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PubMed1986

Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae.

We determined the nucleotide sequence, mapped the 5' and 3' mRNA termini, and examined the regulation of the RAD2 gene of Saccharomyces cerevisiae. A long open reading frame within the RAD2 transcribed region encodes a protein of 1,031 amino acids with a calculated molecular weight of 117,847. A disruption of the RAD2 gene that deletes the 78 carboxyl terminal codons results in loss of RAD2 function. The 5' ends of RAD2 mRNA show considerable heterogeneity, mapping 5 to 62 nucleotides upstream of the first ATG codon of the long RAD2 open reading frame. The longest RAD2 transcripts also contain a short open reading frame of 37 codons that precedes and overlaps the 5' end of the long RAD2 open reading frame. The RAD2 3' mRNA end maps 171 nucleotides downstream of the TAA termination codon and 20 nucleotides downstream from a 12-base-pair inverted repeat that might function in transcript termination. Northern blot analysis showed a ninefold increase in steady-state levels of RAD2 mRNA after treatment of yeast cells with UV light. The 5' flanking region of the RAD2 gene contains several direct and inverted repeats and a 44-nucleotide-long purine-rich tract. The sequence T G G A G G C A T T A A found at position -167 to -156 in the RAD2 gene is similar to a sequence present in the 5' flanking regions of the RAD7 and RAD10 genes.

Amino Acid Sequence↗

PubMed1993

A single-site mutation in the XPAC gene alters photoproduct recognition.

The XPAC (xeroderma pigmentosum group A complementing) gene, which is located on chromosome 9, carries a variety of point mutations in XP group A patients. We investigated the role of the XPAC gene product in excision repair by generating revertants of an XP group A cell line (XP12RO) that have increased resistance to ultraviolet light. One of these cell lines, XP129, can repair (6-4) pyrimidine-pyrimidone photoproducts normally but has reduced repair of cyclobutane dimers, as in XP12RO. Sequence analysis of cDNA from the XPAC gene indicated that XP12RO contains a termination codon at amino acid position 207, resulting in a reduced amount of mRNA and no detectable protein. In the revertant XP129 line, this termination codon has been mutated further and now encodes glycine in one allele instead of the wild-type arginine. The mRNA level detected by allele-specific polymerase chain reaction amplification was greater for the reverted sequence than for the chain-terminating sequence. These observations indicated that a point mutation resulting in a mis-sense mutation in the XPAC gene and altered expression of the XPAC protein can alter the substrate specificity of the excision repair system, and imply that the XPAC gene product plays an important role in photoproduct recognition.

Amino Acid Sequence↗

PubMed1988

Mapping and characterization of mutations induced by benzo[a]pyrene diol epoxide at dihydrofolate reductase locus in CHO cells.

Chinese hamster ovary cells were mutagenized with benzo[a]pyrene diol epoxide (BPDE), an aromatic hydrocarbon carcinogen, and mutants at the dihydrofolate reductase (dhfr) locus were isolated. Of 15 mutants analyzed by Southern blotting, one contained a large deletion that spanned all six exons of the 25-kb dhfr gene; the remaining mutants exhibited no detectable changes. Three of these putative point mutations were localized by the loss of a restriction site: a SacI site in exon III, an MspI site in exon III, and a KpnI site in exon VI. The affected regions in two of these mutants were cloned and sequenced. The SacI- mutant was caused by a G:C----T:A transversion resulting in an amber termination codon. In the MspI- mutant, the deletion of a single C:G resulted in a frameshift and a downstream ochre termination codon. On the basis of overlapping restriction site sequences, the KpnI- mutant was deduced to be a splicing mutant involving the most 3' G in intron V. The location of these and the remaining 11 putative point mutations was sought using RNA heteroduplex mapping. Mismatched bases between riboprobes complementary to wild-type dhfr mRNA and mutant mRNA molecules were detected in 10 of the 14 mutants analyzed. These mutations mapped to four of the six exons or exon splice sites. Surprisingly, over half of these mutants exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA.

Animals↗

PubMed1981

Gene and mRNA for precursor polypeptide VI from adenovirus type 2.

We present a 1,040-base-pair-long sequences of adenoviruses type 2 DNA which encodes the complete gene for precursor polypeptide VI (pVI). pVI consists of 250 amino acids amounting to a molecular weight of 26,990. The proteolytic cleavage maturing pVI to virion polypeptide VI removes 33 amino acids from the amino-terminal end of the polypeptide, thus giving the mature polypeptide VI a molecular weight of 23,400. The UAA stop codon terminating pVI translation is separated by 84 nucleotides from the initiator triplet for the hexon gene. Both polypeptides are encoded by the same translational reading frame, suggesting the evolution of pVI and hexon as separate proteins by the introduction of a termination codon and selection of a new splice acceptor site in an ancestral fused polypeptide chain. The splice site where the common tripartite leader is attached to the pVI mRNA precedes the initiator codon for pVI translation by one nucleotide and forms, together with other late splice acceptor sites, a late adenovirus consensus acceptor site. We also demonstrate that the 3' end of the mRNA's belonging to the L2 3'-cotermination family is located only 31 nucleotides upstream from the splice junction of the pVI mRNA. Furthermore, we show that four novel polypeptides of molecular weights 80,000, 39,000, 36,000, and 10,500 are encoded by region L2.

Adenoviruses, Human↗

PubMed1999

Functional anatomy of herpes simplex virus 1 overlapping genes encoding infected-cell protein 22 and US1.5 protein.

Earlier studies have shown that (i) the coding domain of the alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and a protein, US1.5, which is initiated from methionine 147 of ICP22 and which is colinear with the remaining portion of that protein; (ii) posttranslational processing of ICP22 mediated largely by the viral protein kinase UL13 yields several isoforms differing in electrophoretic mobility; and (iii) mutants lacking the carboxyl-terminal half of the ICP22 and therefore DeltaUS1.5 are avirulent and fail to express normal levels of subsets of both alpha (e.g., ICP0) or gamma2 (e.g., US11 and UL38) proteins. We have generated and analyzed two sets of recombinant viruses. The first lacked portions of or all of the sequences expressed solely by ICP22. The second set lacked 10 to 40 3'-terminal codons of ICP22 and US1. 5. The results were as follows. (i) In cells infected with mutants lacking amino-terminal sequences, translation initiation begins at methionine 147. The resulting protein cannot be differentiated in mobility from authentic US1.5, and its posttranslational processing is mediated by the UL13 protein kinase. (ii) Expression of US11 and UL38 genes by mutants carrying only the US1.5 gene is similar to that of wild-type parent virus. (iii) Mutants which express only US1. 5 protein are avirulent in mice. (iv) The coding sequences Met147 to Met171 are essential for posttranslational processing of the US1.5 protein. (v) ICP22 made by mutants lacking 15 or fewer of the 3'-terminal codons are posttranslationally processed whereas those lacking 18 or more codons are not processed. (vi) Wild-type and mutant ICP22 proteins localized in both nucleus and cytoplasm irrespective of posttranslational processing. We conclude that ICP22 encodes two sets of functions, one in the amino terminus unique to ICP22 and one shared by ICP22 and US1.5. These functions are required for viral replication in experimental animals. US1.5 protein must be posttranslationally modified by the UL13 protein kinase to enable expression of a subset of late genes exemplified by UL38 and US11. Posttranslational processing is determined by two sets of sequences, at the amino terminus and at the carboxyl terminus of US1.5, respectively, a finding consistent with the hypothesis that both domains interact with protein partners for specific functions.

Amino Acid Sequence↗

PubMed1992

A deletion in the ornithine aminotransferase gene in gyrate atrophy.

Gyrate atrophy (GA) is an autosomal recessive chorioretinal degenerative disease of the eye caused by an inborn defect of the nuclear encoded mitochondrial enzyme ornithine aminotransferase (OAT). We have described previously a GA patient with a 5.0-kilobase pair truncated EcoRI OAT gene fragment and the absence of OAT mRNA on Northern blot analysis. Cloning and sequencing analysis of the truncated gene fragment revealed a 1,072-base pair (bp) deletion including the entire exon 6, starting in intron 5, 172 bp upstream of exon 6 and ending in intron 6, 772 bp downstream of exon 6. A short direct repeat sequence (AGGAGC), resembling the sequence shown to cause DNA polymerase alpha to pause, and sequences capable of forming hairpin loops were both present at the 5' and 3' break-points of the deletion. Reverse transcription-polymerase chain reaction amplification of the patient's RNA with OAT primers yielded DNA fragments of two different sizes, consistent with a low level expression of OAT mRNA. Direct sequencing of the smaller fragment demonstrated the complete absence of exon 6 sequence in the mRNA predicted from the deletion, causing a reading frame shift which results in a premature termination codon at position 192. The mutation in the other allele has been demonstrated by polymerase chain reaction, denaturing gradient gel electrophoresis, and direct sequencing also to be a premature termination codon in exon 6. The absence of detectable OAT mRNA in this patient is consistent with these premature termination mutations because they have been shown to decrease the level of mRNA, especially if present early in the coding sequence.

Amino Acid Sequence↗

PubMed1998

Cloning and characterization of the gene encoding human osteoprotegerin/osteoclastogenesis-inhibitory factor.

The human osteoprotegerin (OPG)-osteoclastogenesis-inhibitory factor (OCIF) gene has been cloned and characterized. The OPG-OCIF gene is a single-copy gene consisting of five exons, and spans 29 kb of the human genome. All the exon/intron boundaries comply with the GT/AG rule. The translation-termination codon is present in exon 5 and a typical poly(A)-addition signal resides 173-nucleotides downstream of the translation-termination codon. A major transcription-initiation site is present 67-nucleotides upstream of the initiation ATG codon. Two minor sites are present further upstream. The 4.2-kb and 6.5-kb transcripts detected in IMR-90 cells were found to contain the 3'-half of intron 2 and the entire intron 2, respectively. In the OPG-OCIF gene, a single intron divides the stretch that encode four Cys-rich motifs, implying diversity from the other members of the tumor necrosis factor receptor (TNFR) family. Two death domain homologous regions (DDHs) present in tandem in OPG-OCIF are encoded separately by exons 4 and 5. The conservation of amino-acid sequences suggests that exon 4 is produced by a duplication of a portion of exon 5.

Alternative Splicing↗

PubMed1996

Two new mutations in the sterol 27-hydroxylase gene in two families lead to cerebrotendinous xanthomatosis.

This report concerns two new mutations in the sterol 27-hydroxylase gene in two patients with cerebrotendinous xanthomatosis (CTX). In a Surinam-Creole patient (patient A), a G deletion on position cDNA 546/547 in exon 3 led to a frameshift and the introduction of a premature termination codon. In a Dutch patient (patient B), a C-->T transition at position 496 in exon 3 also led to a premature termination codon. Patient A was homozygous for the mutation, whereas patient B was compound heterozygous, a C-->T transition also being found in exon 6 at position 1204. The two new mutations were confirmed by restriction analysis with the restriction enzymes FokI and MaeI, respectively.

Adult↗

PubMed1980

A UGA termination suppression tRNATrp active in rabbit reticulocytes.

A tRNATrp was purified from rabbit reticulocytes which suppresses the UGA termination codon of beta-haemoglobin mRNA. Evidence is presented that the beta-haemoglobin readthrough protein is found in reticulocyte translations and intact cells. Some natural readthrough proteins perform essential functions; they are synthesised through suppression of UGA or UAG but not UAA termination codons.

Animals↗

PubMed1993

Complete nucleotide sequence and organization of the bipartite RNA genome of soil-borne wheat mosaic virus.

The complete nucleotide sequences of RNAs 1 and 2 of soil-borne wheat mosaic virus (SBWMV), type member of the furovirus group, were determined. RNA 1 is 7099 nucleotides (nt) and encodes a 150-kDa protein from the 5' end region, the UGA termination codon of which can be partially read through to produce a 209-kDa protein, and a 37-kDa protein in the 3' end region. The C-terminal region of the 150-kDa protein contains an NTP-binding helicase motif and the readthrough region, an RNA polymerase motif, indicating that these two overlapping proteins may form an RNA replication complex similar to those of tobamo- and tobraviruses. The 37-kDa protein has sequence similarity with the cell-to-cell transport protein of dianthoviruses. RNA 2 is 3593 nt and, from the 5' end region, encodes the 19-kDa capsid protein, whose UGA termination codon can be partially suppressed to produce an 84-kDa readthrough protein and, at the 3' proximity, a 19-kDa protein which is rich in cysteine residues. The 28K (kilodaltons, as estimated by SDS-PAGE) protein, previously considered as another capsid readthrough product, is apparently initiated at an in-frame non-AUG codon upstream from the capsid protein gene. In both RNAs 1 and 2, the 5' terminus is capped, and the 3' untranslated region possibly forms internal consecutive pseudoknots as found in tobamovirus RNA as well as a terminal tRNA-like structure similar to tymovirus RNA. An amino acid sequence comparison of RNA replicase genes indicates that, phylogenetically, SBWMV belongs to a cluster formed by tobamo-, tobra-, and hordeiviruses. Differences in the 3' end structure and in the cell-to-cell movement protein, and the distant phylogeny of the RNA replicase genes of SBWMV and beet necrotic yellow vein virus, suggest that the furoviruses should be divided into at least two groups.

Amino Acid Sequence↗

PubMed2005

Dominant and recessive COL6A1 mutations in Ullrich scleroatonic muscular dystrophy.

In this study, we characterized five Ullrich scleroatonic muscular dystrophy patients (two Italians, one Belgian, and two Turks) with a clinical phenotype showing different degrees of severity, all carrying mutations localized in COL6A1. We sequenced the three entire COL6 complementary DNA. Three of five patients have recessive mutations: two patients (P1and P3) have homozygous single-nucleotide deletions, one in exon 9 and one in exon 22; one patient (P2) has a homozygous single-nucleotide substitution leading to a premature termination codon in exon 31. The nonsense mutation of P2 also causes a partial skipping of exon 31 with the formation of a premature termination codon in exon 32 in 15% of the total COL6A1 messenger RNA. The remaining two patients carry a heterozygous glycine substitution in exons 9 and 10 inside the triple-helix region; both are dominant mutations because the missense mutations are absent in the DNA of their respective parents. As for the three homozygous recessive mutations, the apparently healthy consanguineous parents all carry a heterozygous mutated allele. Here, for the first time, we report a genotype-phenotype correlation demonstrating that heterozygous glycine substitutions in the triple-helix domain of COL6A1 are dominant and responsible for a milder Ullrich scleroatonic muscular dystrophy phenotype, and that recessive mutations in COL6A1 correlate with more severe clinical and biochemical Ullrich scleroatonic muscular dystrophy phenotypes.

Adolescent↗

PubMed2003

[A novel Krit-1 mutation in Han family with cerebral cavernous malformation].

OBJECTIVE: To detect the mutations of Krit-1 gene that cause familial cerebral cavernous malformation (CCM) in the Han ethnic origin. METHODS: The subjects were hospitalized in the Department of Neurosurgery, Tiantan Hospital affiliated to Capital University of Medical Sciences. Two families (A and B) and 8 apparently sporadic individuals affected with CCM were screened for mutations of Krit-1 gene. Members of the family CCM have a wide range in age of onset with seizures, headaches and skin lesions. The gene was screened by PCR amplification of 16 exons and mutation was detected by direct sequencing. RESULTS: In family A samples, analysis of the Krit-1 gene revealed a new point mutation in exon 14 [a heterozygous C to G transition at nucleotide 1 289 (counting from the start codon or nt 2 308 counting from the first nt of the mRNA, aligned according to Gene Bank AF388384)] which predicts the substitution of a premature termination codon for Serine at codon 430 (S430X), belonging a nonsense point mutation. No mutation was identified in one of family A members as well as in any of the sporadic individuals with the exception of a single nucleotide polymorphism. CONCLUSIONS: Report the first family in the Han with CCM having a novel mutation in the CCM1 gene on the continent of Asia. The newly identified mutation creates a premature termination codon and is predicted to produce a truncated Krev1 interaction-trapped 1 protein, KRIT1. This result allows efficient presymptomatic molecular diagnosis.

Adult↗

PubMed1994

Identification of two novel mutations in the methylmalonyl-CoA mutase gene with decreased levels of mutant mRNA in methylmalonic acidemia.

Genetic defects in the methylmalonyl-CoA mutase (MCM) gene result in methylmalonic acidemia which is inherited as an autosomal recessive disease. We investigated fibroblast cultures obtained from two Japanese patients with MCM deficiency. MCM mRNA was not detected by Northern blot analysis, suggesting that MCM mRNA was markedly decreased. Reverse transcription/polymerase chain reaction (RT-PCR) of MCM mRNA followed by analysis on a fluorescent fragment analyzer indicated that the level of MCM mRNA in these fibroblasts was less than 1% of normal controls. This minute amount of MCM mRNA was successfully amplified by nested RT-PCR and subjected to primary structure analysis. Sequence analysis revealed two novel mutations: a G-to-T substitution at nucleotide position 425 and a 2 bp deletion at nucleotide positions 769 and 770. The first mutation (G425T) resulted in the substitution of a termination codon for glutamic acid at amino acid position 117. The second mutation (769 delta CA) resulted in a frame shift which created a premature termination codon 508 amino acid upstream of the C-terminus of the protein. Patient 1 was homozygous for G425T and patient 2 was a compound heterozygote for G425T and 769 delta CA. Our report is the first to identify MCM mutations that affect the stability of MCM mRNA. An analysis of 16 Japanese patients revealed the presence of G425T in six patients, suggesting a relatively high incidence of the mutation among Japanese patients. This is in sharp contrast to a previous report describing diverse heterogeneity of MCM mutations among Caucasians.

Amino Acid Metabolism, Inborn Errors↗

PubMed1992

Cloning, mapping, and sequencing of 3' and its flanking region of bovine alpha-s1 casein gene.

A positive clone was isolated from a bovine genomic library in EMBL3 with the probe of alpha-s1 casein gene cDNA. The results from both restriction endonuclease digestion and Southern hybridization probed with the upstream and downstream fragments from the termination codon of bovine alpha-s1 casein gene cDNA suggested that the clone contained 3.5 kb downstream and 10.2 kb upstream fragments from the termination codon of alpha-s1 casein gene. Some divergences in restriction endonucleases sites, a few deletions, and many point mutations were found in introns and exons when compared with those which were published.

Animals↗

PubMed1989

Alternative mRNA splicing generates the two ribulosebisphosphate carboxylase/oxygenase activase polypeptides in spinach and Arabidopsis.

Sequence analysis of ribulosebisphosphate carboxylase/oxygenase (rubisco) activase cDNA and genomic clones isolated from spinach and Arabidopsis thaliana indicates that the two polypeptides of rubisco activase arise from alternative splicing of a common pre-mRNA. In spinach, two 5' splice sites are used in processing a single 137-nucleotide intron near the 3' end of the primary transcript. This intron was either removed completely or, alternatively, the first 22 nucleotides of the intervening sequence were retained in the mature rubisco activase mRNA. The 22-nucleotide auxiliary exon contains an in-frame ochre termination codon and leads to the synthesis of a 41-kilodalton polypeptide. Removal of the entire 137-nucleotide intervening sequence results in the synthesis of a larger 45-kilodalton polypeptide. Thus, alternative splicing of the spinach rubisco activase mRNA results in the synthesis of two polypeptides that are identical except for 37 additional amino acids at the C terminus of the 45-kilodalton polypeptide. This conclusion was confirmed by Cleveland peptide mapping and by N-terminal and C-terminal amino acid sequence analyses of both purified polypeptides. This method of producing the two rubisco activase polypeptides may be an evolutionarily conserved feature in higher plants because a nearly identical process occurs in the production of the two rubisco activase polypeptides in Arabidopsis. In Arabidopsis, an alternatively spliced intron resides at precisely the same position as the alternatively spliced intron in spinach and results in the synthesis of 44-kilodalton and 47-kilodalton rubisco activase polypeptides. In contrast to spinach, however, the retained portion of the intervening sequence does not contain an in-frame termination codon. Rather, a shift in reading frame leads to termination of translation of the smaller polypeptide within the coding region of the larger polypeptide.

Amino Acid Sequence↗

PubMed2003

Identification of a new G-to-A transition mutation at nucleotide position 129 of the Xrcc4 gene in ionizing radiation-hypersensitive mutant LX830 cells.

The mouse lymphoma cell line LX830 is an X-ray-hypersensitive mutant. Complementation tests between LX830 cells and radiation-sensitive mutants of M10 (Xrcc4 deficient cells) or SX10 (DNA ligase IV deficient cells) cells showed that M10 cells did not complement LX830 cells, but SX10 cells did, suggesting that LX830 cells would belong to the X-ray-cross complementation group (XRCC4). A sequence analysis of Xrcc4 cDNA in LX830 cells disclosed a transition of G to A at nucleotide position 129, which resulted in a change of tryptophan (43) to a termination codon. Transfection of the mouse Xrcc4 cDNA rescued the X-ray sensitivity of the mutant cells. LX830 is an Xrcc4-deficient cell line bearing a termination codon in exon 2 of the Xrcc4 gene and no wild-type Xrcc4 gene.

Amino Acid Sequence↗

PubMed1992

Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo.

Animals↗

PubMed1981

Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus.

Deletions of polyoma virus DNA around the region that codes for the C-terminus of the viral middle T-antigen were created using a transforming fragment (BamH I/EcoR I) of viral DNA cloned in the plasmid vector pAT153. These species were recloned and assayed for their ability to transform Rat-1 cells in culture. Our results showed that whereas the DNA sequence between the presumed translational termination codon for the viral middle T-antigen and the single viral EcoR I site could be removed with no apparent effect on transformation, the removal of the termination codon itself or any amino acid coding sequences of this protein caused a drastic decrease in the transforming ability of the DNA. Transfection of Rat-1 cells with plasmids that contained viral DNA with deletions which corresponded to the last fourteen or more amino acids of the middle T-antigen never gave rise to cellular transformation.

Amino Acid Sequence↗