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PubMed1994

Studies on FGF-2: nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin.

Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3' to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1991

Transcription of Clostridium thermocellum endoglucanase genes celF and celD.

Transcripts of the Clostridium thermocellum endoglucanase genes celF and celD, encoding endoglucanases F and D, respectively, were characterized. The size of the mRNAs was about 2.35 kb for celF and 2.1 kb for celD, indicating monocistronic transcription of both genes. A unique 5' end, located 218 bp upstream from the initiation codon, was found for celF mRNA. No convincing homology could be identified between the sequence upstream from the celF 5' end and other procaryotic promoters. Two 5' ends, located 124 and 294 bp upstream from the initiation codon, were mapped for celD mRNA. The -10 and the -35 sequences preceding the ATG-distal 5' end of celD mRNA were homologous to the consensus sequence of Bacillus subtilis sigma 43 promoters. The sequence upstream from the ATG-proximal 5' end had some similarity with the -10 sequence of B. subtilis sigma 28 promoters. During growth on cellobiose, the 5' end of celD transcripts was found predominantly at the -124 site during the late exponential phase but almost exclusively at the -294 site during the early stationary phase. The kinetics of appearance of celA, celC, celD, and celF mRNA was followed by dot blot analysis. Transcripts of celA, celD, and celF were detected during late exponential and early stationary phase. In contrast, the celC transcript was detected almost exclusively during early stationary phase. Since growth was limited by the availability of cellobiose, the results suggest that the genes are regulated by a mechanism analogous to catabolite repression.

Amino Acid Sequence↗

PubMed1990

Analysis of the sequences flanking the translational start sites of Plasmodium falciparum.

The 5' and 3' regions adjacent to the initiation codon in 22 Plasmodium falciparum sequences were examined. A 5' consensus sequence (AAAA/ATG) was found. Although P. falciparum non-translated DNA is A-rich, A occurred significantly more frequently in the 4 positions preceding the initiation ATG than in adjacent non-translated DNA, suggesting that this consensus sequence has functional significance in the initiation of translation. This region has similarities with the equivalent sequences in yeast and Drosophila but differs markedly from that in vertebrates. No significant bias in nucleotide frequencies was found 3' to the initiation codon.

Animals↗

PubMed1977

Role of DNA sequences in genetic recombination in the iso-1-cytochrome c gene of yeast. II. Comparison of mutants altered at the same and nearby base pairs.

X-ray-induced mitotic recombination rates and spontaneous meiotic recombination rates have been determined in two-point crosses of various defined cyc1 mutants of the yeast Saccharomyces cerevisiae. All but one of the 17 cyc1 mutants chosen for this study contained either the addition, deletion or substitution of single base-pairs located within a defined segment of the gene that corresponds to the 11 amino acid residues at the amino terminus of iso-1-cytochrome c; approximately half of these mutants had alterations of the AUG initiation codon, some at the same base pair. Up to 66-fold differences in X-ray-induced recombination rates were observed when the same cyc1 mutant was crossed to cyc1 mutants having different alterations in the AUG initiation codon; over a ten-fold difference was observed in series of homologous crosses involving mutants with different changes at the same base-pair. Recombination rates that were associated with specific cyc1 mutants co-segregated with the particular alleles following meiosis, and comparable recombination patterns were also observed for independently isolated, identical mutations. With the mutants used in this study, the frequencies of meiotic recombination did not differ as markedly, suggesting a dissimilar dependence on specific DNA sequences for these two modes of recombination. These disproportionalities of recombination rates suggest that the nature of the mismatched bases influences the recombination process, but not in a way that can be simply interpreted.

Base Sequence↗

PubMed1993

Modulation of human prorenin gene expression by antisense oligonucleotides in transfected CHO cells.

Four phosphorothioate oligonucleotides whose sequences are complementary to the 5' untranslated region, the initiation codon or the coding region of human prorenin mRNA, were studied for their capacity to inhibit gene expression in stably transfected Chinese hamster ovary (CHO) cells constitutively producing human prorenin. In contrast to oligomers complementary to the initiation codon and the coding region, antisense oligomers directed towards the 5' untranslated region have no inhibitory effects. The intracellular delivery of a biotinylated phosphorothioate oligonucleotide (biotin-CATCCATGCTTCCCTC) was monitored in immunofluorescence studies. In the absence of a cationic liposome preparation, Lipofectin, the oligomer failed to penetrate the cells. In the presence of Lipofectin, the 35S-labelled oligomer entered the cells and was distributed in proportions of 54% to the nuclei and 35% to the cytosol. The effects of regular oligonucleotides and of 3'-end and/or 5'-end-modified phosphodiester oligonucleotides on prorenin production were tested. Terminal modification by biotinylation at the 5'-end and/or 3'-dodecyl esterification stabilized oligonucleotides towards exonucleases, but did not translate into a significant inhibition of prorenin production and did not improve the intracellular delivery and or stability of the oligomers. We have shown that it is possible to inhibit prorenin production intracellularly using specific antisense oligonucleotides. Stability and delivery are crucial factors in the design of potent and specific compounds directed at prorenin mRNA.

Animals↗

PubMed1999

Functional and transcriptional analyses of a fengycin synthetase gene, fenC, from Bacillus subtilis.

A 37-kb DNA fragment containing five fengycin synthetase genes, including fenC, fenD, fenE, fenA, and fenB, was cloned and sequenced. Among these genes, fenC encodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa. This protein contains two amino acid activation modules, FenC1 and FenC2, which activate L-glutamic acid and L-ornithine, respectively. Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon of fenC, implying that a promoter is located upstream from the start site. Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon of fenC. Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth. The promoter is active during the log phase, and the activity reaches the highest level during the late log phase. The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase. The results of this investigation also suggest that the transcription of fenC is positively regulated during the late log phase. Results presented herein provide further insight into fengycin synthesis by B. subtilis F29-3.

Adenine↗

PubMed1995

p10 genes of Bombyx mori nuclear polyhedrosis virus and Autographa californica multiple nuclear polyhedrosis virus.

EcoR I-P fragment has been cloned from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) genomic DNA and used as a probe. 0.5-kb and 1.1-kb fragments including p10 gene from Bombyx mori nuclear polyhedrosis virus (BmNPV) have been hybridized. The p10 ORF was located in the EcoR I-R fragment. Initiation codon ATG of p10 from BmNPV has been mutated by PCR, and the ATG region became a Bgl II site. A novel transfer vector pBmAcPV-1 has been constructed using both the p10 5'-flanking region whose initiation codon ATG has been mutated with BmNPV and the p10 3'-flanking region of AcMNPV. The vector can recombine with not only AcMNPV DNA to express foreign gene in Sf cells, but also BmNPV DNA to express foreign gene in Bm cells. CAT gene was expressed at high level in Bm cells under the control of the mutated p10 promoter of BmNPV.

Amino Acid Sequence↗

PubMed1981

DNA sequence of a mutation in the leader region of the yeast iso-1-cytochrome c mRNA.

We have constructed a plasmid that selectively integrates adjacent to the CYC1 locus, which determines iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Different CYC1 alleles can be conveniently recovered by digestion of total DNA from transformed strains with BgI II, a restriction endonuclease that does not cut the vector or the CYC1 gene, followed by transformation of Escherichia coli, selecting the ampicillin resistance gene carried on the original vector. This procedure was used to clone the cyc1-362 gene, which contains an alteration in front of the AUG initiation codon. The cyc1-362 mutational causes a deficiency of the iso-1-cytochrome c protein but still allows transcription of the iso-1-cytochrome c mRNA. DNA sequence analysis showed that the cyc1-362 mutation consisted of two single-base-pair substitutions, producing an A leads to G change 18 nucleotides and a G leads to A change 30 nucleotides in front of the AUG initiation codon in the mRNA. The A leads to G change at position -18 resulted in the creation of an AUG triplet, which is proximal to the normal initiation site and out of phase with the normal reading frame. The deficiency of iso-1-cytochrome c is most simply explained by assuming that translation initiates at the more proximal abnormal AUG site but not at the normal AUG site.

Base Sequence↗

PubMed1984

In vitro template activity of 0.3 mRNA from wild type and initiation mutants of bacteriophage T7.

Bacteriophage T7 0.3 mRNA synthesised and processed in vitro has been purified starting from the DNA of T7+ as well as from that of two initiation mutants of T7 (CR17 with a U----C transition in the initiation codon and CR35b whose potential Shine and Dalgarno (S-D) interaction is interrupted by a G----A transition). These mRNAs were used as templates to direct the binding of fMet-tRNA and the synthesis of 0.3 protein in both E. coli and wheat germ cell-free systems. The initiation codon mutant displayed approximately 50% inhibition of fMet-tRNA binding and 0.3 protein synthesis in both systems. The S-D sequence mutant, on the other hand, was found to be less affected than the initiation triplet mutant (20%-40% inhibition) in both fMet-tRNA binding and template activity in the E. coli system. In the wheat germ system, which does not make use of the S-D interaction, however, this mutant displayed normal template activity suggesting that the inhibition obtained in the E. coli system, albeit slight, is due to the impairment of the S-D interaction and not to an alteration of the mRNA secondary or tertiary structure caused by the base substitution.

Cell-Free System↗

PubMed1995

Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli.

The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage lambda-4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacteria↗

PubMed1992

Culture conditions differentially affect the translation of individual Escherichia coli mRNAs.

Our aim is to investigate whether changes in growth conditions can differentially affect the initiation of translation from individual Escherichia coli mRNAs that are not subjected to specific translational control. As a model system, we have constructed a series of point-mutated lacZ genes which differ in their Shine-Dalgarno (SD) sequence, their initiator codon, or the secondary structure around these elements. Alterations in growth conditions produced large (up to 8-fold) changes in the relative expression from these genes, which, we argue, stem from changes in their relative efficiencies of translation initiation. In particular, compared to genes bearing mutations outside the SD or initiator codon, genes mutated in these elements experience a significant decrease in their expression when cells are grown in minimal rather than rich medium; at 42 degrees C rather than 37 degrees C; or under amino acid starvation. We discuss the mechanisms underlying these effects, and evocate their possible generality.

Base Sequence↗

PubMed1993

Organization and transcription of the principal sigma gene (rpoDA) of Pseudomonas aeruginosa PAO1: involvement of a sigma 32-like RNA polymerase in rpoDA gene expression.

S1 nuclease mapping and Northern (RNA) hybridization revealed that the rpoDA gene encoding the principal sigma subunit of Pseudomonas aeruginosa PAO1 is transcribed as a monocistronic mRNA of 2 kb and that the transcription from the rpoDA promoter (PC) starts 32 bases upstream from the first nucleotide of the initiation codon during the steady-state growth condition at a low temperature (30 degrees C). The transcript terminates 31 bases downstream from the last nucleotide of the termination codon. When the growth temperature was shifted to 42 degrees C, the synthesis of rpoDA mRNA from a heat shock promoter was transiently induced, although transcription was still occurring from PC during the heat shock period. The transcription initiation site of the heat shock promoter (PHS) is located about 220 bases upstream of the initiation codon of rpoDA. In addition, both promoters were utilized in vitro by RNA polymerase partially purified from heat-shocked cells of P. aeruginosa PAO1. When the rpoDA was introduced into Escherichia coli, the transcription patterns of rpoDA at 30 and 42 degrees C were similar to those observed for P. aeruginosa. These results suggested that the transcription of rpoDA in P. aeruginosa is regulated by the principal RNA polymerase and the heat shock RNA polymerase in response to the environmental temperature.

Base Sequence↗

PubMed1991

Separate promoters control transcription of the human aminopeptidase N gene in myeloid and intestinal epithelial cells.

Aminopeptidase N is a membrane-bound metalloprotease expressed on the surface of normal and malignant human myeloid cells, fibroblasts, hepatocytes, and the epithelial cells that form brush borders of the small intestine and kidney. Northern blot analysis of RNA extracted from these tissues revealed two distinct aminopeptidase N transcripts: a 3.7-kilobase (kb) transcript expressed by normal monocytes, myeloid leukemia cells, and fibroblasts and a 3.4-kb transcript expressed by intestinal epithelium and kidney cells. In intestinal epithelial cells, transcripts originated 47 base pairs upstream from the initiation codon and 22 base pairs downstream from a TATA box. By contrast, the longer transcripts found in myeloid cells and fibroblasts originated from several sites clustered in an upstream exon located 8 kb from the exon containing the initiation codon. Functional promoter activity was demonstrated by fusing sequences approximately 1 kb upstream from each transcription origin to bacterial reporter genes and transfecting the resultant constructs into murine NIH-3T3 fibroblasts. A novel feature of this system is that regulatory elements of the epithelial cell promoter, including the TATA box and transcription origin, are included within the 5'-untranslated region of the longer myeloid cell transcript. Both aminopeptidase N transcripts encode the same polypeptide, indicating that the physically distinct promoters must have evolved to regulate expression of this cell-surface peptidase by cells of different tissues.

Aminopeptidases↗

PubMed1999

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator.

An Escherichia coli-mycobacterial shuttle vector, pJCluc, containing a luciferase reporter gene, was constructed and used to analyse the Mycobacterium tuberculosis katG promoter. A 1.9 kb region immediately upstream of katG promoted expression of the luciferase gene in E. coli and Mycobacterium smegmatis. A smaller promoter fragment (559 bp) promoted expression with equal efficiency, and was used in all further studies. Two transcription start sites were mapped by primer extension analysis to 47 and 56 bp upstream of the GTG initiation codon. Putative promoters associated with these show similarity to previously identified mycobacterial promoters. Deletions in the promoter fragment, introduced with BAL-31 nuclease and restriction endonucleases, revealed that a region between 559 and 448 bp upstream of the translation initiation codon, designated the upstream activator region (UAR), is essential for promoter activity in E. coli, and is required for optimal activity in M. smegmatis. The katG UAR was also able to increase expression from the Mycobacterium paratuberculosis P(AN) promoter 15-fold in E. coli and 12-fold in M. smegmatis. An alternative promoter is active in deletion constructs in which either the UAR or the katG promoters identified here are absent. Expression from the katG promoter peaks during late exponential phase, and declines during stationary phase. The promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. The promoter constructs exhibited similar activities in Mycobacterium bovis BCG as they did in M. smegmatis.

Bacterial Proteins↗

PubMed1999

Post-transcriptional control of bacteriophage T4 gene 25 expression: mRNA secondary structure that enhances translational initiation.

Secondary structure of the mRNA in the translational initiation region is an important determinant of translation efficiency. However, the secondary structures that enhance or facilitate translation initiation are rare. We have previously proposed that such structure may exist in the case of bacteriophage T4 gene 25 translational initiation region, which contains three potential Shine-Dalgarno sequences (SD1, SD2, and SD3) with a spacing of 8, 17, and 27 nucleotides from the initiation codon of this gene, respectively. We now present results that clearly demonstrate the existence of a hairpin structure that includes SD1 and SD2 sequences and brings the SD3, the most typical of these Shine-Dalgarno sequences, to a favourable spacing with the initiation codon of gene 25. Using a phage T7 expression system, we show that mutations that prevent the formation of hairpin structure or eliminate the SD3 sequence result in a decreased level of gp25 synthesis. Double mutation in base-pair V restores the level of gene 25 expression that was decreased by either of the two mutations (C-to-G and G-to-C) alone, as predicted by an effect attributable to mRNA secondary structure. We introduced the mutations into the bacteriophage T4 by plasmid-phage recombination. Changes in the plaque and burst sizes of T4 mutants, carrying single and double mutations in the translational initiation region of gene 25, strongly suggest that the predicted mRNA secondary structure controls (enhances) the level of gene 25 expression in vivo. Hybridization of total cellular RNA with a gene 25 specific probe indicated that secondary structure or mutations in the translational initiation region do not notably affect the 25 mRNA stability. Immunoblot analysis of gp25 in Escherichia coli cells infected by T4 mutants showed that mRNA secondary structure increases the level of gp25 synthesis by three- to fourfold. Since the secondary structure increases the level of gp25 synthesis and does not affect mRNA stability, we conclude that this structure enhances translation initiation. We discuss some features of two secondary structures in the translational initiation regions of T4 genes 25 and 38.

Base Sequence↗

PubMed1994

Footprinting mRNA-ribosome complexes with chemical probes.

We footprinted the interaction of model mRNAs with 30S ribosomal subunits in the presence or absence of tRNA(fMet) or tRNA(Phe) using chemical probes directed at the sugar-phosphate backbone or bases of the mRNAs. When bound to the 30S subunits in the presence of tRNA(fMet), the sugar-phosphate backbones of gene 32 mRNA and 022 mRNA are protected from hydroxyl radical attack within a region of about 54 nucleotides bounded by positions -35 (+/- 2) and +19, extending to position +22 when tRNA(Phe) is used. In 70S ribosomes, protection is extended in the 5' direction to about position -39 (+/- 2). In the absence of tRNA, the 30S subunit protects only nucleotides -35 (+/- 2) to +5. Introduction of a stable tetraloop hairpin between positions +10 and +11 of gene 32 mRNA does not interfere with tRNA(fMet)-dependent binding of the mRNA to 30S subunits, but results in loss of protection of the sugar-phosphate backbone of the mRNA downstream of position +5. Using base-specific probes, we find that the Shine-Dalgarno sequence (A-12, A-11, G-10 and G-9) and the initiation codon (A+1, U+2 and G+3) of gene 32 mRNA are strongly protected by 30S subunits in the presence of initiator tRNA. In the presence of tRNA(Phe), the same Shine-Dalgarno bases are protected, as are U+4, U+5 and U+6 of the phenylalanine codon. Interestingly, A-1, immediately preceding the initiation codon, is protected in the complex with 30S subunits and initiator tRNA, while U+2 and G+3 are protected in the complex with tRNA(Phe) in the absence of initiator tRNA. Additionally, specific bases upstream from the Shine-Dalgarno region (U-33, G-32 and U-22) as well as 3' to the initiation codon (G+11) are protected by 30S subunits in the presence of either tRNA. These results imply that the mRNA binding site of the 30S subunit covers about 54-57 nucleotides and are consistent with the possibility that the ribosome interacts with mRNA along its sugar-phosphate backbone.

Bacteriophage T7↗

PubMed1999

Structure of the human VIPR2 gene for vasoactive intestinal peptide receptor type 2.

The VPAC(2) (vasoactive intestinal peptide (VIP)(2)) receptor is a seven-transmembrane spanning G protein-coupled receptor which responds similarly to VIP and pituitary adenylate cyclase activating polypeptide (PACAP) in stimulating cAMP production. Recently, we reported the localisation of the human VPAC(2) receptor gene (VIPR2) to chromosome 7q36.3 (Mackay, M. et al. (1996) Genomics 37, 345-353). Here, we describe the characterisation of the VIPR2 gene structure and promoter region. The VIPR2 gene is encoded by 13 exons, the initiator codon of the 438 amino acid open reading frame is located in exon 1 and the termination signal and a poly-adenylation signal sequence are located in exon 13. The 5' untranslated region extends 187 bp upstream of the initiator codon and is extremely GC-rich (80%). The poly-adenylation signal is located 2416 bp downstream of the stop codon. Intron sizes range from 68 bp (intron 11) to 45 kb (intron 4) and the human gene spans 117 kb.

5' Untranslated Regions↗

PubMed1998

Human galactocerebrosidase gene: promoter analysis of the 5'-flanking region and structural organization.

Galactocerebrosidase (GALC; EC 3.2.1.46) is a lysosomal enzyme which hydrolyzes several galactolipids and the deficiency of GALC is responsible for Krabbe disease. Recently, we cloned cDNAs for human and murine GALC. In this study we characterized the genomic organization and the promoter of the human gene. The gene was about 60 kb in length and consisted of 17 exons as reported by Luzi et al. DNA sequence analysis showed that the 5'-flanking region of the first exon was GC-rich and had not typical TATA-box but ten GC-box-like sequences within a 200 bp sequence upstream from the initiation codon. Another inframe ATG, which has better Kozak consensus sequence, was found at 48 bp upstream to the first ATG reported]. Promoter analysis using a luciferase assay in COS 7 cells showed that the -149 to -112 nucleotide (from the initiation codon A) region has dominant promoter activity. In this region three GC-box-like sequence and one YY1 binding site were detected. Primer extension revealed several transcription start sites within the region of -146 to -103 nucleotide. In this study we firstly demonstrated that the YY1 binding site and subsequent GC-box-like sequences could be a promoter in a housekeeping gene.

Animals↗