pair formation at the region where the two pseudoknot stems are thought to stack co-axially is not a pre-requisite for efficient frameshifting. The stems, however, must be in close proximity to generate a functional structure. In general, the removal of a single base-pair contact in either stem is sufficient to reduce or abolish frameshifting. No primary sequence determinants in the stems or loops appear to be involved in the frameshift process; as long as the overall structure is maintained, frameshifting is highly efficient. Thus, small insertions into the pseudoknot loops and a deletion in loop 2 that reduced its length to the predicted functional minimum did not influence frameshifting. However, a large insertion (467 nucleotides) into loop 2 abolished frameshifting. A simple stem-loop structure with a base-paired stem of the same length and nucleotide composition as the stacked stems of the pseudoknot could not functionally replace the pseudoknot, suggesting that some particular conformational feature of the pseudoknot determines its ability to promote frameshifting.

Base Sequence↗

Molecular phylogeny of the carnivora (mammalia): assessing the impact of increased sampling on resolving enigmatic relationships.

This study analyzed 76 species of Carnivora using a concatenated sequence of 6243 bp from six genes (nuclear TR-i-I, TBG, and IRBP; mitochondrial ND2, CYTB, and 12S rRNA), representing the most comprehensive sampling yet undertaken for reconstructing the phylogeny of this clade. Maximum parsimony and Bayesian methods were remarkably congruent in topologies observed and in nodal support measures. We recovered all of the higher level carnivoran clades that had been robustly supported in previous analyses (by analyses of morphological and molecular data), including the monophyly of Caniformia, Feliformia, Arctoidea, Pinnipedia, Musteloidea, Procyonidae + Mustelidae sensu stricto, and a clade of (Hyaenidae + (Herpestidae + Malagasy carnivorans)). All of the traditional "families," with the exception of Viverridae and Mustelidae, were robustly supported as monophyletic groups. We further have determined the relative positions of the major lineages within the Caniformia, which previous studies could not resolve, including the first robust support for the phylogenetic position of marine carnivorans (Pinnipedia) within the Arctoidea (as the sister-group to musteloids [sensu lato], with ursids as their sister group). Within the pinnipeds, Odobenidae (walrus) was more closely allied with otariids (sea lions/fur seals) than with phocids ("true" seals). In addition, we recovered a monophyletic clade of skunks and stink badgers (Mephitidae) and resolved the topology of musteloid interrelationships as: Ailurus (Mephitidae (Procyonidae, Mustelidae [sensu stricto])). This pattern of interrelationships of living caniforms suggests a novel inference that large body size may have been the primitive condition for Arctoidea, with secondary size reduction evolving later in some musteloids. Within Mustelidae, Bayesian analyses are unambiguous in supporting otter monophyly (Lutrinae), and in both MP and Bayesian analyses Martes is paraphyletic with respect to Gulo and Eira, as has been observed in some previous molecular studies. Within Feliformia, we have confirmed that Nandinia is the outgroup to all other extant feliforms, and that the Malagasy Carnivora are a monophyletic clade closely allied with the mongooses (Herpestidae [sensu stricto]). Although the monophyly of each of the three major feliform clades (Viverridae sensu stricto, Felidae, and the clade of Hyaenidae + (Herpestidae + Malagasy carnivorans)) is robust in all of our analyses, the relative phylogenetic positions of these three lineages is not resolvable at present. Our analyses document the monophyly of the "social mongooses," strengthening evidence for a single origin of eusociality within the Herpestidae. For a single caniform node, the position of pinnipeds relative to Ursidae and Musteloidea, parsimony analyses of data for the entire Carnivora did not replicate the robust support observed for both parsimony and Bayesian analyses of the caniform ingroup alone. More detailed analyses and these results demonstrate that outgroup choice can have a considerable effect on the strength of support for a particular topology. Therefore, the use of exemplar taxa as proxies for entire clades with diverse evolutionary histories should be approached with caution. The Bayesian analysis likelihood functions generally were better able to reconstruct phylogenetic relationships (increased resolution and more robust support for various nodes) than parsimony analyses when incompletely sampled taxa were included. Bayesian analyses were not immune, however, to the effects of missing data; lower resolution and support in those analyses likely arise from non-overlap of gene sequence data among less well-sampled taxa. These issues are a concern for similar studies, in which different gene sequences are concatenated in an effort to increase resolving power.

Animals↗

Transcription factor GATA-5 selectively up-regulates mucin gene expression.

PURPOSE: Overlapping expression patterns of epithelial mucins, MUC1-MUC6, and transcription factor GATA-5 were reported previously. However, the functional relationship between them is poorly understood. The aim of the current study is to elucidate whether or not expression of mucin genes is regulated by GATA-5. METHODS: GATA-5 was transiently overexpressed in COS-7 and 293T cells by plasmid transfection and/or adenovirus infection. GATA-5 expression was confirmed by Western blot analysis. Expression of mucin genes was studied by reverse-transcription-PCR. Reporter gene assays were employed to analyze the effect of GATA-5 on the promoter activity of mucin genes. RESULTS: mRNA levels of MUC2, MUC3, and MUC4 were increased, whereas those of MUC1, MUC5AC, MUC5B, and MUC6 remained unchanged upon the overexpression of GATA-5 in both COS-7 and 293T cells. By means of luciferase assay, GATA-5 was found to activate the promoters of the human MUC2 and MUC4 genes. GATA-5 lacking the zinc finger domain impaired these functions. CONCLUSIONS: These findings indicate that GATA-5 may play important roles in the regulation of mucin expression and gastrointestinal epithelial cell differentiation.

Adenoviridae↗

X-linked mental retardation with dystonic movements of the hands (PRTS): revisited.

A family with a syndrome of mental retardation, dystonic movements of the hands, and dysarthria (MIM no. 309510) was described and mapped to Xp22 by Partington et al. (Am J Med Genet 1988; 30:251-262). The original localization encompassed the distal half of the short arm of the X chromosome, with a peak lod score of 2.1 at the DXS41 locus. The gene localization for this disorder (PRTS) has now been refined using recently characterized dinucleotide repeat markers. The PRTS gene maps between DXS365 and DXS28, an interval estimated to be less than 15 cM. A peak lod score of 3.01 at a recombination fraction of zero was generated by 2-point linkage analysis with the marker DXS989. Dystonic movements may be progressive and could be overlooked in children. Clinical assessments of affected men who are mentally retarded should be critically evaluated for this manifestation, where they belong to families in which the gene localization overlaps with PRTS.

Child, Preschool↗

HLA genes associated with rheumatoid arthritis. Identification of susceptibility alleles using specific oligonucleotide probes.

We examined the association of individual HLA genes with rheumatoid arthritis (RA), using oligonucleotide probes that identified both DR4-associated and non-DR4-associated genes. Two distinct HLA-DR beta alleles (Dw4 and Dw14) were found in DR4+ RA patients compared with controls (Dw4 50% versus 17%; Dw14 35% versus 5%; total DR4 73% versus 30%), indicating that these 2 alleles are independent susceptibility genes. Remarkably, the majority of the DR4- RA patients also demonstrated a linear DNA sequence, apparently "shuffled" between different susceptibility alleles, identified with an oligonucleotide probe to a key portion of the Dw14 gene.

Adult↗

Spectrum of clinical features in Muckle-Wells syndrome and response to anakinra.

OBJECTIVE: Mutations in the NALP3/CIAS1/PYPAF1 gene are associated with the autoinflammatory diseases Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), and neonatal-onset multisystem inflammatory disease (NOMID), which is also known as chronic infantile neurologic, cutaneous, articular (CINCA) syndrome. Molecular studies suggest that NALP3 is involved in the processing of interleukin-1beta (IL-1beta), prompting us to investigate whether IL-1 blockade may be therapeutic in patients with MWS. METHODS: We reviewed the clinical features of 3 members of a family, all of whom had MWS associated with the NALP3 variant V200M (also designated V198M), and evaluated the response of their inflammatory disease to treatment with the recombinant human IL-1 receptor antagonist anakinra. The subjects kept a diary of symptoms and underwent fortnightly clinical and laboratory assessments, including measurement of the serum amyloid A protein concentration. RESULTS: Each subject had fever, rashes, arthralgia, conjunctivitis, sensorineural deafness, and an intense acute-phase response characteristic of MWS. However, additional features were identified, including exacerbation of their disease by cold and neurologic manifestations, that have hitherto been described only in FCAS and NOMID, respectively. Clinical and serologic evidence of active inflammatory disease resolved rapidly and completely during treatment with anakinra. CONCLUSION: The remarkable response of MWS to anakinra suggests that IL-1beta has a fundamental role in the pathogenesis of inflammation associated with mutations in the NALP3 gene, and supports study of IL-1 inhibition in patients with NOMID/CINCA syndrome or FCAS. The clinical features of the various syndromes associated with mutations in the NALP3 gene may overlap to a greater extent than has previously been recognized.

Acute-Phase Reaction↗

Cloning and expression of the Cdx family from the frog Xenopus tropicalis.

The caudal-related (Cdx) homeodomain transcription factors have a conserved role in the development of posterior structures in both vertebrates and invertebrates. A particularly interesting finding is that Cdx proteins have an important function in the regulation of expression from a subset of Hox genes. In this study, we report the cloning of cDNAs from the Cdx genes of the amphibian Xenopus tropicalis. Xenopus tropicalis is a diploid species, related to the commonly used laboratory animal Xenopus laevis, and has attracted attention recently as a potential genetic model for animal development. The Xenopus tropicalis cDNAs, Xtcad1, Xtcad2, and Xtcad3, show between 88 and 94% sequence identity with their Xenopus laevis orthologues. This finding corresponds to between 90 and 95% identity at the level of derived amino acid sequence. We also present a detailed description of Xtcad1, Xtcad2, and Xtcad3 expression during normal development. In common with the Cdx genes of other vertebrates, the Xenopus tropicalis Cdx genes show overlapping and dynamic patterns of expression in posterior regions of the embryo through the early stages of development.

Amino Acid Sequence↗

Identification of rare polymerase variants of hepatitis B virus using a two-stage PCR with peptide nucleic acid clamping.

Emergence of lamivudine-resistant variants, with amino acid substitutions in the Tyr-Met-Asp-Asp (YMDD) motif of hepatitis B virus (HBV) reverse transcriptase, is a serious problem in antiviral therapy. Presence of YMDD motif variants in patients who had never been treated with lamivudine has been reported recently. However, no analysis of nucleotide and amino acid sequences of these variants has been performed. In the present study, using polymerase chain reaction (PCR) with peptide nucleic acid (PNA) clamping, we detected many new variants, such as Tyr-Arg-Asp-Asp (YRDD), Tyr-Met-Asp-Asn (YMDN). Many of them had stop codon(s) in overlapping HBs gene. Although the biological activity of these HBV polymerase variants remains to be determined, our results showed that numerous quasispecies are created during virus replication. A typical lamivudine-resistant Tyr-Val-Asp-Asp (YVDD) variant was detected in only one of 62 (1.6%) anti-HBe patients with HBV infection before administration of lamivudine. This variant did not have the L528M mutation, which is often associated with YVDD variants, and lamivudine therapy in this patient suppressed HBV replication. Thus, care should be taken when interpreting the results of detection of YMDD variants, especially when the sensitivity of the assay is very high. Amplification of rare variants by PCR with PNA seems a useful tool to examine the emergence of drug-resistant variants as well as naturally occurring mutants, such as the hepatitis B e antigen (HBeAg) stop codon and vaccine escape mutants. Examination of rare variants should enhance the understanding of the mechanism for emergence of drug-resistant HBV variants and help in developing strategies for new antiviral drugs.

Adult↗

YMDD mutants in patients with chronic hepatitis B before treatment are not selected by lamivudine.

Hepatitis B virus (HBV) mutants with mutations in the YMDD motif of viral DNA polymerase/reverse transcriptase are described in patients infected with HBV who have not received lamivudine therapy, but their pathogenetic potential is not clear. These mutants were detected by the polymerase chain reaction with peptide nucleic acid clamping in pretreatment sera from two patients who later received lamivudine. One patient developed acute exacerbation with hepatic encephalopathy and received lamivudine along with plasma exchange, which were effective on his illness. YIDD mutants were detected in all three pretreatment sera and both posttreatment sera from him. HBV DNA clones from pretreatment and posttreatment sera, however, did not have the same amino acid sequence. In the other patient who developed severe breakthrough hepatitis after receiving lamivudine, YIDD mutants were detected in two pretreatment and two posttreatment sera. When amino acid sequences of HBV DNA clones with the YIDD mutation were compared before and after he received lamivudine, however, they were not in accord. Hence, YIDD mutants in both patients with chronic hepatitis B before treatment were not selected by lamivudine after they had been placed on it. Numerous amino acid conversions were detected in HBV DNA clones with YIDD mutations, and some of them created stop codons in the overlapping S gene sequence. In Conclusion, HBV mutants with mutations in the YMDD motif in patients before treatment would not be selected by lamivudine or induce breakthrough hepatitis, and some of these would not be replication-competent due to stop codons in the S gene.

Adult↗

Micronodular thymoma: an epithelial tumour with abnormal chemokine expression setting the stage for lymphoma development.

The aetiology of primary B-cell lymphomas of the thymus is enigmatic. Although thymic follicular lymphoid hyperplasia (TFH) is commonly associated with myasthenia gravis (MG), lymphoma is not a complication of this condition. The present paper reports a high frequency of monoclonal B-cell populations (6 of 18 cases; 33%) in micronodular thymoma (MNT), a peculiar thymic epithelial neoplasm with a B-cell-rich stroma, while B cells were consistently polyclonal in TFH (25 cases) and other types of thymomas (15 cases) (p < 0.001). An intratumoural lymphoma could be identified in three of the six monoclonal MNTs. Sequencing of the monoclonal IgH chain revealed partially overlapping VDJ gene usage in MNT and thymic mucosa-associated lymphoid tissue (MALT) lymphomas. The neoplastic epithelium of MNTs, but not of TFH and other types of thymoma, expressed high levels of dendritic cell, T-cell, and B-cell chemoattractants, such as CCL18, CCR6, and CCL20. It is concluded that abnormal chemokine expression in an epithelial tumour, MNT, can promote the recruitment of MALT, the emergence of monoclonal B cells, and, eventually, the subsequent development of mediastinal lymphomas. More generally, the concept that expression of a 'high-risk' spectrum of chemokines due to local or genetic factors may interfere with B-cell homeostasis and may contribute to MALT lymphoma development in chronic inflammatory states is proposed.

Adult↗

Upstream stimulatory factor 1 (USF1) suppresses induction of CYP1A1 mRNA by 3-methylcholanthrene (MC) in HepG2 cells.

In this study, an endogenous factor(s) involved in the suppression of the induction of CYP1A1 was studied. Analyzing the sequences, we found that the sequence of xenobiotic responsive element (XRE) in the upstream region of the human CYP1A1 gene was overlapped with that of the upstream stimulatory factor 1 (USF1)-binding site in mouse metallothionein I promoter. In fact, a gel shift assay using a specific competitor or mutant probes showed that the core sequence of human XRE was specifically recognized by USF1. The amount of USF1 in the nuclear extracts from HepG2 cells was smaller than that from rat and rabbit livers as assayed by the binding to XRE. To determine whether or not USF1 could inhibit the interaction of aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (Arnt) complex with XRE, we transfected USF1-SR alpha expression vector into HepG2 cells. The results showed that no interaction of AhR/Arnt complex with XRE occurred even when the cells were treated with 2,3,7,8-tetrachlorodibenzofuran (TCDF). Furthermore, the S1 nuclease protection assay showed that the induction of CYP1A1 mRNA by 3-methylcholanthrene (MC) was depressed by the transfection of USF1-SR alpha into HepG2 cells. Thus, it is highly possible that USF1 negatively regulates the induction of CYP1A1 in humans.

Animals↗

Isolation and characterization of the DLAD/Dlad genes, which lie head-to-head with the genes for urate oxidase.

We previously found that a novel DNA endonuclease named DLAD (DNase II-Like Acid DNase) is specifically expressed in murine liver. Here, we describe the isolation and characterization of the human DLAD and mouse Dlad genes. Both DLAD and Dlad consist of 6 exons. DLAD encodes a 361 amino acid protein sharing 34.6% amino acid identity with human DNase II. Although a recombinant protein for the putative human DLAD has a divalent cation-independent acid DNase activity, expression of the DLAD mRNA containing the entire open reading frame was not detected in any human tissues tested, except for lung, in which a short 1.1 kb transcript lacking the first two exons is expressed. Interestingly, sequence analysis of Dlad showed that the 1st exon of the urate oxidase gene, Uox, is located on the opposite strand in its 5'-flanking region. The head-to-head organization of DLAD and UOX is conserved in the human genomic sequence. Promoter analysis revealed that the intergenic region between Dlad and Uox has promoter activity for both the Dlad and Uox directions, however, the corresponding human genomic fragment has promoter activity only for DLAD. It is known that murine Uox is expressed only in the liver, whereas human UOX has been inactivated as a pseudogene. On the basis of these results, the expression of DLAD/Dlad and UOX/Uox is suggested to be coordinated by a common regulatory mechanism(s), and the balance between the two enzymes is thought to be important for maintaining the purine nucleotide pool in the liver.

5' Flanking Region↗

The Charcot-Marie-Tooth binary repeat contains a gene transcribed from the opposite strand of a partially duplicated region of the COX10 gene.

Misalignment between the two elements of the CMT1A-REP binary repeat on chromosome 17p11.2-p12 causes two inherited peripheral neuropathies, Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies. This binary repeat contains repetitive DNA elements that include LINES, SINES, medium reiteration frequency repeats, and a transposon-like element. The COX10 gene has been mapped 10 kb centromeric to the distal CMT1A-REP element, and a portion of this gene is present in both the proximal and the distal CMT1A-REP elements. We report the isolation and characterization of a novel cDNA (C170RF1), which maps centromeric to and partially within the proximal CMT1A-REP element. Part of C170RF1 is transcribed from the opposite strand of the COX10 partial gene duplication present in the proximal CMT1A-REP element. This finding shows that C170RF1 and COX10 are being transcribed from opposite strands of identical DNA sequences that are separated by 1.5 Mb in the genome. RT-PCR analysis showed the proximal transcript was expressed in skeletal muscle. Sequence analysis identified an open reading frame encoding a 199-amino-acid protein. Zoo blot analysis showed that the transcript is conserved in nonhuman primates. The presence of a binary repeat contributes to the instability of this region of chromosome 17, yet two CMT1A-REP elements are present in the chimpanzee and all human populations. The presence of expressed sequences in both elements of the CMT1A-REP binary repeat could explain the maintenance of this repeat in humans.

Adult↗

The novel gene, gamma2-COP (COPG2), in the 7q32 imprinted domain escapes genomic imprinting.

The gene MEST (or PEG1) on chromosome 7q32 is paternally expressed in human fetal tissues as a result of genomic imprinting. Since some imprinted genes are clustered, we speculated that an imprinted gene cluster might exist at 7q32. We have sought to isolate additional human genes close to MEST and to characterize their allelic expression patterns. Here, we report the biallelic expression of the gene, gamma2-COP (coatomer protein complex, subunit gamma 2, HUGO-approved symbol COPG2), and monoallelic expression of the transcript, CIT1, which is located in intron 20 of gamma2-COP. Recently, gamma2-COP was reported to be a novel imprinted gene that overlaps the 3'-untranslated region (3'-UTR) of MEST in a tail-to-tail orientation. However, our results revealed biallelic expression in all fetal tissues and adult blood lymphocytes. On the other hand, CIT1 was an antisense transcript of gamma2-COP intron 20 and was expressed from the paternal allele in all fetal tissues examined. Adult blood lymphocytes showed biallelic expression. We identified additional MEST 3'-UTR sequence, which overlaps the last four exons and introns of gamma2-COP. This additional MEST 3'-UTR may complicate analysis of gamma2-COP imprinting. Our data indicate that the region containing MEST at 7q32 is an imprinted domain, but gamma2-COP adjacent to MEST escapes genomic imprinting.

3' Untranslated Regions↗

IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif.

Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA. OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB. This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif. OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro. The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper. We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction. Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function. Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats. Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB[1-149], that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex". Preincubation of OrfAB[1-149] with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA. Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats.

Amino Acid Sequence↗

Sequence and gene expression analyses of plasmid pHPM8 from Helicobacter pylori reveal the presence of two operons with putative roles in plasmid replication and antibiotic activity.

The DNA sequence of a 7.8-kb Helicobacter pylori plasmid, pHPM8, was determined. Six open reading frames (ORFs) were present. Ribonuclease protection studies showed that ORF1/ORF2 and ORF3/ORF4 genes are organized in operons possibly involved in plasmid replication and in production of a peptide with antibiotic activity, respectively. Finding areas of pHPM8 with a high level of identity to H. pylori chromosomal DNA supported the hypothesis that recombination occurs between plasmids and the chromosome of H. pylori.

Amino Acid Sequence↗