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PubMed1997

Characterization of Post-Transcriptionally Suppressed Transgene Expression That Confers Resistance to Tobacco Etch Virus Infection in Tobacco.

Tobacco lines expressing transgenes that encode tobacco etch virus (TEV) coat protein (CP) mRNA with or without nonsense codons give rise to TEV-resistant tissues that have reduced levels of TEV CP mRNA while maintaining high levels of transgene transcriptional activity. Two phenotypes for virus resistance in the lines containing the transgene have been described: immune (no virus infection) and recovery (initial systemic symptoms followed by gradual recovery over several weeks). Here, we show that at early times in development, immune lines are susceptible to TEV infection and accumulate full-length CP mRNA. Therefore, immune lines also exhibit meiotic resetting, as is seen in the recovery lines, providing molecular evidence for a common mechanism of gene silencing and virus resistance in both cases. We also investigated the characteristics of two sets of low molecular weight RNAs that appear only in silenced tissue. One set has nearly intact 5[prime] ends, lacks poly(A) tails, and is associated with polyribosomes; the second set contains the 3[prime] end of the mRNA. Treating silenced leaf tissue with cycloheximide resulted in decreased levels of full-length mRNA and an increase in the levels of the low molecular weight RNAs, supporting a cytoplasmic decay mechanism that does not require ongoing translation. Surprisingly, mRNA from the transgene containing nonsense codons was associated with more ribosomes than expected, possibly resulting from translation from a start codon downstream of the introduced translational stop codons. We present a hypothesis for transgene/viral RNA degradation in which RNA degradation occurs in the cytoplasm while in association with polyribosomes.

Journal Article↗

PubMed1985

Synthesis of Escherichia coli carbamoylphosphate synthetase initiates at a UUG codon.

The ribosome binding region of the messenger RNA for the Escherichia coli carA gene contains two adjacent putative translational start codons, UUG and AUU, both of them unusual. By Edman degradation and mass spectrometry of purified carA protein, we show that only UUG is used in vivo. Translation initiation at UUG in carA appears about half as efficient as at AUG in lacZ.

Amino Acid Sequence↗

PubMed1985

Cryptic plasmid of Neisseria gonorrhoeae: complete nucleotide sequence and genetic organization.

The naturally occurring cryptic plasmid pJD1 of Neisseria gonorrhoeae is 4,207 base pairs long and is found in about 96% of gonococcal strains. The total probable coding capacity of pJD1 was determined from the complete nucleotide sequence by using computational probes to identify open reading frames with similar codon usage and by screening for the presence of ribosomal binding sites before the start codons. Candidates for promoters and terminators were also found in the sequence. Based on these findings, we propose a model for the genetic organization of the plasmid. The model predicts two transcriptional units, each composed of five compactly spaced genes. A promoter of one of the transcripts was shown to function in Escherichia coli, and the products of three of the five genes in this operon were identified in minicell expression experiments. Of these, the cppA gene encoded a 9-kilodalton protein, and the cppB and cppC genes both coded for 24-kilodalton proteins. No expression of the other transcriptional unit was detected, but two genes in this operon were expressed in minicells when transcribed from an E. coli promoter. The experimental data were consistent with the model.

Amino Acid Sequence↗

PubMed2002

Transcription of Xanthomonas campestris prt1 gene encoding protease 1 increases during stationary phase and requires global transcription factor Clp.

Xanthomonas campestris pv. campestris produces three proteases, Prt1, Prt2, and Prt3, the first two of which are involved in pathogenicity. In this study, nucleotide A 84 nt upstream of the prt1 start codon, which is 8 nt downstream of the -10 sequence, was determined as the transcription start site by the 5(') RACE (rapid amplification of cDNA ends) method. Using Pprt1-lacZ transcriptional fusion constructs for assays, several interesting characteristics of prt1 promoter were revealed. The expression is inducible by LB medium or casein proteins and involves the global transcription factor Clp (cyclic AMP receptor protein-like protein). The region containing bp -392 to -80 relative to the prt1 translation initiation codon is required for maximal expression, in which bp -392 to -207 responds to the Clp-mediated regulation and the induction. In presence of inducers and the clp wild-type background, the levels of expression continue to increase following cell growth until 30 h after the cultures entering stationary phase. Since prt1 promoter shows no response to stressful conditions and neither growth nor cell viability is affected by prt1 mutation, Prt1 appears to be a secondary metabolite of X. campestris pv. campestris.

5' Flanking Region↗

PubMed1989

Aspergillus oryzae has two nearly identical Taka-amylase genes, each containing eight introns.

cDNA and genomic DNA for two nearly identical genes, AmyI and AmyII, coding for the enzyme Taka-amylase A (TA-A) of the fungus Aspergillus oryzae have been cloned and characterized. These genes are apparently unlinked, differing by only 3 nucleotides (nt) out of the 2720 nt that span the coding regions. The 617-nt 5'-flanking regions differ only at nt -372 (T or A) from the putative ATG start codon and contain four sets of short, inverted repeats (IR) upstream from the putative TATAAA box at nt -100 and the transcription start point at nt -69. The coding regions consist of 499 codons disrupted by eight intervening sequences. The putative proenzymes differ by only two amino acids (aa) and consist of the 478-aa extracellular enzyme plus a 21-aa hydrophobic leader sequence. Except for the replacement site changes in codons 35 (Arg----Gln) and 151 (Phe----Leu), the identity of the two genes continues downstream for 58 nt past the TGA stop codon before diverging. Exon 9 codes for 94 of the 98 aa of the domain B of mature TA-A. Little conservation of TA-A exons was found when these exons were aligned with those of human amylase. The genes are flanked by at least 6 to 10 kb of unrelated chromosomal nucleotide sequence. The Amy genes are co-expressed, since mRNA (cDNA) specific to the 3'-UTR of both genes was recovered from mycelia grown on starch, a known inducer of TA-A biosynthesis. The 3'-UTRs of cDNAs related to AmyI are shorter (128 nt and 145 nt) than those of AmyII (179 nt and 297 nt). The AmyI specific 3'-UTR is characterized by the absence of IR sequences and the presence of a putative 'AATAAA' polyadenylation signal.

Amino Acid Sequence↗

PubMed1996

Systematic screening for mutations in the human serotonin 1F receptor gene in patients with bipolar affective disorder and schizophrenia.

Using single strand conformational analysis we screened the complete coding sequence of the serotonin 1F (5-HT1F) receptor gene for the presence of DNA sequence variation in a sample of 137 unrelated individuals including 45 schizophrenic patients, 46 bipolar patients, as well as 46 healthy controls. We detected only three rare sequence variants which are characterized by single base pair substitutions, namely a silent T-->A transversion in the third position of codon 261 (encoding isoleucine), a silent C-->T transition in the third position of codon 176 (encoding histidine), and an C-->T transition in position -78 upstream from the start codon. The lack of significant mutations in patients suffering from schizophrenia and bipolar affective disorder indicates that the 5-HT1F receptor is not commonly involved in the etiology of these diseases.

Base Sequence↗

PubMed1990

Role of tRNA modification in translational fidelity.

In transfer RNA many different modified nucleosides are found, especially in the anticodon region. In this region, pseudouridine (psi) is found in positions 38, 39 or 40 in a subset of tRNA species, 2-methylthio-6-hydroxyisopentenyladenosine (ms2io6A) is found in position 37 in tRNAs that read codons starting with U and 1-methylguanosine (m1G) is found in position 37 in tRNAs reading codons of the UCCNG type. We have used the mutants hisT, miaA and miaB and trmD, which are deficient in the biosynthesis of psi, ms2io6A, and m1G, respectively, to study the functional aspects of the respective modified nucleosides. We have shown: (1) Presence of psi improved the cellular growth rate, the polypeptide step-time, and the efficiency of an amber suppressor, but did not appreciably sense the codon context. (2) Presence of ms2io6A improved the cellular growth rate, the polypeptide step-time and the efficiency of several amber suppressor tRNAs. It also had a profound effect on the codon context sensitivity of the tRNA. (3) Presence of m1G improved the cellular growth rate and the polypeptide steptime and also prevented the tRNA from shifting the reading frame. Thus, these three modified nucleosides present in the anticodon region have apparently different functions.

Anticodon↗

PubMed2001

Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: additional domains for membrane attachment?

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a membrane integrated glycoprotein, which physiologically performs the interconversion of active and inactive glucocorticoid hormones and which also participates in xenobiotic carbonyl compound detoxification. Since 11beta-HSD 1 is fixed to the endoplasmic reticulum (ER) with a N-terminal membrane spanning domain, the enzyme is very difficult to purify in an active state. Upon expression experiments in Escherichia coli, 11beta-HSD 1 turns out to be hardly soluble without detergents. This study describes attempts to increase the solubility of 11beta-HSD 1 via mutagenesis experiments by generating several truncated forms expressed in E. coli and the yeast Pichia pastoris. Furthermore, we investigated if the codon for methionine 31 in human 11beta-HSD 1 could serve as an alternative start codon, thereby leading to a soluble form of the enzyme, which lacks the membrane spanning segment. Our results show that deletion of the hydrophobic membrane spanning domain did not alter the solubility of the enzyme. In contrast, the enzyme remained bound to the ER membrane even without the N-terminal membrane anchor. However, activity could not be found, neither with the truncated protein expressed in E. coli nor with that expressed in P. pastoris. Hydrophobicity plots proved the hydrophobic nature of 11beta-HSD 1 and indicated the existence of additional membrane attachment sites within its primary structure.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

PubMed1997

Cloning, sequence and expression of the pel gene from an Amycolata sp.

The pel gene from an Amycolata sp. encoding a pectate lyase (EC 4.2.2.2) was isolated by activity screening a genomic DNA library in Streptomyces lividans TK24. Subsequent subcloning and sequencing of a 2.3 kb BamHI BglII fragment revealed an open reading frame of 930 nt corresponding to a protein of 29,660 Da. The overall G + C content for the coding region was 65%, with a strong G + C preference in the third (wobble) codon position (93%). A putative ribosome-binding site 5'-GGGAG-3' preceded the translational start codon by 7 base pairs. The Amycolata pectate lyase contains a signal peptide of 26 amino acids, that is cleaved after the sequence Ala-Thr-Ala. The size of the deduced protein as well as its N-terminal amino-acid sequence match the wild-type pectate lyase from the Amycolata sp. Expression of the pel gene in S. lividans TK24 resulted in high pectate lyase activity in the culture supernatant, concomitant with the appearance of a dominant protein band on a sodium dodecyl polyacrylamide gel at 30 kDa. No pectate lyase activity was detected in E. coli BL21 with the pel gene under the strong T7 promotor. The deduced amino-acid sequence showed 40% identity with PelE from Erwinia chrysanthemi and the pectate lyase from Glomerella cingulata. The Amycolata pectate lyase clearly belongs to the pectate lyase superfamily, sharing all functional amino acids and likely has a similar structural topology as Pels from Erwinia chrysanthemi and Bacillus subtilis.

Actinomycetales↗

PubMed2001

An isoform-specific inhibitory domain regulates the LHX3 LIM homeodomain factor holoprotein and the production of a functional alternate translation form.

The LHX3 LIM homeodomain transcription factor is required for pituitary development and motor neuron specification. The Lhx3 gene encodes two isoforms, LHX3a and LHX3b, that differ in their amino-terminal sequences. Humans and mice with defective Lhx3 genes are deficient in gonadotrope, lactotrope, somatotrope, and thyrotrope pituitary cells. We show that, whereas Lhx3b is highly expressed in these Lhx3-dependent cell types, high levels of Lhx3a expression are restricted to alpha glycoprotein subunit-expressing thyrotropes and gonadotropes. Cross-species comparison reveals the LHX3b-specific domain is more conserved than the LHX3a-specific domain. We demonstrate that the LHX3b-specific domain is a transferable inhibitor that reduces gene activation and DNA binding by homeodomain proteins. In addition, we identify a novel LHX3 protein (M2-LHX3) and determine that this molecule is generated by an internal translation initiation codon. The LHX3a- and LHX3b-specific coding sequences regulate differential usage of this internal start codon. Further, we identify the major activation domain of LHX3 in the carboxyl terminus of the molecule. M2-LHX3 is active because it retains this domain and binds DNA better than LHX3a or LHX3b. Other LIM homeodomain genes, including Lhx4, generate similar truncated proteins. These studies describe how transcriptional regulatory genes can generate multiple functional proteins.

Amino Acid Sequence↗

PubMed1995

A novel human c-sis mRNA species is transcribed from a promoter in c-sis intron 1 and contains the code for an alternative PDGF B-like protein.

The human platelet-derived growth factor (PDGF) B chain precursor is usually translated from a 3.5 kb c-sis/PDGF B gene transcript. The first exon of the c-sis/gene contains the code for the signal peptide of the PDGF B chain precursor, preceded by a 1 kb long untranslated sequence with potent translation inhibitory activity. In this paper we show that a novel 2.6 kb c-sis mRNA present in the human choriocarcinoma cell line JEG-3 initiates at an alternative exon 1, which we refer to as exon 1a. The 90 bp long exon 1a is located in the center of the first intron of the gene. It coincides with a very pronounced DNase-I-hypersensitive site and is preceded by a functional promoter. Of the three ATG codons present in exon 1a, the third one perfectly matches the criteria of a consensus start codon. It initiates an open reading frame that is continuous with the code for the PDGF B chain precursor but lacks the code for a signal peptide. We conclude that this novel 2.6 kb c-sis mRNA species lacks the strong translation inhibitory potential of the regular exon 1 and contains the code for a PDGF B-like protein that may be targeted to the cell nucleus.

Alternative Splicing↗

PubMed1980

Nucleotide sequence of non-initiator methionine tRNA from Bacillus subtilis.

Non-initiator methionine tRNA (tRNAMet) was purified from Bacillus subtilis W 168 by a consecutive use of several column chromatographic systems. The nucleotide sequence was determined to be p-G-G-C-G-G-U-G-U-A-G-C-U-C-A-G-C-G-G-C-D-A-G-A-G-C-G-U-A-C-G-G-U-U-C-A-U-m6A-C-C-C -G-U-G-A-G-G(m7G)-U(D)-C-G-G-G-G-G-T-psi-C-G-A-U-C-C-C-C-U-C-C-G-C-C-G-C-U-A-C- C-A-OH. The nucleosides of G46 and U47 were partially modified to m7G and D, respectively. The nucleotide sequence shows a unique feature that the position adjacent to 3'-end of the anticodon C-A-U is occupied by m6A, not by t6A, although the tRNAMet belongs to a groups of tRNAs which recognize codons starting with A.

Anticodon↗

PubMed2000

Analysis of the dormancy-inducible narK2 promoter in Mycobacterium bovis BCG.

Upon depletion of oxygen, the obligate aerobe mycobacteria switch from growth to a state of non-replicating persistence or dormancy. Here, we report the first functional analysis of a dormancy-dependent mycobacterial promoter in Mycobacterium bovis BCG. Promoter probing using a 'lacZ reporter detected a dormancy-inducible promoter activity upstream of the coding sequence for the putative nitrite extrusion protein NarK2. Primer extension analysis mapped a transcriptional start point 47 bp upstream of the narK2 start codon. Deletion analysis revealed that the sequence -222 to -133 bp upstream from the transcriptional start point was required for basal and dormancy-inducible reporter expression. The sequence +1 to +47 downstream of the transcriptional start point had a strong inhibitory effect on the level of dormancy-induced beta-galactosidase activity. The identification of apparent activating and inhibiting regions suggests that the narK2 promoter is at least under dual control.

Anaerobiosis↗

PubMed2001

New vascular endothelial growth factor isoform generated by internal ribosome entry site-driven CUG translation initiation.

We recently demonstrated that the very long 5'-untranslated region (5'-UTR) of the vascular endothelial growth factor (VEGF) mRNA contains two independent internal ribosome entry sites (IRES A and B). In the human sequence, four potential CUG translation initiation codons are located in between these IRES and are in frame with the classical AUG start codon. By in vitro translation and COS-7 cell transfections, we demonstrate that a high mol wt VEGF isoform [called large VEGF (L-VEGF)] is generated by an alternative translation initiation process, which occurs at the first of these CUG codons. Using a bicistronic strategy, we show that the upstream IRES B controls the translation initiation of L-VEGF. This isoform is 206 amino acids longer than the classical AUG-initiated form. With a specific antibody raised against this NH2 extension, we show that the L-VEGF is present in different mouse tissues or in transfected COS-7 cells. We also demonstrate that L-VEGF is cleaved into two fragments: a 23-kDa NH2-specific fragment and a fragment with an apparent size similar to that of the classical AUG-initiated form. This cleavage requires the integrity of a hydrophobic sequence located in the central part of the L-VEGF molecule. This sequence actually plays the role of signal peptide in the classical AUG-initiated form. The AUG-initiated form and the COOH cleavage product of the L-VEGF are both secreted. In contrast, the large isoform and its NH2 fragment present an intracellular localization. These data unravel a further level of complexity in the regulation of VEGF expression.

5' Untranslated Regions↗

PubMed1990

IL-2 receptor (Tac antigen) protein expression is down-regulated by the 5'-untranslated region of the mRNA.

We have investigated the regulatory role of the 5'-untranslated region (5'-UTR) of the IL-2R mRNA. We noticed a region of striking homology (92%) between the human and bovine IL-2R cDNA in a stretch of 26 nucleotides located in the 5'-UTR. Within this 26 nucleotide region is an AUG that is out of frame with the IL-2R coding sequence. The murine IL-2R cDNA has an 11 bp direct repeat in the 5'-UTR that includes an upstream AUG, and this sequence is identical to the translational start site for the murine IL-2R protein. These observations raised the possibility that the two upstream AUG start codons might down regulate translation of the IL-2R by acting as false translational start sites. To investigate the possibility of IL-2R translational control, we examined sucrose gradient polysome profiles from Con A stimulated murine CD4+ splenocytes. The IL-2R mRNA was found in the portion of the gradient where free RNA, mono, and disomes migrate, whereas actively translated mRNA (lymphokines and beta-actin) were found in the portion of the gradient that contained the polysome fractions. This finding was consistent with translational down-regulation of the IL-2R mediated by ribosomal binding to the 5'-UTR start sites. We next examined cells transfected with the IL-2R cDNA that had the 5'-UTR deleted and compared protein expression to cells transfected with the full length construct. Flow microfluorometry analysis of cell surface IL-2R expression, showed that a clone transfected with the 5'-UTR deletion expressed five- to six-fold more IL-2R than a clone transfected with the full-length construct, even though both clones produced equivalent IL-2R mRNA levels. In clones transfected with the full length construct, the IL-2R mRNA associated with a reduced number of ribosomes compared to the mRNA in the deleted construct clones. These results indicate that sequences in the 5'-UTR of IL-2R mRNA lead to a decrease in the amount of ribosomes bound per IL-2R RNA molecule, and suggest that the level of IL-2R expression can thus be translationally down-regulated. This is the first growth factor receptor shown to be posttranscriptionally controlled at the translational level, and these findings have important implications for IL-2R synthesis and cell surface expression of this immunologically active cell surface receptor.

Animals↗

PubMed1995

Mutations in GCD11, the structural gene for eIF-2 gamma in yeast, alter translational regulation of GCN4 and the selection of the start site for protein synthesis.

Translation initiation factor 2 (eIF-2) in eukaryotic organisms is composed of three non-identical subunits, alpha, beta and gamma. In a previous report, we identified GCD11 as an essential gene encoding the gamma subunit of eIF-2 in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of yeast eIF-2 gamma displays remarkable similarity to bacterial elongation factor Tu, including the presence of sequence elements conserved in all known guanine nucleotide binding proteins. We have identified the molecular defects present in seven unique alleles of GCD11 characterized by a partial loss of function. Three of these mutations result in amino acid substitutions within the putative GTP binding domain of eIF-2 gamma. We show that the gcd11 mutations specifically alter regulation of GCN4 expression at the translational level, without altering the scanning mechanism for protein synthesis initiation. Six of the mutant alleles presumably alter the function of eIF-2 gamma, rather than its abundance. A single allele, gcd11-R510H, suppresses a mutant his4 allele that lacks a functional AUG start codon. The latter result indicates that the gamma subunit of eIF-2 participates in recognition of the start site for protein synthesis, a role previously demonstrated in yeast for eIF-2 alpha and eIF-2 beta.

Alleles↗

PubMed1993

Small circular plasmids of the eukaryote Dictyostelium purpureum define two novel plasmid families.

Two novel groups of circular, nuclear plasmids were discovered in the simple eukaryote Dictyostelium purpureum. They define two new Dictyostelium plasmid families each containing three members: Dpp1A, Dpp1B, and Dpp2 in the Dpp1 family, and Dpp3, Dpp4, and Dpp5 in the Dpp3 family. These plasmids are among the smallest known, ranging in size from 1309 bp (Dpp1A and Dpp1B) to 1961 bp (Dpp4). Family members are very similar. The most distantly related members of the Dpp1 family (Dpp1A and Dpp2) are 89% identical, while the most distantly related members of the Dpp3 family (Dpp3 and Dpp4) are 91% identical. No sequence similarity is found between these plasmid families, or to any other known plasmid or chromosomal DNA sequence. A 72-bp inverted repeat present in one copy in Dpp1A and Dpp1B is tandemly repeated in Dpp2. The Dpp3 family contains a region of 102-160 nucleotides rich in short, overlapping direct sequence repeats. This region is present once in Dpp3 and Dpp5 and is tandemly repeated in Dpp4. The repeat structures in the Dpp1 and Dpp3 families are relatively rich in GC base pairs (29-41%) in comparison to the unique sequence regions of the plasmids (16-22%). The longest open reading frame (ORF) beginning with an AUG codon in these plasmids is 168 bp in the Dpp3 family, although longer ORFs without AUG start codons (up to 201 bp) also exist. Northern blot analysis did not detect any plasmid-specific transcripts in total RNA prepared from vegetative cells carrying Dpp1A or Dpp3.

Animals↗

PubMed2001

Cloning, characterization, and phylogenetic analysis of a shrimp white spot syndrome virus gene that encodes a protein kinase.

An open reading frame (ORF) that encodes a 715-amino-acid polypeptide was found in an 8421-bp EcoRI fragment of the shrimp white spot syndrome virus (WSSV) genome. The polypeptide shows significant homology to eukaryotic serine/threonine protein kinase (PK) and contains the major conserved subdomains for eukaryotic protein kinases. Coupled in vitro transcription and translation generated a protein having an apparent molecular mass of about 87 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For transcriptional analysis of the pk gene, total RNA was isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with pk-specific riboprobe found a major and a minor transcript of 2.7 and 5.7 kb, respectively. Rapid amplification of the 5' cDNA ends of the major 2.7-kb pk transcript showed that there were two transcriptional initiation sites located at nucleotide residues -38(G) and -39(G) relative to the ATG translational start codon. Temporal expression analysis by RT-PCR indicated that the transcription of the pk gene started 2 h after infection and continued for at least 60 h. Phylogenetic analysis showed that WSSV protein kinase does not have any close relatives and does not fall into any of the major protein kinase groups.

3' Untranslated Regions↗