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PubMed1984

Effects of alterations in the translation control region on bacterial gene expression: use of cat gene constructs transcribed from the lac promoter as a model system.

The region controlling translation of the cat gene, which codes for chloramphenicol acetyltransferase, has been varied structurally in a series of plasmids that place the gene under control of the lac promoter. These plasmid constructs have enabled study of the structural features that affect the efficiency of mRNA translation. Altering the potential for secondary structure formation within the translation control region caused a tenfold variation in the synthesis of CAT enzyme, whereas varying the distance between the Shine-Dalgarno sequence (SD) and the translation start codon from 7 to 13 bases did not significantly affect the yield of CAT. If the SD was situated in a region of mRNA that is capable of base pairing, the efficiency of translation was decreased; however, the translation start codon, AUG, can initiate translation efficiently even when located in a segment capable of duplex formation. Overlapping of the cat translation control region by translation initiated upstream markedly affected initiation of translation within the cat gene: out-to-frame overlapping translation reduced CAT production by 90%; in-frame overlapping translation prevented detectable initiation of protein synthesis at the cat gene translation start codon, and yielded only fusion proteins. The enzymatic activity of such proteins was influenced by the length of the adventitious peptide segment added to the amino-terminus of the CAT polypeptide.

Acetyltransferases↗

PubMed1999

Identification of novel polymorphisms within the promoter region of the human beta2 adrenergic receptor gene.

By screening the 1470 bp 5' to the start codon of the human beta2 adrenergic receptor gene, we have identified a total of eight polymorphisms (-20 T-->C, -47 T-->C, -367 T-->C, -468 C-->G, -654 G-->A, -1023 G-->A, -1343 A-->G and -1429 T-->A c.f. beta2 adrenergic receptor start codon). Transient transfection of 5' flanking deletion luciferase reporter constructs demonstrated the majority of activity of the human beta2 adrenergic gene 5' flanking region to be present within a 549 bp fragment immediately upstream from the start codon. Because of linkage disequilibrium, some combinations of polymorphisms were particularly frequent. We transiently transfected COS-7 cells with luciferase constructs under the control of the 549 bp of 5' flanking DNA containing the two most frequent extended haplotypes in this region. Luciferase activity was significantly reduced in cells transfected with the 'mutant' construct (-20C, -47C, -367C, -468G) c.f. the 'wild-type' construct (-20T, -47T, -367T, -468C). These data suggest that polymorphisms have the potential to alter human beta2 adrenergic receptor gene expression.

Animals↗

PubMed2003

Non-randomness in Shine-Dalgarno regions: links to gene characteristics.

A probabilistic approach to the study of the Shine-Dalgarno region was used to identify the most non-random positions based on parsing of genomes in four species: Escherichia coli, Bacillus subtilis, the AT-rich Clostridium perfringens, and the GC-rich Streptomyces coelicolor. The compositional non-randomness shows a clear peak centered around 9-11 nucleotides upstream of the start codon. This peak was in all species associated with guanine as the most abundant nucleotide, flanked by guanine in the closest proximity and adenines farther away (cytosine in case of S. coelicolor). Using contingency tables, the nucleotides in the Shine-Dalgarno region were shown to have a strong association to the choice of start codons. We also show that gene characteristics such as length, aromaticity, and lipophilicity are related to the nucleotide at this peak position upstream of the start codon.

Conserved Sequence↗

PubMed2001

Analysis of transcriptional control mechanisms of capsule expression in Neisseria meningitidis.

The major virulence factor which contributes to the survival of Neisseria meningitidis in the blood stream and the cerebrospinal fluid is the capsular polysaccharide. Expression of the capsule genes of N. meningitidis serogroups B, C, W-135 and Y is controlled by an intergenic region separating the capsule biosynthesis operon (siaA-D) and the capsule transport operon (ctrA-D). To further investigate capsule expression in N. meningitidis we amplified and sequenced the intergenic region of 42 meningococcal isolates of different serogroups. Sequence variations were found mainly in a repeat region preceding the siaA start codon. Correlation between sequence variation and serogroup could not be observed. To measure the transcriptional and translational activity of the respective intergenic regions we performed transcriptional and translational fusions with the lacZ gene integrated into the chromosome of N. meningitidis. Sequence variations preceding the siaA start codon had no effect on beta-galactosidase activity. Different in vitro growth conditions such as temperature, glucose concentration, osmolarity, pH and iron concentration also did not influence beta-galactosidase activity. Sequential deletions of the intergenic region showed that an Up-like element adjacent to the predicted -35 box is necessary for full transcriptional activity. The deletion of the untranslated region preceding the siaA start codon led to a threefold higher beta-galactosidase activity compared with the full-length construct suggesting that the respective region may be involved in capsule regulation.

Bacterial Capsules↗

PubMed1992

Expression of a downstream gene from a bicistronic transcription unit in transgenic tobacco plants.

We have constructed a set of plasmids carrying an artificial compact stop-start codon sequence, TGATGTAACATGA, between an upstream open reading frame, terminating at one of the stop codons, and a downstream kanamycin-resistance (KmR)-encoding gene (nptII) initiating at the second ATG. These plasmids were introduced into tobacco protoplasts by direct gene transfer. The efficiency of expression of the downstream nptII gene was measured by scoring the number of KmR transformants. With a closer distance between the functional stop and start codons, a tendency to less efficient expression of nptII was found. The integration and expression of both genes as a bicistronic transcription unit were verified by Southern- and Northern-blot analyses. A possible application of the compact stop-start codon sequence for insertional mutagenesis is discussed.

Base Sequence↗

PubMed1991

Analysis of barley nitrate reductase cDNA and genomic clones.

Barley nitrate reductase cDNA and genomic clones were isolated by homology with the barley nitrate reductase cDNA clone bNRp10 and sequenced. This is the first reported analysis of a full-length nitrate reductase gene and its corresponding cDNA in the same species. The longest cDNA clone extends to within 9 bp of the ATG start codon and the sequence is similar to that reported for the higher plant NR sequences. As expected, the amino acid sequence of barley nitrate reductase is more related closely to the rice (84% homology) than to the Arabidopsis (62%) sequence. Four different polyA addition sites were identified from sequence analysis of nine barley NR cDNA clones. A 7.3 kb region of a genomic recombinant lambda clone was subcloned as two contiguous BamHI fragments into p Bluescript, designated pMJ7 and pMJ8, and sequenced. These clones include the entire nitrate reductase coding region, one large intron, 2.7 kb of untranslated sequence 5' to the translation start codon and 0.25 kb 3' to the translation termination codon. The mRNA cap site was identified as a cytosine, 111 bases upstream of the ATG translation start codon. The putative CAAT and TATA boxes were identified at -115 and -33 bp, respectively, with the mRNA cap site designated as +1. The barley nitrate reductase gene coding region strongly favors G or C in the third codon position.

Base Sequence↗

PubMed2000

Kv3.3 potassium channels in lens epithelium and corneal endothelium.

Human Kv3.3/KCNC3 is a Shaw-type potassium channel that has been mapped to chromosome 19q13.3-13.4. Complete mouse and rat Kv3.3 cDNA coding sequences have been published, yet the human Kv3.3 cDNA has remained incomplete for years. We report here for the first time the amino acid sequence for hKv3.3 and the electrophysiological behavior of the encoded channel in transiently transfected mammalian cells. In addition, we report the occurrence of Kv3.3 message in rabbit corneal endothelial cells and the properties of the currents when the corneal channel is expressed. The hKv3.3 gene is highly GC-rich (69%) and contains numerous GC runs which made DNA sequencing and PCR amplification especially problematic. The full-length sequence contains two possible start codons. The encoded 757 amino acid hKv3. 3 protein is about 93% identical to mouse and rat Kv3.3 in the first 659 amino acids before the C-terminal domains diverge greatly as a result of alternative splicing. The rabbit cornea Kv3.3 is a close sequence match for hKv3.3 even in the C-terminal domain. However, we have not yet found a cornea sequence which contains the first potential start codon from hKv3.3. Electrophysiologically, the hKv3. 3 channel produces an A-current although expression of constructs which lack the 5' region of the first start codon inactivate much more slowly than full-length constructs. This short hKv3.3 construct also shows changes in activation.

Amino Acid Sequence↗

PubMed1987

Altered translation of the uncC gene coding for the epsilon subunit of the F1F0-ATPase of Escherichia coli.

The nucleotide sequence of the previously described uncC424 allele was determined and found to be the same as that of a wild-type uncC gene. However, a G----A change occurred 7 nucleotides upstream from the translation start codon, changing the putative Shine-Dalgarno sequence from GAGG to GAAG. Four revertant strains were examined. In one revertant, which had normal growth and membrane properties, a single base deletion had occurred to re-form the Shine-Dalgarno sequence GAGG 1 nucleotide closer to the translation start codon. A second revertant had a single base deletion in the preceding uncD gene, causing an extension of the beta subunit by 6 amino acids and an increase, presumably by translational coupling, in the amount of epsilon subunit. The third and fourth revertant strains were phenotypically similar and had either C----T or G----T changes 18 or 19 nucleotides, respectively, upstream from the translation start codon.

Base Sequence↗

PubMed1989

Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin.

A genomic DNA clone encoding oryzacystatin (Oc), a cysteine proteinase inhibitor (cystatin) of rice, was isolated from a lambda EMBL3 phage library constructed with Sau3AI partial digests of rice chromosomal DNA, by screening with an oc cDNA as a probe. The restriction map of the isolated DNA fragment was consistent with the pattern of the genomic Southern-blot analysis using a cDNA probe, and consequently, the gene is considered to be a single-copy gene. The oc gene is about 1.4 kb long and composed of three exons and two introns. The first intron (336 bp) intervenes between Ala-38 and Asp-39. The second intron (372 bp) exists in the 3'-noncoding region at the G residue next to the stop codon. S1 nuclease mapping showed the major transcription start point (tsp) at A, 104 bp upstream from the start codon (ATG). Typical CAT and TATA box sequences were found in the 5'-upstream region of the tsp. The nucleotide sequences around the TATA box, the tsp, the start codon, and the stop codon essentially matched the consensus sequences of other higher plant genes. The intron boundaries of the oc gene were quite different from those of the human kininogen-encoding gene and the human salivary cystatin (cystatin S)-encoding gene.

Amino Acid Sequence↗

PubMed2003

Analysis of the Brugia malayi HSP70 promoter using a homologous transient transfection system.

Biolistic transient transfection of Brugia malayi embryos with constructs driving the expression of a luciferase reporter gene was used to identify regions of the upstream sequence of the heat shock protein 70 (HSP70) gene of B. malayi necessary for transgene expression. Analysis of 1160 nucleotides upstream of the start codon of the HSP70 gene identified several potentially important elements, including putative CAAT and TATA boxes, a core promoter domain, a polypurine stretch, and a spliced leader addition site. Nested deletion analysis of the HSP70 upstream domain mapped the promoter of the HSP70 gene to the region 396 to 31 nucleotides upstream of the start codon. This encompassed the putative CAAT and TATA boxes, and putative core promoter. Deletion of the putative CAAT box did not result in any diminution of reporter activity, while constructs in which the TATA box or core promoter were deleted retained roughly half of the activity of the undeleted construct. Unlike the native gene, transcripts derived from constructs containing the HSP70 upstream sequences were not trans-spliced. However, incorporation of the 495 nucleotides downstream of the start codon (encompassing exon 1, intron 1 and part of exon 2) resulted in the production of transcripts that were correctly cis- and trans-spliced. Similarly, a construct containing the 495 downstream nucleotides in which most of exon 1 was deleted, was correctly cis- and trans-spliced. This finding suggests that downstream intron sequences in addition to the splice leader addition site are necessary for trans-splicing in B. malayi.

Alternative Splicing↗

PubMed1999

Promoter architecture of the Porphyromonas gingivalis fimbrillin gene.

Porphyromonas gingivalis fimbriae can mediate adherence to many of the available substrates in the oral cavity. Expression of P. gingivalis fimbriae is regulated at the transcriptional level by environmental signals, such as temperature and hemin concentration. The arrangement of the upstream promoter and regulatory sequences required for transcription and control of the fimbrial structural gene (fimA) was investigated. Primer extension analysis demonstrated that the transcriptional start site of the fimA gene is located 41 bp upstream from the translational start codon. A region (upf) spanning 648 bp upstream of the start codon to 44 bp downstream of the translational start site was cloned upstream of a promoterless lacZ reporter gene. A series of deletion and base substitution mutations were then generated in the upf region. The constructs were introduced into the chromosome of P. gingivalis, and promoter activity measured by assaying levels of beta-galactosidase. The results showed that fimA contains sequences resembling sigma70 promoter consensus sequences, consisting of a -10 region (TATGAC) located at -18 to -23 and a -35 region (TTGTTG) located at -41 to -46 from the transcriptional start point. The AT-rich upstream sequences spanning bases -48 to -85 and bases -90 to -240 were required for full expression of the fimA gene, indicating the existence of positive regulation regions. Moreover, the -48 to -64 region may constitute an UP element, contributing to promoter activity in P. gingivalis. Thus, our data suggest that the P. gingivalis fimA gene has a transcription complex consisting of -10 and -35 sequences, an UP element, and additional AT-rich upstream regulatory sequences.

Bacterial Proteins↗

PubMed2006

Analysis of the targeting sequences of an iron-containing superoxide dismutase (SOD) of the dinoflagellate Lingulodinium polyedrum suggests function in multiple cellular compartments.

One of the proteins targeted to the peridinin plastid of the dinoflagellate Lingulodinium polyedrum is the iron-containing superoxide dismutase (LpSOD). Like dinoflagellate plastid proteins of class II, LpSOD carries a bipartite presequence comprising a signal peptide followed by a transit peptide. Our bioinformatic studies suggest that its signal peptide is atypical, however, and that the entire presequence may function as a mitochondrial targeting signal. It is possible that LpSOD represents a new class of proteins in algae with complex plastids, which are co-targeted to the plastid and mitochondrion. In addition to the ambiguous N-terminal targeting signal, LpSOD contains a potential type-1 peroxisome-targeting signal (PTS1) located at its C-terminus. In accordance with a peroxisome localization of this dismutase, its mRNA has two in-frame AUG codons. Our bioinformatic analyses indicate that the first start codon resides in a much weaker oligonucleotide context than the second one. This suggests that synthesis of the plastid/mitochondrion-targeted and peroxisome-targeted isoforms could proceed through so-called leaky scanning. Moreover, our results show that expression of the two isoforms could be regulated by a 'hairpin' structure located between the first and second start codons.

Animals↗

PubMed2004

Control of the vascular endothelial growth factor internal ribosome entry site (IRES) activity and translation initiation by alternatively spliced coding sequences.

The vascular endothelial growth factor-A (VEGF) gene locus contains eight exons that span 14 kb. Alternative splicing generates multiple, different mRNAs that in turn translate into at least five protein isoforms. While the canonical AUG start codon is located at position 1039 in exon 1, there also exists an upstream, in-frame CUG initiation codon that drives expression of L-VEGF, containing an additional 180 amino acids. Two separate internal ribosome entry sites (IRES) regulate the activity of each initiation codon. Thus the 5'-UTR of VEGF, which comprises the majority of exon 1, consists of IRES B, the CUG, IRES A, and the AUG, from 5' to 3'. Previously, it has been shown that IRES B regulates initiation at the CUG and IRES A regulates AUG usage. In this study, we have found evidence that the exon content of the VEGF mRNA, determined through alternative splicing, controls IRES A activity. While the CUG is most efficient at initiating translation, transcripts that lack both exons 6 and 7 and therefore contain an exon 5/8 junction lack AUG-initiated translation. The process of splicing is not responsible for this start codon selection since transfection of genomic and cDNA VEGF sequences give the same expression pattern. We hypothesize that long range tertiary interactions in the VEGF mRNA regulate IRES activity and thus control start codon selection. This is the first report describing the influence of alternatively spliced coding sequences on codon selection by modulating IRES activity.

5' Untranslated Regions↗

PubMed2002

Molecular cloning and sequencing of cDNA encoding for human FA-1 antigen.

The cDNA encoding for the human FA-1 sperm antigen was cloned and sequenced from the in-house constructed subtractive human testis cDNA expression library in lambda Ziplox using the FA-1 monoclonal antibody (mAb). The full--length sequence was obtained by using the 5' rapid amplification of 5'-cDNA end (5'-RACE) procedure. It is 1,576-bp long, and has an open reading frame (ORF) of 283 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 57 and the stop codon TAG at nt 906. It has two termination codons at the 5' end before the ATG start codon. The translated protein has a calculated molecular weight of 32.1 kDa and estimated isoelectric point (pI) of 11.59. It has one potential N-linked glycosylation site and one tyrosine phosphorylation site, besides several O-linked glycosylation and serine and threonine phosphorylation sites. Hydrophilicity analysis of the deduced aa sequence showed it to be a membrane-anchored protein. Extensive computer search in the database did not identify any known nt/aa sequence having homology with FA-1 cDNA or deduced aa, indicating it to be a novel gene. The Northern blot and reverse transcription-polymerase chain reaction (RT-PCR)-Southern blot analyses indicated the testis-specific expression of FA-1 antigen at the mRNA level. The ORF of the FA-1 was subcloned into pGEX- 1lambda T for expression. The expressed FA-1 recombinant protein had a molecular size of approximately 40 kDa, and was recognized by the FA-1 mAb, and not by the myeloma control Ig. The rabbit antibodies (Ab) raised against the recombinant (r) FA-1 antigen recognized the rFA-1 antigen as well as the native (n) FA-1 antigen. The rFA-1 Ab specifically recognized a protein band of approximately 50 kDa in human testis extract in the Western blot involving 11 types of human tissue extracts, indicating the testis-specific expression of FA-1 at the protein level. The Ab showed binding with live and methanol-fixed human sperm at the post-acrosomal, mid-piece, and tail regions. The Ab caused a significant (P < 0.001) and concentration-dependent inhibition of human sperm capacitation/acrosome reaction by blocking tyrosine phosphorylation of the FA-1 antigen. The sperm-specific human FA-1 recombinant antigen may find applications in immunocontraception, and diagnosis and treatment of immunoinfertility in humans.

Acrosome Reaction↗

PubMed1993

Transcriptional analysis of the Chlamydia trachomatis plasmid pCT identifies temporally regulated transcripts, anti-sense RNA and sigma 70-selected promoters.

We analysed transcription of the DNA region immediately downstream of the origin of replication in the chlamydial plasmid pCT. This region comprises two convergent open reading frames (ORF7, ORF8), encoding putative polypeptides that are homologous to each other and with C-terminal domains typical of the phage integrase family of proteins. Northern blot and RNA 5' end mapping analyses indicated that both ORFs were transcribed in the late phase of the chlamydial replicative cycle. RNA mapping showed the presence of a transcript starting 31 nucleotides (nt) before the ATG start codon of ORF7, and two temporally regulated transcripts starting 59 and 89 nt upstream of the ATG start codon of ORF8. Two abundant RNA species of 225 and 415 nt were also identified as overlapping anti-sense transcripts (AS-RNAs), complementary to the 3' end of ORF8 mRNA, with identical 5' ends but different 3' ends. In vitro and in vivo experiments in Escherichia coli showed that the sigma 70-RNA polymerase complex was capable of initiating RNA synthesis at the same sites as observed in Chlamydia trachomatis for ORF7 and AS-RNA transcripts, but was not able to transcribe ORF8. In accord with this, sequences at -10 and -35 nt upstream of the RNA 5' ends resemble sigma 70 consensus promoters in the case of ORF7 and AS, but not in the case of the two ORF8 transcripts. Therefore, transcription of ORF7 and ORF8 is controlled by different types of promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Optimization of the hygromycin B resistance-conferring gene as a dominant selectable marker in mammalian cells.

The HyR gene, conferring resistance to hygromycin B (Hy), has been modified for optimal expression in mammalian cells. Modifications to the HyR gene and its expression cassette include: (1) removal of all upstream start codons, (2) conversion of the region around the start codon to the consensus sequence associated with efficient translation initiation, and (3) removal of downstream splice donor and acceptor sequences. The resulting HyR gene is an efficient dominant selectable marker that is useful for studies requiring resistance from a low-copy-number gene driven by a promoter of moderate strength. The HyR gene was also tested for its compatibility with BPV vectors. Mouse C127 cells harboring pHyR-BPV plasmids exhibited properties of BPV-transformed cells and were resistant to toxic levels of Hy. The vectors were stable as episomes and present in high copy. The HyR gene thus joins the NmR (neo) gene as the only dominant selectable markers that are known to be compatible with BPV replication.

Animals↗

PubMed2003

Effect of translational signals on mRNA decay in Bacillus subtilis.

A 254-nucleotide model mRNA, designated deltaermC mRNA, was used to study the effects of translational signals and ribosome transit on mRNA decay in Bacillus subtilis. DeltaermC mRNA features a strong ribosome-binding site (RBS) and a 62-amino-acid-encoding open reading frame, followed by a transcription terminator structure. Inactivation of the RBS or the start codon resulted in a fourfold decrease in the mRNA half-life, demonstrating the importance of ternary complex formation for mRNA stability. Data for the decay of deltaermC mRNAs with stop codons at positions increasingly proximal to the translational start site showed that actual translation--even the formation of the first peptide bond--was not important for stability. The half-life of an untranslated 3.2-kb deltaermC-lacZ fusion RNA was similar to that of a translated deltaermC-lacZ mRNA, indicating that the translation of even a longer RNA was not required for wild-type stability. The data are consistent with a model in which ribosome binding and the formation of the ternary complex interfere with a 5'-end-dependent activity, possibly a 5'-binding endonuclease, which is required for the initiation of mRNA decay. This model is supported by the finding that increasing the distance from the 5' end to the start codon resulted in a 2.5-fold decrease in the mRNA half-life. These results underscore the importance of the 5' end to mRNA stability in B. subtilis.

Bacillus subtilis↗

PubMed2006

A weak blood group A phenotype caused by a translation-initiator mutation in the ABO gene.

BACKGROUND: Weak blood group A and B phenotypes are correlated with ABO glycosyltransferases exhibiting single-amino-acid changes and/or C-terminal modifications. STUDY DESIGN AND METHODS: A healthy donor diagnosed as having weak A antigen expression and his two children were subjected to extensive ABO typing. HeLa cells were used to transfect ABO expression plasmids. RESULTS: The donor's red blood cells were type A(weak)B and his serum sample contained weakly reactive anti-A(1) antibodies. A single T>C transition identified at the +2 position of the start codon of an ABO*A101 allele predicted the disruption of this methionine codon. In the transfection studies, a significant reduction of A activity was observed on HeLa cells transfected with a plasmid containing the variant ABO*A allele. Coexpression of the respective antithetical ABO*B101 wild-type construct further reduced cell surface A antigen expression. Similar expression results were obtained with ABO constructs in which the Met(1) start codon and five alternative start sites at codons 20, 26, 43, 53, and 69 had successively been interrupted. CONCLUSION: The donor's weak blood group A phenotype most likely resulted from expression of an N-truncated A transferase triggered by alternative translation start sites in the transmembrane domain or stem region.

ABO Blood-Group System↗