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PubMed1996

Premature nonsense codons decrease the stability of phytohemagglutinin mRNA in a position-dependent manner.

Premature termination of translation has often been associated with decreased mRNA accumulation in plants, but the affected step in gene expression has not been identified. To investigate this problem, the expression of wild-type and mutant alleles of the bean phytohemagglutinin (PHA) gene has been examined in tobacco cells and transgenic plants. Measurement of mRNA decay rates in stably transformed cell lines demonstrated that premature nonsense codons markedly destabilized the mRNA. This decreased stability was also reflected by decreased accumulation of transcripts containing premature nonsense codons in transgenic plants. The positional dependence of the nonsense codon effect was evaluated by introducing premature nonsense codons at different distances from the PHA AUG start codon. Transcripts with nonsense codons about 20, 40 or 60% of the way through the normal PHA coding region yielded highly unstable mRNAs, whereas a transcript with a nonsense codon at 80% was as stable as wild-type. The ability to recognize and rapidly degrade certain transcripts with early nonsense codons could provide plant cells with a means to minimize the production of wasteful and possible deleterious truncated proteins.

Alleles↗

PubMed2001

Trichinella spiralis mtDNA: a nematode mitochondrial genome that encodes a putative ATP8 and normally structured tRNAS and has a gene arrangement relatable to those of coelomate metazoans.

The complete mitochondrial DNA (mtDNA) of the nematode Trichinella spiralis has been amplified in four overlapping fragments and 16,656 bp of its sequence has been determined. This sequence contains the 37 genes typical of metazoan mtDNAs, including a putative atp8, which is absent from all other nematode mtDNAs examined. The genes are transcribed from both mtDNA strands and have an arrangement relatable to those of coelomate metazoans, but not to those of secernentean nematodes. All protein genes appear to initiate with ATN codons, typical for metazoans. Neither TTG nor GTT start codons, inferred for several genes of other nematodes, were found. The 22 T. spiralis tRNA genes fall into three categories: (i) those with the potential to form conventional "cloverleaf" secondary structures, (ii) those with TPsiC arm + variable arm replacement loops, and (iii) those with DHU-arm replacement loops. Mt-tRNA(R) has a 5'-UCG-3' anticodon, as in most other metazoans, instead of the very unusual 5'-ACG-3' present in the secernentean nematodes. The sequence also contains a large repeat region that is polymorphic in size at the population and/or individual level.

Adenosine Triphosphatases↗

PubMed1999

Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase.

The Corynebacterium glutamicum ack and pta genes encoding the acetate-activating enzymes acetate kinase and phosphotransacetylase were isolated, subcloned on a plasmid and re-introduced into Corynebacterium glutamicum. Relative to the wild-type, the recombinant strains showed about tenfold higher specific activities of both enzymes. Sequence analysis of a 3657 bp DNA fragment revealed that the ack and pta genes are contiguous in the corynebacterial chromosome, with pta upstream and the last nucleotide of the pta stop codon (TAA) overlapping the first of the ack start codon (ATG). The predicted gene product of pta consists of 329 amino acids (Mr 35242), that of ack consists of 397 amino acids (Mr 43098) and the amino acid sequences of the two polypeptides show up to 60 % (phosphotransacetylase) and 53% (acetate kinase) identity in comparison with respective enzymes from other organisms. Northern (RNA) blot hybridizations using pta- and ack-specific probes and transcriptional cat fusion experiments revealed that the two genes are transcribed as a 2.5 kb bicistronic mRNA and that the expression of this operon is induced when Corynebacterium glutamicum grows on acetate instead of glucose as a carbon source. Directed inactivation of the chromosomal pta and ack genes led to the absence of detectable phosphotransacetylase and acetate kinase activity in the respective mutants and to their inability to grow on acetate. These data indicate that no isoenzymes of acetate kinase and phosphotransacetylase are present in Corynebacterium glutamicum and that a functional acetate kinase/phosphotransacetylase pathway is essential for growth of this organism on acetate.

Acetate Kinase↗

PubMed1996

Role of an upstream open reading frame in mediating arginine-specific translational control in Neurospora crassa.

The Neurospora crassa arg-2 transcript contains an upstream open reading frame (uORF) specifying a 24-residue leader peptide and is subject to a novel form of negative translational regulation in response to arginine. The role of the arg-2 uORF in arginine-specific negative regulation was investigated by using translational fusions of wild-type and mutant arg-2 sequences to the Escherichia coli lacZ reporter gene specifying beta-galactosidase. The wild-type uORF conferred Arg-specific regulation on the reporter gene in N. crassa, but mutated or truncated uORFs did not, as determined by measurements of beta-galactosidase activity produced in N. crassa strains expressing arg-2-lacZ fusion genes. All effects on reporter gene expression were posttranscriptional, as determined by measurement of RNA levels. Both sequence-dependent and sequence-independent effects of uORFs were observed. Genes containing the wild-type uORF or a 21-codon mutated uORF showed reduced translation in comparison with that of a gene lacking a uORF. Both uORF-containing transcripts showed reduced association with polysomes relative to transcripts lacking a uORF, but only the transcript with the wild-type uORF showed a reduced average number of ribosomes associated with it in response to arginine addition. Direct translational fusions between uORF sequences and lacZ sequences indicated that the uORF is translated. Overlapping the uORF with the lacZ initiation codon indicated that ribosome reinitiation at a downstream start codon is not integral to uORF-mediated, Arg-specific translational regulation. These studies provide direct biochemical evidence for arg-2 uORF function in translational control.

Arginine↗

PubMed1995

Translational enhancement of FGF-2 by eIF-4 factors, and alternate utilization of CUG and AUG codons for translation initiation.

The FGF-2 mRNA of most mammals contains a long and inhibitory 5' UTR in addition to at least two CUG codons upstream and in frame with the AUG start codon. These CUGs are used for translation initiation to generate several polypeptides. Cells overexpressing the translation initiation factor 4E produce and secrete large amounts of FGF-2, and particularly the largest, CUG1-initiated form. Overexpression of FGF-2 is due to a translational enhancement of its mRNA, as indicated by its association with large polyribosomes in contrast to control cells, where it partitions mostly in fractions lighter than 80 S or small polyribosomes. Breast carcinomas expressing elevated eIF-4E also exhibited the large FGF-2 isoforms, which could play an important role in tumor angiogenesis. Translation of in vitro transcribed rat FGF-2 in reticulocyte lysates resulted in synthesis of four polypeptides, of similar size to those observed in vivo. Addition of purified eIF-4F preferentially increased translation of CUG1- and AUG-initiated isoforms. Since the different isoforms of FGF-2 may have different roles and intracellular distribution, the effects of the eIF-4 factors on FGF-2 expression could be important for the control of cell proliferation and differentiation.

Animals↗

PubMed1998

Molecular characterization of type E Clostridium botulinum and comparison to other types of Clostridium botulinum.

Determination of nucleotide sequence upstream to the neurotoxin binding protein (NBP) gene of type E Clostridium botulinum has revealed an open reading frame whose stop codon is only 18 bp apart from the start codon of the NBP gene. Amino acid sequence derived from the corresponding nucleotide sequence suggested the existence of the open reading frame as a 47.8 kDa protein (P-48). Protein data bank search revealed that the 47.8 kDa protein has 80% sequence identity to P-47 of type F C. botulinum. The gene organization of type E. Clostridium botulinum was predicted and compared to other types of C. botulinum. In type E C. botulinum, genes for the P-48, the neurotoxin binding protein and the neurotoxin form an operon which was similar to that of type F C. botulinum. However, type E C. botulinum has a P-18 gene instead of P-21 gene observed in type F C. botulinum, both located upstream to their respective P-48/P-47 gene.

Amino Acid Sequence↗

PubMed1990

Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

The gene coding for the key glycolytic enzyme fructose-1,6-diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.

Amino Acid Sequence↗

PubMed1993

Identification of a new hepatitis B virus (HBV) genotype from Brazil that expresses HBV surface antigen subtype adw4.

The complete genome of a hepatitis B virus (HBV) from Brazil that expressed the subtype adw4 of HBV surface antigen (HBsAg) was cloned and sequenced. The genome, termed w4B, consists of 3215 bp. The overall genetic organization of typical hepadnaviruses with four open reading frames including the preC region was found to be conserved. When comparing the w4B sequence with 19 complete HBV genomes it was, however, found to be more divergent (15%) than any other HBV sequence thus far reported. Until now, no more than 11% divergence has been reported. Distinct from the five known HBV genotypes A to E, w4B made up a new, sixth genotype. The importance of the conserved third start codon in the HBV X gene became apparent in isolate w4B. By mutation, this ATG was out of frame, and by what appears to have been a linked mutation, a new start site two codons downstream was re-established. The significance of several other mutations is discussed.

Amino Acid Sequence↗

PubMed1998

Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans.

Arthrobacter nicotinovorans is a Gram-positive aerobic soil bacterium able to grow on nicotine as its sole source of carbon and nitrogen. The initial steps of nicotine catabolism are catalyzed by nicotine dehydrogenase, the l- and d-specific 6-hydroxynicotine oxidases, and ketone dehydrogenase. The genes encoding these enzymes reside on a 160 kb plasmid, pAO1. The cccDNA of this plasmid was isolated in high purity and reasonable yield. It served as template material for the construction of a lambda-phage DNA library of the plasmid. The genes coding for 6-hydroxy-l-nicotine oxidase and for the subunits of the heterotrimeric ketone dehydrogenase were identified, subcloned and sequenced. The 6-hlno gene was identified as a 1278 bp open reading frame; its regulatory elements were also recognized. The derived primary structure of the monomer of apo-6-hydroxy-l-nicotine oxidase (46,264.5 Da) agrees with the data obtained by partial amino acid sequencing. 6-Hydroxy-l-nicotine oxidase and 6-hydroxy-d-nicotine oxidase were expressed in Escherichia coli and obtained in a state of high purity and crystallized. Ketone dehydrogenase (KDH) was found to be a heterotrimer with subunits of molecular mass 89,021.71, 26,778.65 and 17,638.88. The genes of KDH-A and KDH-B are juxtaposed; the A of the stop codon of KDH-A is used in the start codon of KDH-B, eliciting a frame shift. KDH-C is separated from KDH-A by 281 bp.

Arthrobacter↗

PubMed1997

A novel process of inosine 5'-monophosphate production using overexpressed guanosine/inosine kinase.

A novel process for producing inosine 5'-monophosphate (5'-IMP) has been demonstrated. The process consists of two sequential bioreactions; the first is a fermentation of inosine by a mutant of Corynebacterium ammoniagenes, and the second is a unique phosphorylating reaction of inosine by guanosine/inosine kinase (GIKase). GIKase was produced by an Escherichia coli recombinant strain, MC1000(pIK75), which overexpressed the enzyme up to 50% of the total cellular protein. The overproducing plasmid, pIK75, which was randomly screened out from deletion plasmids with various lengths of intermediate sequence between the E. coli trpL Shine-Dalgarno sequence, derived from the vector plasmid, and the start codon of the GIKase structural gene. In pIK75, the start ATG was placed 16 bp downstream of the trpL Shine-Dalgarno sequence under the control of the E. coli trp promoter. Fermentation of inosine and its phosphorylation were sequentially performed in a 5-1 jar fermenter. At the end of inosine fermentation by C. ammoniagenes KY13761, culture broth of MC1000(pIK75) was mixed with that of KY13761 to start the phosphorylating reaction. Inosine in the reaction mixture was stoichiometrically phosphorylated, and 91 mM 5'-IMP accumulated in a 12-h reaction. This new biological process has advantages over traditional methods for producing 5'-IMP.

Base Sequence↗

PubMed1989

Cloning and nucleotide sequence of ovine prolactin cDNA.

A cDNA expression library was constructed in the lambda gt 11 phage vector using ovine (o) pituitary mRNA. The clone, pOP1, carrying a 934-bp insert contains an open reading frame beginning with the first nucleotide (nt) and ending with the stop codon TAA at nt position 781. Two potential translation start codons (ATGs) are present in the 5' region of this cDNA. Translation initiation could occur at the 5' proximal ATG at nt position 61. The nucleotide sequence around this ATG (TCCATGG), resembles the optimum sequence context for translation initiation by the eukaryotic ribosomes, as defined by mutational analysis [Kozak, Cell 44 (1986) 283-292)], with its substitution of the A at -3 of the consensus sequence by a T residue in this clone. Translation initiated at this codon could potentially code for the entire pre-prolactin (pre-PRL) molecule. The 3'-untranslated region is 154 nt long and contains a polyadenylation signal AATAAA. The deduced amino acid sequence agrees in totality with the published amino acid sequence of the mature hormone. The present study reports on the nucleotide sequence of o-PRL mRNA and the deduced amino acid sequence in the signal peptide of the hormone.

Amino Acid Sequence↗

PubMed1994

Structure and promoter activity of a stress and developmentally regulated polyubiquitin-encoding gene of Nicotiana tabacum.

A polyubiquitin-encoding gene was identified from a Nicotiana tabacum genomic library using a specific probe spanning the 3' untranslated region of the corresponding cDNA. The gene, Ubi.U4, is expressed in various amounts in the whole plant, except in just-fully-expanded leaves. Genomic blots indicate that it originates from N. tomentosiformis. Sequence analyses reveal that the gene consists of four ubiquitin monomers extended by a fifth truncated subunit. It is disrupted by a single 457-bp intron in close proximity to the start codon of translation. Primer extension experiments localized the transcription start point (tsp). Transient gene expression in N. tabacum protoplasts indicates that the deletion of the intron has no significant influence on gene expression. Mutagenesis on putative cis-regulatory elements indicates at least three important motifs in the proximal promoter: an 'ACGT' core element, an A + T-rich sequence and a less clearly defined cis-element located between bp -162 and -113.

Amino Acid Sequence↗

PubMed1995

Cloning and sequencing of a secY homolog from Streptomyces scabies.

The complete DNA sequence of the Streptomyces scabies (Ss) secY homolog and partial sequences of adjacent upstream and downstream open reading frames (ORFs) have been determined. The nucleotide sequence of a 2-kb region predicts a polypeptide of 437 amino acids in length with homology to the SecY protein family. The Ss secY homolog lies upstream from a sequence that has homology to the adenylate kinase gene (adk) family. The translational stop codon of the putative SecY ORF overlaps the predicted start codon for the Adk ORF. Another ORF that lies upstream from the secY homolog has sequence similarity to the genes that code for the L15 r-protein. Within the 243-bp intergenic region between the L15 and SecY coding sequences, the presence of a streptomycete-like promoter sequence and an 18-bp inverted repeat suggests that the secY homolog and the adjacent downstream sequences may be transcribed independently of the L15 coding sequence. Transcript analysis indicates that the secY homolog is expressed in both Ss and Streptomyces lividans. The proposed gene and transcript organization of the L15-SecY-Adk coding regions in the Ss clone resembles that of Micrococcus luteus which, like the streptomycetes, has a G+C-rich genome.

Adenylate Kinase↗

PubMed1996

Protein-encoding genes in the sulfothermophilic archaea Sulfolobus and Pyrococcus.

A number of unrelated protein-encoding genes from sulfothermophilic archaea, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Pyrococcus furiosus and Pyrococcus woesei, has been analyzed. In the Sulfolobus genus, the content of A + T is significantly higher than that of C + G and the base usage follows the order, A > T > G > C. In Pyrococcus, the A + T content is also higher than that of C + G, but with lower values; in the order of base usage, G precedes T. The codon usage of these sulfothermophiles has been determined; alternative start codons are frequently used in both genera; codon preferences reflect the rich A + T composition of the corresponding genomes; for both genera the codon bias is particularly evident within the different arginine triplets, where AGA and AGG are predominant. From the similarities in the codon usage, close taxonomic relationships become evident within the Sulfolobus or the Pyrococcus genus; a lower, but significant similarity is also clear between these genera. The synonymous codon usage of these sulfothermophiles shows similarities with that of Saccharomyces cerevisiae and bovine mitochondria, whereas clear divergences are observed with the halophilic archaeal genus, Halobacterium, or the eubacterium, Escherichia coli. The unrelated proteins of the considered sulfothermophiles have been analyzed for the content of hydrophobic residues; the comparison with mesophiles reveals a significant increase in the average hydrophobicity of amino acid residues. This finding could indicate a mechanism of adaptation of proteins in organisms living under extreme environments. It is noteworthy that an opposite trend, i.e. a decreased average hydrophobicity, occurs in unrelated halophilic proteins.

Animals↗

PubMed2005

Functional expression of the rat organic anion transporter 1 (rOAT1) in Saccharomyces cerevisiae.

Organic anion transporter 1 (OAT1) is localized in the basolateral membrane of the proximal tubule in the kidney and plays an essential role in eliminating a wide range of organic anions, preventing their toxic effects on the body. Structural and functional studies of the transporter would be greatly assisted by inexpensive and rapid expression in the yeast Saccharomyces cerevisiae. The gene encoding rat OAT1 (rOAT1) contains many yeast non-preferred codons at the N-terminus and so was modified by fusion of the favored codon sequence of a hemagglutinin (HA) epitope preceding the start codon. The modified gene was cloned into several yeast expression plasmids, both integrative and multicopy, with either ADH1 promoter or GAL1 promoter in order to find a suitable expression system. Compared with the wild type gene, a substantial increase in rOAT1 expression was achieved by modification in the translational initiation region, suggesting that the codon chosen at the N-terminus influenced its expression. The highest inducible expression of rOAT1 was obtained under GAL1 promoter in 2 mu plasmid. A large fraction of rOAT1 was glycosylated in yeast, unaffected by growth temperature. The recombinant yeast expressing rOAT1 showed an increase in the uptake of p-aminohippurate (PAH) and this showed a positive correlation with rOAT1 expression level. Location of rOAT1 predominantly in the yeast plasma membrane confirmed correct processing. The importance of glycosylation for rOAT1 targeting was also shown. To our knowledge, this is the first successful functional expression of rOAT1 in the yeast S. cerevisiae.

Amino Acid Sequence↗

PubMed2006

Cloning and characterization of the astaxanthin biosynthesis gene cluster from the marine bacterium Paracoccus haeundaensis.

Carotenoids are important natural pigments produced by many microorganisms and plants. In a previous study, we isolated and characterized a marine bacterium, Paracoccus haeundaensis, which produces carotenoids, mainly astaxanthin. In the present study, a carotenoid biosynthesis gene cluster involved in the production of astaxanthin was isolated and characterized from this marine bacterium. The cluster contained six carotenogenic genes, five of which, with the same orientation, were designated crtW, crtZ, crtY, crtI, and crtB, and one of which, with the opposite orientation, was designated crtE. The crtW, crtZ, crtY, crtI, crtB, and crtE genes contained 726, 486, 1158, 1503, 912, and 879 base pairs, respectively, and encoded polypeptides of 242, 162, 386, 501, 304, and 293 amino acid residues, respectively. The stop codon of each crt gene, except crtE, overlapped the start codon of the following crt gene. Comparisons of the structures and nucleotide sequences of the crt genes showed that the signature domains of the carotenoid biosynthesis genes are highly conserved. The molecular phylogenetic tree analysis showed the evolutionary relationships among the carotenoid biosynthesis genes of various organisms. An expression vector (pCR-XR-TOPO-Crt-full vector) containing the astaxanthin biosynthesis genes was constructed and transformed into Escherichia coli BL21(DE3) Codon Plus cells. The E. coli transformants produced 400 microg astaxanthin per gram of dry cell weight.

Base Sequence↗

PubMed2003

Molecular characterization of an inducible gentisate 1,2-dioxygenase gene, xlnE, from Pseudomonas alcaligenes NCIMB 9867.

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enables the degradation of xylenols and cresols via gentisate. Previous reports had indicated that one set of enzymes is constitutively expressed whereas the other set is strictly inducible by aromatic hydrocarbon substrates. The gene encoding gentisate 1,2-dioxygenase (GDO), the enzyme that catalyzes the cleavage of the gentisate aromatic ring, was cloned from strain P25X. The GDO gene, designated xlnE, is 1,044 bp, and is part of a 5.4 kb operon which consists of six genes, xlnEFGHID. Transcription of this operon was driven by a sigma 70-type promoter, PxlnE, located 123 bp upstream of the xlnE start codon. Primer extension analysis showed that the xlnE transcription start point is located at the -87 adenine residue. In a P25X xlnE knockout mutant, GDO activity could only be detected when cells were grown in the presence of aromatic substrates, suggesting that xlnE encodes for the constitutive copy of GDO. This was verified by constructing a P25X strain with xlnE transcriptionally fused to a promoterless catechol 2,3-dioxygenase gene. In this strain, catechol 2,3-dioxygenase activity was detected in cells that were grown in the absence of aromatic inducers. However, catechol 2,3-dioxygenase activity increased up to four fold when these cells were grown in the presence of aromatic substrates, in particular 3-hydroxybenzoate. Thus, xlnE is in fact, inducible and the constitutive activity observed under non-inducing conditions was due to its relatively high basal levels of expression.

Amino Acid Sequence↗

PubMed1997

The polyhomeotic locus of Drosophila melanogaster is transcriptionally and post-transcriptionally regulated during embryogenesis.

The polyhomeotic (ph) locus of Drosophila is a complex locus essential for the maintenance of segmental identity. Genetic analysis suggested that two independent units contribute to ph function. Comparison of genomic sequence shows that the ph locus has been duplicated, and that it contains proximal and distal transcription units. The proximal transcription unit encodes two embryonic mRNAs of 6.4 and 6.1 kb, and the distal unit encodes a 6.4-kb embryonic mRNA. The proximal and distal transcription units are differentially regulated at the mRNA level during development as shown by developmental Northern analysis. The distal protein is very similar to the proximal product, except for the absence of an amino terminal region, and a small region near the carboxy terminus. The long open reading frame in the distal cDNA does not begin with an ATG codon, and an internal ATG is used for a start codon. We show that the proximal protein occurs in two forms that are developmentally regulated, and that probably arise from use of two different initiator methionine codons. We find no evidence for differential binding of proximal and distal products to polytene chromosomes. Nevertheless, we show that mutations in the proximal and distal proteins have differing effects on regulation of a reporter under the control of a regulatory region from bithoraxoid, suggesting that ph proximal and distal proteins have different functions. These results show that the ph locus undergoes complex developmental regulation, and suggest that Polycomb group regulation may be more dynamic than anticipated.

Animals↗