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PubMed1991

Beta-tubulins are encoded by at least four genes in the brown alga Ectocarpus variabilis.

Complementary DNA clones of two mRNA species that encode beta-tubulin in the brown alga Ectocarpus variabilis have been isolated. Sequence analysis revealed that the encoded proteins are very similar in primary structure to homologues in other eukaryotes, and differ from each other at six of 447 amino acid residues. The beta 6 message shows a preference for C- or G-terminated codons, using only 49 codons. The beta 5 message has a lesser codon bias, and makes a minor contribution to the beta-tubulin mRNA pool. Southern analysis of E. variabilis DNA demonstrated a beta-tubulin gene family of at least four members.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases.

A cDNA library from freshly isolated protoplasts was differentially screened using cDNAs from mesophyll cells, stressed leaf strips and cell suspension cultures. One of the selected clones, 6P229, turned out to encode a putative polypeptide showing homology to the btuE periplasmic protein of Escherichia coli and to animal selenium-dependent glutathione peroxidases. A major difference was that the putative selenocysteine in the active site was not encoded by the termination codon TGA. The 6P229 gene was found to be expressed in germinating seeds, in apex and in flowers, as well as in stressed tissues. This pattern of expression would be consistent with a key role in cellular metabolism such as defense against oxidative stresses.

Amino Acid Sequence↗

PubMed1994

Acute intermittent porphyria caused by a single base insertion of C in exon 15 of the porphobilinogen deaminase gene that results in a frame shift and premature stopping of translation.

A single base insertion of C in exon 15 of the porphobilinogen deaminase (PBG-D) gene was observed in a patient with acute intermittent porphyria (AIP) by polymerase chain reaction (PCR)-direct sequencing analysis. The insertion locates between positions -22 and -21 from the translation termination codon TAA, causes a frame shift, and results in a stop codon located 4 codons downstream from the insertion (premature stopping of translation). The mutation generates an MspI recognition site, which can be used, in turn, to detect the mutant allele. Analysis of the cDNA fragments amplified by PCR revealed the existence of the abnormal PBG-D mRNA from the mutant allele in the patient.

Amino Acid Sequence↗

PubMed1990

Extent and high frequency of a short conversion between the human A gamma and G gamma fetal globin genes.

Southern blotting and DNA sequencing after polymerase chain reaction (PCR) amplification provide evidence for the frequent occurrence (in 7 out of 24 chromosomes) of a short conversion G gamma----A gamma in the 3' end of the human fetal A gamma globin gene. This short conversion is characterized by the presence, 3 nucleotides downstream from the termination codon of the A gamma gene, of the TCAC sequence that is normally present at the equivalent position at the 3' end of the G gamma gene; it is therefore identical to a conversion already described. Interestingly, we have found that this conversion is associated with the presence of the HindIII polymorphic restriction site in the A gamma IVS2, occupying an equivalent position in both the G gamma and A gamma genes. Our observations strengthen the hypothesis that the presence of the HindIII polymorphic restriction site in A gamma IVS2 and the presence of the sequence TCAC at the 3' end of the A gamma gene might be the result of a single conversion event.

Base Sequence↗

PubMedDate not supplied

Splicing defect of the glycoasparaginase gene in two Japanese siblings with apartylglucosaminuria.

Molecular analysis of the glycoasparaginase gene was performed on two Japanese siblings with aspartylglucosaminuria. The cDNA from one patient contained 7 additional bases between exons 3 and 4 (3'-terminal sequence of intron 3). This insertion resulted in a frame shift, and a termination codon appeared at amino acid 146. Amplification and sequencing of genomic DNA detected a single base transition (A-->G) at the 5' side adjacent to the insertion sequence. This mutation created a consensus AG dinucleotide in the splice acceptor site, and produced almost exclusively an abnormal mRNA containing the insertion by alternative splicing. The calculation of the sample score of the acceptor site supported this analytical result. BsmAI restriction site analysis of amplified cDNA and genomic DNA showed that these patients were homozygotes for this mutation. We conclude that the splicing defect in intron 3 causes glycoasparaginase deficiency in these patients.

Asian People↗

PubMed1991

The nucleotide sequence and evolution of ovine MHC class II B genes: DQB and DRB.

The nucleotide sequences of one Ovar-DQB gene, excluding exon 1 and parts of the introns, and one Ovar-DRB pseudogene are presented. The structure of the Ovar-DQB gene is typical of a major histocompatibility complex (MHC) class II B gene and demonstrates considerable sequence similarity with that of humans including such characteristics as the less common polyadenylation signal, ATTAAA. The ovine sequence has a typical 5' acceptor splice signal for exon 5, thus potentially encoding a full length cytoplasmic tail. The Ovar-DRB gene identified in this study was found to be a pseudogene, lacking a defined exon 2 and containing premature termination codons in both exons 3 and 4. The 3' donor splice site of exon 3 is also atypical. A purine-pyrimidine microsatellite repeat, (dC.dA)15, in the 3' region of the pseudogene may be a hotspot for recombination within the ovine DR subregion.

Amino Acid Sequence↗

PubMed1991

MHC gene Q8/9d of the BALB/cJ mouse strain cannot encode a Qa-2,3 class I antigen.

We have determined the nucleotide sequence of Q8/9d gene of the BALB/c strain of mice, isolated from Steinmetz's cosmid library. As for all other class I genes of the Qa region, the Q8/9d gene spans approximately 4.7 kilobases (kb) and consists of seven exons and six introns. A seven bases deletion in exon 3 results in the occurrence of an early termination codon. Thus the Q8/9d gene cannot encode a normal class I protein. Comparison of the nucleotide sequence of the Q8/9d gene with that of other class I MHC genes revealed a stronger homology to Q7 and Q8 than to K, D, L, TL, and other Q genes. However, the gene cannot originate from a mere fusion between Q8 and Q9 genes except if the ancestor to putative Q8d was markedly different from the present Q8b gene. Using polymerase chain reaction (PCR) technology, we have confirmed the presence of a Q8/9 gene, identical to that present in cosmid 46.1, in the genome of BALB/cJ (Qa-2low). Finally, it has been reported that cDNA clone 94-A, which codes for a Qa-2 antigen, could derive from a transcript of gene Q8/9d. The nucleotide sequences of gene Q8/9d and of cDNA clone 94-A are distinctly different in their 5' regions, in spite of an almost perfect matching in their 3' regions. Thus, clone 94-A cannot derive from an mRNA transcribed from the Q8/9d gene.

Animals↗

PubMed1991

Factors determining the frequency of plasmid cointegrate formation mediated by insertion sequence IS3 from Escherichia coli.

Transposition events mediated by plasmidborne copies of the insertion sequence IS3 of Escherichia coli are difficult to detect because of a low frequency of cointegrate formation. We found that cointegration activity could be strongly enhanced by using plasmid constructions in which a second IS3 element, disabled by a large deletion, was placed adjacent to an intact IS3 copy. Attempts to construct plasmids containing two adjacent intact IS3 copies were unsuccessful, probably because of instability. Transpositional hyperactivity of tandemly duplicated IS sequences was previously described for spontaneous duplications of IS21 and IS30 and may well be a more general phenomenon. The frequency of cointegration events was also strongly increased in an E. coli strain deficient in Dam methylation, suggesting that IS3, like some other Dam site-containing IS elements, is regulated by the Dam methylation system. Insertion sites were strongly clustered within the target lambda repressor gene: however no sequence specificity determinants could be identified. All insertions analyzed carried the IS element in the same orientation; target sequence duplications were mostly 3 bp, but in some cases 4 bp long. To obtain information about the roles of the open reading frames (ORFs) in IS3, we constructed plasmid-borne mutant elements in which potentially functional reading frames were inactivated by site-directed mutations; the mutants were introduced into partial tandem constructions and tested in cointegration assays. Mutations inactivating the putative initiation condons of ORF I and II in the intact element reduced insertion activity to less than 4% of the wild type, whereas the introduction of a termination codon into ORF IV had no effect on cointegration frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1990

Cotranscription of the wild-type chloroplast atpE gene encoding the CF1/CF0 epsilon subunit with the 3' half of the rps7 gene in Chlamydomonas reinhardtii and characterization of frameshift mutations in atpE.

We have characterized two independently isolated point mutants in Chlamydomonas reinhardtii, ac-u-a-1-15 and FUD 17, mapping to the chloroplast ac-u-a locus which corresponds to the atpE gene. Both mutants have a single A:T base pair deletion in a sequence of 6 A:T base pairs at nucleotide positions 102 to 107. This causes a frameshift, altering the coding sequence for the next 8 amino acids and creating a termination codon at amino acid position 44, 98 amino acids from the C-terminus of the protein. Assembly of the ATP synthase is impaired in the mutants; less than 5% of the wild-type level of alpha and beta subunits and no gamma or epsilon subunits are associated with thylakoid membranes of the mutants. The genes encoding the beta and epsilon subunits of the chloroplast ATP synthase from C. reinhardtii are not cotranscribed, in contrast to all other photosynthetic organisms examined to date. Four transcripts, of approximately 1.7, 2.9, 3.3 and 7.0 x 10(3) nucleotides (nt), are found for the atpE gene. S1 nuclease mapping of the 1.7 x 10(3) nt transcript shows that the atpE gene message is preceded by a leader of about 1250 nt. DNA sequence analysis of this region revealed a 159 bp open reading frame corresponding to the 3' half of the rps7 gene, encoding the S7 protein of the small subunit of the chloroplast ribosome. Only the 5' portion of this gene is located in the opposite unique sequence region of the C. reinhardtii chloroplast genome where the rps7 gene was previously mapped by heterologous hybridization.

Amino Acid Sequence↗

PubMed1990

Construction of expression vectors for the gram-negative bacterium Zymomonas mobilis.

A set of vectors was constructed for the cloning and expression of heterologous genes in the Gram-negative bacterium Zymomonas mobilis under the control of the pdc promoter of Z. mobilis. The vectors pPTZ1, pPTZ3, and pPTZ4 are based on the cryptic Z. mobilis plasmid pZM02 and on parts of the Escherichia coli plasmids pKK223-3 and pBR322 together with the multiple cloning site of phage M13mp18. DNA fragments can be readily inserted immediately downstream from the pdc promoter at unique restriction sites for KpnI, XbaI and PstI in pPTZ1 and additionally for SmaI and BamHI in pPTZ3. In pPTZ4, the 5' terminal codons of pdc were deleted allowing the formation of gene fusions. Expression of a promoterless chloramphenicol acetyltransferase gene (cat) controlled by the pdc gene promoter resulted in enzyme activities of up to 5.5 U/mg total cell protein in Z. mobilis cells.

Base Sequence↗

PubMed1992

Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis.

The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9-192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 bp long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.

Amino Acid Sequence↗

PubMed1979

Construction and characterization of a plasmid coding for a fragment of the Escherichia coli recA protein.

The E. coli recA gene was cloned from the phage lambda precA into the vector pBR313. A plasmid, pJL3, was also isolated by cloning a portion of the recA gene into the vector pBR322. pJL3 coded for a fragment of the recA protein 34 Kd (kilodaltons) in size (compared to 40 Kd for the intact protein). This fragment was antigenically related to the recA protein and its synthesis was subject to the same controls as that of the recA protein. The fragment did not express any detectable recA function. When wild-type cells with pJL3 were treated with nalidixic acid, the 34 Kd fragment and the beta-lactamase, made from a gene located downstream from the recA segment, were expressed at very high levels. Moreover, in these cells the rate of synthesis of intact recA protein from the chromosome was inhibited about 2-fold, relative to other chromosomal proteins, when compared to wild-type cells with the pBR322 vector. High level expression of the recA protein fragment and/or the beta-lactamase appeared to be lethal. The size of the 34 Kd fragment, taken together with the location of chain-termination codons in pJL3, localizes the regulatory region of the recA gene within 100 base pairs.

Bacterial Proteins↗

PubMed1979

Nucleotide sequence of small ColE1 derivatives: structure of the regions essential for autonomous replication and colicin E1 immunity.

A small ColE1 derivative, pAO2, which replicates like the original ColE1 and confers immunity to colicin E1 on its host cell has been constructed from a quarter region of ColE1 DNA (Oka, 1978). The entire nucleotide sequence of pAO2 (1,613 base pairs) was determined based on its fine cleavage map. The sequence of a similar plasmid, pAO3, carrying additional 70 base pairs was also deduced. The sequence in the region covering the replication initiation site on these plasmids was consistent with those reported for ColE1 by Tomizawa et al. (1977) and by Bastia (1977). DNA sequences indispensable for autonomous replication were examined by constructing plasmids from various restriction fragments of pAO2 DNA. As a result, a region of 436 base pairs was found to contain sufficient information to permit replication. The occurrence of initiation and termination codons and of the ribosome-binding sequence on pAO2 DNA suggests that a polypeptide chain consisting of 113 amino acid residues may be encoded by the region in which the colicin E1 immunity gene has been mapped.

Base Sequence↗

PubMed1992

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Previous genetic analyses indicated that translational frameshifting in the--1 direction occurs within the run of six adenines in the sequence 5'-TTAAAAAACTC-3' at nucleotide positions 305-315 in IS 1, where the two out-of-phase reading frames insA and B'-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84-87. IS 1 mutants with a 1 bp insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84-87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-Lys-Lys-Leu. An IS 1 mutant with the DNA segment 5'-CTTAAAAACTC-3' at positions 305-315 carrying the termination codon TAA in the B'-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The beta-galactosidase activity specified by several IS 1-lacZ fusion plasmids, in which B'-insB is in-frame with lacZ, showed that the region 292-377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu-Lys-Lys-Leu encoded by the DNA segment 5'-TTAAAAAACTC-3', indicating that--1 frameshifting does occur within the run of adenines.

Adenine↗

PubMed1995

Active transcription of the pseudogene for subunit 7 of the NADH dehydrogenase in Marchantia polymorpha mitochondria.

A pseudogene, psi nad7, which has significant sequence similarity (66.7% amino acid identity) with the bovine nuclear gene for a 49 kDa subunit of the NADH dehydrogenase (NADH:ubiquinone oxidoreductase, EC 1.6.99.3), has been identified on the mitochondrial genome of the liverwort Marchantia polymorpha. The predicted coding region, which includes six termination codons, is actively transcribed into RNA molecules of 16 and 9.6 kb in length, but RNA splicing products were not detected in the liverwort mitochondria. Genomic DNA blot analysis and RNA blot analysis using poly(A)+ RNA suggest that a structurally related nuclear gene encodes the mitochondrial ND7 polypeptide. These results imply that this psi nad7 is a relic of a gene transfer event from the mitochondrial genome into the nuclear genome during mitochondrial evolution in M. polymorpha.

Amino Acid Sequence↗

PubMed1994

An atpE-specific promoter within the coding region of the atpB gene in tobacco chloroplast DNA.

The atpB and atpE genes encode beta and epsilon subunits, respectively, of chloroplast ATP synthase and are co-transcribed in the plant species so far studied. In tobacco, an atpB gene-specific probe hybridizes to 2.7- and 2.3-kb transcripts. In addition to these, a probe from the atpE coding region hybridizes also to a 1.0-kb transcript. The 5' end of the atpE-specific transcript has been mapped 430/431 nt upstream of the atpE translation initiation site, within the coding region of the atpB gene. In-vitro capping revealed that this transcript results from a primary transcriptional event and is also characterized by -10 and -35 canonical sequences in the 5' region. It has been found to share a common 3' end with the bi-cistronic transcripts that has been mapped within the coding region of the divergently transcribed trnM gene, approximately 236 nt downstream from the atpE termination codon. Interestingly, this transcript accumulates only in leaves and not in proplastid-containing cultured (BY-2) cells, indicating that, unless it is preferentially degraded in BY-2 cells, its expression might be transcriptionally controlled.

Base Sequence↗

PubMed1987

Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12.

The nucleotide sequence of the putP gene coding for the proline carrier in Escherichia coli has been determined and the amino acid sequence of the proline carrier deduced from it. The proline carrier is predicted to consist of 502 amino acids, resulting in a molecular weight of 54,343. The predicted protein is very hydrophobic (70% nonpolar amino acids), and its hydropathy profile suggests that it is composed of 12 hydrophobic segments with a mean length of 24.4 residues/segment. If these segments are assumed to be alpha-helical, the mean length of each domain corresponds to the thickness of the hydrophobic core of the membrane. Potential promoter, catabolite gene activator protein (CAP) binding sites and several palindromic sequences, which might be regulatory regions by the putA gene product, were also found in the 5' flanking region of the postulated putP gene. A typical rho-independent transcription termination signal was found after the terminator codon of the putP gene.

Amino Acid Sequence↗

PubMed1988

Rice mitochondrial genome contains a rearranged chloroplast gene cluster.

We have previously reported the isolation and partial sequence analysis of a rice mitochondrial DNA fragment (6.9 kb) which contains a transferred copy of a chloroplast gene cluster coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL), beta and epsilon subunits of ATPase (atpB and atpE), methionine tRNA (trnM) and valine tRNA (trnV). We have now completely sequenced this 6.9 kb fragment and found it to also contain a sequence homologous to the chloroplast gene coding for the ribosomal protein L2 (rpl2), beginning at a site 430 bp downstream from the termination codon of rbcL. In the chloroplast genome, two copies of rpl2 are located at distances of 20 kb and 40 kb, respectively, from rbcL. We have sequenced these two copies of rice chloroplast rpl2 and found their sequences to be identical. In addition, a 151 bp sequence located upstream of the chloroplast rpl2 coding region is also found in the 3' noncoding region of chloroplast rbcL and other as yet undefined locations in the rice chloroplast genome. Hybridization analysis revealed that this 151 bp repeat sequence identified in rice is also present in several copies in 11 other plant species we have examined. Findings from these studies suggest that the translocation of rpl2 to the rbcL gene cluster found in the rice mitochondrial genome might have occurred through homologous recombination between the 151 bp repeat sequence present in both rpl2 and rbcL.

Base Sequence↗