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PubMed1994

Isolation of a pea (Pisum sativum) seed lipoxygenase promoter by inverse polymerase chain reaction and characterization of its expression in transgenic tobacco.

Part of the 5'-flanking sequence of a pea (Pisum sativum) lipoxygenase (LOX) gene was cloned, after amplification from genomic DNA by inverse polymerase chain reaction. Translational and transcriptional fusions of 818 bp of the 5'-flanking region and its deletion derivatives (-513 and -356) were made to a beta-glucuronidase (GUS)-coding sequence and introduced into tobacco. Analysis of T1 transformants showed that the 818 bp 5'-flanking sequence drove GUS expression in seeds that was temporally regulated in a fashion similar to the accumulation of LOX mRNA in developing pea seeds. Contrary to expectations, however, expression of the 818 bp promoter-GUS fusion was not seed-specific; GUS activity was highest in leaves and also present in stems and, to a lesser extent, roots. Deletion analyses identified the region between -818 and -513 as essential for high-level, temporally regulated expression in seeds and also indicated that the sequence between -513 and -356 plays a negative role in leaf/stem, but not seed, expression. Comparison of translational and transcriptional fusions indicated that the LOX initiation codon was used more efficiently than the GUS initiation codon by the tobacco leaf translational apparatus.

Base Sequence↗
PubMedDate not supplied

A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1.

Initiation of T-lymphocyte proliferation by mitogen or antigen involves a cascade of gene activation events. Thus, by the time mitogen-activated T cells have reached the G1/S interface, many genes that are transcriptionally silent in G0, like the c-myc, IL-2, IL-2 receptor (IL-2R) and transferrin receptor (TfR) genes, have been transcriptionally activated. To understand the role of the individual genes in the activation process, one must be able to interfere specifically with the expression or function of each particular gene product. In this way, by blocking the IL-2R with an antibody, it has been demonstrated that IL-2/IL-2R interaction is required to induce TfR expression in activated T cells. When the function or expression of intracellular proteins is to be blocked, however, the need to introduce antibodies into the cytoplasm of viable cells, although possible, is a limiting factor. We have taken another approach, namely the exogenous addition to bulk cell cultures of small antisense oligomers. Sequence-specific antisense oligodeoxyribonucleotides have been reported to inhibit intracellular viral replication without interfering with cellular protein synthesis. Similarly, rabbit globin mRNA translation in a cell-free system and in rabbit reticulocytes has been inhibited by oligomers complementary to the globin mRNA initiation codon region. Recently, a pentadecadeoxyribonucleotide complementary to the initiation codon and four downstream codons of human c-myc mRNA was reported to inhibit the proliferation of the human leukaemic cell line HL-60 specifically. We report here that the same c-myc complementary oligonucleotide inhibits mitogen-induced c-myc protein expression in human T lymphocytes and prevents S phase entry. Interestingly, c-myc antisense treatment did not inhibit G0 to G1 traversal as assessed by morphologic blast transformation, transcriptional activation of the IL-2R and TfR genes, or induction of 3H-uridine incorporation.

Cell Division↗
PubMed2000

Sequence-motif analysis of 5'-untranslated region of GB virus-C in Japanese patients.

BACKGROUND: GB Virus C (GBV-C) is considered to belong to the Flaviviridae; however, the structures of the N-terminal end of its putative polyprotein are not well known. The internal ribosomal entry site (IRES) at the 5'-untranslated region of GBV-C and an initiating codon at nucleotides (nt) 552-554 have been proposed. We investigated the validity of this proposal. METHODS: The 5'-untranslated region of GBV-C was amplified from serum samples of 17 Japanese patients. Polymerase chain reaction-amplified products were directly sequenced and the obtained sequences were analysed by comparing them with the IRES structure of other viruses. RESULTS: Fifteen of the 17 (88%) GBV-C strains in our patients were classified as being Asian type. The box-A-like sequence (UUUC) and box-B-like sequence (AUCAUGG) observed in the IRES of picornaviruses were highly conserved in all the strains. Based on pair-wise comparisons with the multiple alignment data, overall sequence divergence for the 5'-terminus was 2.9-12%. When compared with the proposed secondary structure of the IRES model, the sequence divergences of the Asian-type GBV-C were higher at the regions of loop structures and lower at the regions of double-stranded RNA. The AUG codons, except for the one located at nt 552-554, produced truncated polyproteins or were not in-frame with the putative protein. CONCLUSIONS: Our examination of the sequence motif of GBV-C supports the proposal that the GBV-C has common structural motifs for IRES at its 5'-untranslated region and the AUG codon at nt 552-554 may be an initiating codon.

5' Untranslated Regions↗
PubMed2001

Structural and functional studies of CCAAT/enhancer-binding protein epsilon.

CCAAT/enhancer-binding protein (C/EBP) epsilon is a critical transcription factor for differentiation of myeloid cells. Structural and functional relationships of C/EBPepsilon were explored by recombinant protein studies, gene mutation, and transactivation assays. Evidence strongly suggested that C/EBPepsilon does not have disulfide bonds. Transactivation analysis of C/EBPepsilon having mutations of each of three conserved cysteines (C345, C148S, and C280S) indicated that the three mutant proteins had almost the same activity as the wild type. Dimer formation of C/EBPepsilon was not detected using both reducing and non-reducing SDS-polyacrylamide gel electrophoresis with Western blot analysis from either bacterial or mammalian expressed C/EBPepsilon. Furthermore, C/EBPepsilon mutant C280S gave a gel band similar to that for wild type, suggesting that this C-terminal, conserved cysteine is not involved in disulfide bond formation in vivo, even though previous data for C/EBPbeta suggested that dimers may form in vitro utilizing this conserved cysteine residue. Mutational studies of conserved residues in the activating domain 1 (ADM1) and ADM2 of the amino region of the gene indicated that negative charge is critical for transactivational activity of C/EBPepsilon. Mutational analyses of hydrophobic amino acids in ADM1 suggested that these residues do not play a key role in transactivational activity. Further mutational studies indicated that, although the N-terminal 32-amino acid peptide of C/EBPepsilon isoform p32 did not greatly influence the transactivation activity compared with p30 isoform, this peptide does modulate transactivation activity. Domain swapping experiments substituting the ADM1 domain of various C/EBPs for C/EBPepsilon showed that the C/EBPalpha and -delta but not -beta ADM1 markedly enhanced the chimeric C/EBPepsilon transcriptional activity. Based on mutational data and possible mRNA structure, we hypothesized about the effect of mRNA structure on translation of the two major C/EBPepsilon isoforms: p32 and p30. The data suggested a very stable 8-base pair double helical structure with one strand sequence including the initial codon for p32 and complementary strand with the initial codon for p30.

Amino Acid Sequence↗
PubMed1990

Tissue-specific splicing of pro-enkephalin mRNA.

A testis cDNA library derived from 4-6 week-old rats was screened using a 32P-labeled 435 base pair (bp) cDNA probe derived from exon 3 of rat brain proenkephalin (PE). Two positive clones were isolated from an initial screening of approximately 20,000 recombinant bacteriophage plaques. The longest insert (1,500 bp) was sequenced and was found to contain a portion of intron A at the 5'-terminus. Intron B was sliced as it is in the brain, therefore, the domain structure of the cDNA was intron A-exon 2-exon 3, reading in the 5'-3' direction. Since the translational initiator codon is located at the 5'-terminus of exon 2, the reading frame of PE is unaltered, however, within intron A are 4 upstream AUG codons which exist in a favorable context for initiation of translation. It is probable that translation of PE would be inhibited by the presence of these upstream initiator codons, which may explain the discrepancy between the levels of PE mRNA and opioid peptides in the testis. Hypophysectomy prior to the onset of puberty was found to drastically reduce the level of PE mRNA in the testis and epididymus, suggesting that pituitary factors affect the expression of the PE gene in these tissues, either directly or indirectly.

Animals↗
PubMed1996

Genetic analysis of the Mycobacterium smegmatis rpsL promoter.

The DNA sequence of the promoter region of the Mycobacterium smegmatis rpsL gene, which encodes the S12 ribosomal protein, was determined. Primer extension analysis and S1 nuclease protection experiments identified the 5' end of the rpsL mRNA to be 199 bp upstream of the translation initiation codon. The rpsL promoter contained sequences upstream of this start point for transcription that were similar to the canonical hexamers found at the -10 and -35 regions of promoters recognized by Esigma70, the major form of RNA polymerase in Escherichia coli. To define the promoter of the rpsL gene, DNA fragments containing progressive deletions of the upstream region of the rpsL gene were inserted into a plasmid vector containing a promoterless xylE gene. These insertions revealed that the 200 bp of DNA sequence immediately upstream from the translation initiation codon was not essential for promoter function. In addition, 5' deletions removing all but 34 bp upstream of the transcription start point retained greater than 90% promoter activity, suggesting that the -35 hexamer was not essential for promoter activity. To determine which nucleotides were critical for promoter function, oligonucleotide-directed mutagenesis and mutagenic PCR amplification were used to produce point mutations in the region upstream of the start point of transcription. Single base substitutions in the -10 hexamer, but not in the -35 hexamer, severely reduced rpsL promoter activity in vivo. Within the -10 hexamer, nucleotide substitutions causing divergence from the E. Coli sigma70 consensus reduced promoter activity. The DNA sequence immediately upstream from the - 10 hexamer contained the TGn motif described as an extended -10 region in prokaryotic promoters. Mutations in this motif, in combination with a transition at either the -38 or -37 position within the -35 hexamer, severely reduced promoter activity, indicating that in the absence of a functional -35 region, the rpsL promoter is dependent on the TGn sequence upstream from the -10 hexamer. Comparison of the nucleotide sequence of the rpsL promoter region of M. smegmatis with the homologous sequences from Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium tuberculosis showed the presence in these slowly growing mycobacterial species of conserved promoter elements a similar distance upstream of the translation initiation codon of the rpsL gene, but these other mycobacterial promoters did not contain the extended -10 motif.

Base Sequence↗
PubMed1991

Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli.

The phenylalanine-specific permease gene (pheP) of Escherichia coli has been cloned and sequenced. The gene was isolated on a 6-kb Sau3AI fragment from a chromosomal library, and its presence was verified by complementation of a mutant lacking the functional phenylalanine-specific permease. Subcloning from this fragment localized the pheP gene on a 2.7-kb HindIII-HindII fragment. The nucleotide sequence of this 2.7-kb region was determined. An open reading frame was identified which extends from a putative start point of translation (GTG at position 636) to a termination signal (TAA at position 2010). The assignment of the GTG as the initiation codon was verified by site-directed mutagenesis of the initiation codon and by introducing a chain termination mutation into the pheP-lacZ fusion construct. A single initiation site of transcription 30 bp upstream of the start point of translation was identified by the primer extension analysis. The pheP structural gene consists of 1,374 nucleotides specifying a protein of 458 amino acid residues. The PheP protein is very hydrophobic (71% nonpolar residues). A topological model predicted from the sequence analysis defines 12 transmembrane segments. This protein is highly homologous with the AroP (general aromatic transport) system of E. coli (59.6% identity) and to a lesser extent with the yeast permeases CAN1 (arginine), PUT4 (proline), and HIP1 (histidine) of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1996

Both codon context and leader length contribute to efficient expression of two overlapping open reading frames of a cucumber necrosis virus bifunctional subgenomic mRNA.

The importance of codon context and leader length in the translational regulation of p20 and p21 from the bifunctional 0.9-kb subgenomic mRNA cucumber necrosis virus was investigated. Nucleotide substitutions introduced into the -3 and +4 positions of the p21 AUG codon (where the A of the AUG is +1) verified that purines in these positions are favored and demonstrated the similar contribution of the -3 and +4 positions to the efficiency of initiation codon selection in plants. The effect of nucleotide substitutions in the +5 position, most clearly demonstrated when pyrimidines occupy the -3 and +4 positions, also provided direct insight into the influence of the +5 position in plants. The codon context of the upstream p21 initiation codon affected expression from the downstream p20 AUG codon. In addition, an increase in the length of the subgenomic mRNA leader decreased expression from the downstream p20 initiation site. These latter observations are in accordance with the "Kozak rules" for accession of internal AUG codons by leaky ribosomal scanning and provide the first example of an effect of leader length on the efficiency of translation initiation in a plant (viral) mRNA.

Base Sequence↗
PubMed1989

Cloning and comparative mapping of a human class III (chi) alcohol dehydrogenase cDNA.

A cDNA encoding human class III (chi ADH5) alcohol dehydrogenase was isolated, sequenced and used to comparatively map this unusual ADH. In their coding sequences, the three major ADH classes were approximately equisimilar, class II and III ADHs sharing the highest sequence identity (67%). A class III-like ADH was mapped to mouse chromosome 3, site of the ADH gene complex, and synteny of ADH5 with four other ADH loci on human chromosome 4 was confirmed. The nearly full-length 1613 nucleotide cDNA contained 433 nucleotides of 3' nontranslated sequence and two possible initiation sites for translation. A protein of 374 amino acid residues could be synthesized using the potential initiation codon at nucleotide 59. However, use of the likely initiation codon at nucleotide 5 would produce a protein of 392 residues with 19 additional N-terminal residues as compared to the known protein sequence. The derived protein sequence also differs at residue 166, where Tyr is found. This difference, due to a single base substitution, could result from cloning artifact, polymorphism, or two expressed class III ADH genes.

Alcohol Oxidoreductases↗
PubMed1994

Basepairing with 18S ribosomal RNA in internal initiation of translation.

In concert with the translation initiation factors 'trans-acting' factors function specifically during internal initiation on picornaviral mRNAs. Of these trans-acting factors, two have been identified as the La-protein and the polypyrimidine tract binding protein. Within the internal ribosomal entry site on the viral RNA, sequences are present that direct the ribosome to the initiation codon. We suggest that selection of the correct AUG initiation codon occurs through basepairing with a part of 18S ribosomal RNA.

Base Composition↗
PubMed2002

The veA gene activates sexual development in Aspergillus nidulans.

The previously isolated gene complementing the veA1 mutation was confirmed to be the veA gene. The determined nucleotide sequence of the gene demonstrated that there is an open reading frame (ORF) of a 573 amino acid polypeptide. The nucleotide sequence matched some clones of which functions were not assigned yet and the amino acid sequence matched that of Neurospora crassa VeA-related protein with 61% similarity. The nucleotide sequence of the veA1 mutant gene differed from that of the wild type gene by only one nucleotide and the nucleotide G in the initiation codon ATG of the VeA ORF was mutated to the nucleotide T. Then, the mutant ORF may use the 37th methionine codon of the wild type one as a new initiation codon. The veA transcript was present in the conidia and in mycelia cultured for up to 14h and expressed almost constitutively at an increased level throughout the asexual and sexual developmental processes, suggesting that it may act from a relatively early developmental stage. Null mutants of the gene never formed sexual structures, even under conditions where sexual development preferentially occurs in wild types. Over-expressors of the gene formed larger numbers of sexual structures with a much reduced number of conidial heads than a control strain (a veA1 mutant), even under conditions where wild type strains form little sexual structure but form conidial heads very well, such as in the presence of a salt at high concentration. Furthermore, over-expressors could form Hülle cells and cleistothecia, even in a liquid culture. These results indicated that the veA gene is a positive regulator in sexual development and simultaneously a negative one in asexual development.

Amino Acid Sequence↗
PubMed1995

Structure and expression of a rat kappa opioid receptor gene.

A gene encoding the rat kappa opioid receptor (KOR) was cloned and characterized. Results of rat genomic library screening and genomic Southern blot analysis show the gene represented at one copy/haploid genome. Three introns are present within the gene; however, polymerase chain reaction using different sets of primers specifying neighboring exons indicates that alternative splicing does not occur. Using reverse transcription-polymerase chain reaction and primer extension techniques, we are able to demonstrate that two species of transcripts are differentially produced from the KOR gene in a tissue-specific manner. The first transcript that we designate as KOR1 is equivalent to the cDNA sequence reported by other groups and is believed to correspond to KOR subtype 1. KOR1 begins with exon 1 just downstream of two TATA boxes, whereas the second transcript, which we refer to as KORx, begins in intron 1 and thereby retains this intronic sequence in the mature mRNA. Within this intronic sequence there are two potential translation initiation codons that are in-frame with the proposed initiation codon of KOR. The potential open reading frame that starts further upstream in KORx may lead to the translation of a variant KOR protein having a novel peptide sequence at its amino terminus.

Amino Acid Sequence↗
PubMed1990

Evidence that modulation requires sequences downstream of the promoters of two vir-repressed genes of Bordetella pertussis.

Gene expression in Bordetella pertussis is altered by environmental signals in a process called antigenic modulation. In the presence of modulating signals, expression of several known virulence factors and outer membrane proteins is coordinately reduced. From a bank of TnphoA fusions, we have identified five genes whose expression profiles are reciprocal of those of the major virulence determinants; that is, alkaline phosphatase activity is maximal during growth in the presence of the modulators nicotinic acid and MgSO4 (S. Knapp and J. J. Mekalanos, J. Bacteriol. 170:5059-5066, 1988). We have called these loci vir-repressed genes (vrg). Two of these gene fusions (vrg-6 and vrg-18) have been cloned in Escherichia coli, returned on low-copy-number plasmids to several strains of B. pertussis, and found to be regulated similarly to the fusions harbored on the chromosome. Deletions of the two vrg promoters were constructed and returned to B. pertussis. Regulation was maintained even when all but 24 nucleotides upstream of the vrg-18 initiation codon and 60 nucleotides upstream of the vrg-6 initiation codon were deleted, suggesting that cis-acting regulatory elements of these genes lie very near or within the coding region. We observed a 21-base palindromic sequence overlapping an 8-base direct repeat within the signal sequence coding region of vrg-6; insertion of a 6-bp linker in this region abolished regulation. These repetitive sequences are also at the site of greatest primary sequence identify between vrg-6 and vrg-18 and correspond to the signal sequence coding region. We propose models that involve recognition of this region by a vir-regulated gene product.

Alkaline Phosphatase↗
PubMed1986

Localization and fine structure of a vaccinia virus gene encoding an envelope antigen.

The major antigen on the envelope of extracellular vaccinia virus particles is a polypeptide with an apparent molecular weight of 37,000 (p37K; G. Hiller and K. Weber, J. Virol. 55:651-659, 1985). The gene encoding p37K was mapped in the vaccinia virus genome by hybrid selection of RNA followed by in vitro translation. p37K was then identified among the in vitro translation products by immunoprecipitation with a monoclonal antibody. The gene is located close to the right-hand end of the HindIII F fragment. The corresponding region of the DNA was sequenced, and an open reading frame encoding a polypeptide of 41,748 daltons was observed. The 5' end of the mRNA, as defined by nuclease S1 analysis, maps within only a few nucleotides of the translation initiation codon. Examination of the DNA sequence around the putative initiation site of transcription revealed a characteristic sequence, TAAATG, which includes the ATG translation initiation codon and which is conserved in all but one late gene so far analyzed. It is therefore likely that this sequence is an important regulatory signal for late gene expression in vaccinia virus.

Amino Acid Sequence↗
PubMed2000

The joining of ribosomal subunits in eukaryotes requires eIF5B.

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.

Amino Acid Sequence↗
PubMed1995

Unique features of internal initiation of hepatitis C virus RNA translation.

The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.

Animals↗
PubMed2000

Lessons from sequenced genomes. Overlapping genes in Methanococcus jannaschii?

This paper describes our finding on overlapping genes in Methanococcus jannaschii genome. We found that one of the open reading frames (ORFs) within the M. jannaschii genome contains the nucleotide sequence of tRNA(Ser), which raises a serious question of the correctness of the initiation codon assignment for that ORF. We suggest that there are two other possible AUG initiation codons downstream from the TTG triplet, which was initially considered as a translation start site. Only one of the AUG triplets is preceded by the Shine-Dalgarno sequence that seems to be required for binding the ribosome and initiation of translation.

Amino Acid Sequence↗
PubMed1981

The relationship between the SV40 genome and viral mRNA.

Studies of SV40 DNA and mRNA sequences have revealed a surprising complexity in the biogenesis of SV40 RNA. SV40 late mRNA shows heterogeneity in 5' ends, as well as internal splicing patterns. There may be coupling between different splices within the mRNA precursors and splicing is not strictly sequential. Translation may initiate at the second or third downstream AUG triplet, rather than at the first initiation codon of the mRNA. The possibility exists that translation may initiate on the same mRNA at more than one initiation codon. The DNA in proximity to the major 5' ends of late mRNA is not essential for transcription. Deletion mutants removing DNA encoding the segments of the leader sequence of late SV40 mRNA show effects on preferred transcription initiation sites both upstream and downstream. This suggests that relatively long stretches of DNA may play a role in determining the sites of transcription initiation.

Base Sequence↗