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PubMed1989

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation.

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.

Animals↗

PubMed1991

Different promoters of SHV-2 and SHV-2a beta-lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers.

Clinical Klebsiella pneumoniae isolates as well as Escherichia coli transformants producing the beta-lactamases SHV-2 or SHV-2a demonstrate MIC values for cefotaxime of 4 mg l-1 or 64 to greater than 128 mg l-1, respectively. The beta-lactamases differ by one possibly insignificant amino acid exchange at position number 10 of the mature protein; their kinetic parameters are rather similar. The 5' untranslated regions of both corresponding genes show no homology starting 74 nucleotides upstream to the start codon. Hybridization of intragenically annealing oligonucleotides to dot-blotted serial dilutions of total cellular RNA from E. coli transformants harbouring these genes cloned into the same vector plasmid gave a positive signal down to 1.2 micrograms (SHV-2) and 0.32 to 0.16 micrograms (SHV-2a), indicating a four to eight times higher amount of specific transcript in the case of SHV-2a. By primer extension analysis and S1 nuclease digestion the starting point to transcription was located 100 nucleotides (SHV-2) and 50 nucleotides (SHV-2a) in front of the start codon. No other transcripts of different length could be detected after prolonged exposure. Northern blot analysis demonstrated the length of the beta-lactamase mRNA to be about 1.6 kb in both cases, thus comprising a potential open reading frame downstream of the two enzymes' genes. Selective PCR amplification of both promoter regions and of the structural gene of SHV-2 and subsequent combined cloning of each of the promoters and the SHV-2 gene into pBGS19 using a BamHI restriction site introduced by three point mutations into the cloned sequences was employed to transforms E. coli DH5 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor.

Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUG-initiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.

Amino Acid Sequence↗

PubMed1994

Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates.

Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including beta-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low- and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B. fragilis 638 cepA mutant. The results of beta-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.

Amino Acid Sequence↗

PubMed1988

Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity.

The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined. The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein. The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism. BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity. A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified. A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified. The encoded product contains a C terminus homologous to other C. thermocellum endoglucanases.

Amino Acid Sequence↗

PubMed1991

Cytomegalovirus assembly protein nested gene family: four 3'-coterminal transcripts encode four in-frame, overlapping proteins.

The genomic region encoding the assembly protein of simian cytomegalovirus (CMV) strain Colburn has been cloned, sequenced, and found to be organized as a nested set of four in-frame, 3'-coterminal genes, each with its own TATA promoter element and translational start codon, and all using a single 3' polyadenylation signal. The 3' end of the longest open reading frame (1.770 bp) was identical to the 930-bp sequence coding for the assembly protein precursor, as determined from a cDNA clone. The assembly protein coding region of human CMV strain AD169 was similarly organized, suggesting that both viral genomes could give rise to four independently transcribed 3'-coterminal RNAs coding for four overlapping, in-frame, carboxy-coterminal proteins. These predictions were tested and confirmed. Four mRNAs corresponding in size and sequence to those predicted were identified in both human and simian CMV-infected cells by using transcript-specific antisense oligonucleotide probes in Northern (RNA blot) assays. The 5' ends of the three largest of these Colburn transcripts were determined by S1 nuclease protection assays and found to map between the anticipated TATA sequences and corresponding translational start codons. The four predicted overlapping proteins were identified by immunoassays in lysates of simian and human CMV-infected cells by using an antiserum specific for the carboxyl end of the assembly protein precursor. The structural relationship of both sets of proteins was verified by comparing their peptide patterns following protein cleavage at tryptophan residues by N-chlorosuccinimide. The similar organization of the homologous coding regions in other herpesviruses into at least two nested, in-frame, 3'-coterminal genes is discussed.

Amino Acid Sequence↗

PubMed1984

SV40 deletion mutant (d1861) with agnoprotein shortened by four amino acids.

d1861 is an SV40 deletion mutant which was thought to lack the agnoprotein coding region and was used to verify the role of agnoprotein in the life cycle of SV40. In the present study the region flanking the deletion was sequenced and, in contrast to the available information, it was found that d1861 lacks 12 nt in phase, downstream from the AUG start codon of agnoprotein (residues 347-358). Using the runoff protocol with viral transcriptional complexes (VTC), that in vitro elongate the in vivo preinitiated nascent RNA, it was found that in vivo the major initiation site for late transcription is at residue 325, the same as in wild type (WT). In comparison with WT, d1861 encodes information for agnoprotein shortened by four amino acids and it has been identified in d1861 infected cells. However, pulse-chase experiments indicated that the rate of synthesis of d1861 agnoprotein is slower than that of WT agnoprotein and that it has a turnover rate of 1 hr as compared to 3 hr of WT agnoprotein. The reduced rate of synthesis of d1861 agnoprotein can be explained by nuclease S1 analyses in which the major leader of d1861 16 S RNA, that encodes the agnoprotein, appeared in significantly lower amounts as compared to the major leader of WT 16 S RNA. Furthermore, analysis of the potential secondary structures at the 5' end of the leader of d1861 16 S RNA has revealed stable structures in which the start codon of agnoprotein is sequestered in a stem. The involvement of RNA secondary structures in regulating the synthesis of agnoprotein is discussed.

Amino Acid Sequence↗

PubMed1991

Sequence and linkage analysis of the Coxiella burnetii citrate synthase-encoding gene.

The nucleotide (nt) sequence of the Coxiella burnetii citrate synthase-encoding gene (gltA), previously cloned in Escherichia coli, was determined. The nt sequence analysis revealed an open reading frame (ORF) of 1290 bp capable of coding for a protein of 430 amino acids (aa) with a deduced Mr of 48,633. Preceding an ATG start codon, a possible transcription start point (tsp) with homology to the E. coli promoter consensus was detected. A poly-purine-rich region occurred immediately upstream from the gltA reading frame and potentially serves as a ribosome-binding site. Additionally, a G + C-rich region of dyad symmetry 3' to the translational stop codon was found that could possibly function as a Rho-independent transcriptional termination signal. A large, nearly perfect, inverted repeat was identified upstream from the gltA tsp and was shown by Southern analysis to be present in multiple copies in the C. burnetii genome. The deduced aa sequence of C. burnetii GltA was optimally aligned with enzymes from various prokaryotic sources and one eukaryotic source (pig heart). Using perfect aa identity, the C. burnetii enzyme demonstrated the greatest homology with GltA from Acinetobacter anitratum (65%). Although only 26% aa identity was seen with the pig heart enzyme, many of the residues identified in ligand binding appear to be conserved. Sequencing studies of a region centered approx. 5.6 kb upstream from gltA revealed an ORF read with opposite polarity that encodes a peptide highly homologous to the C terminus of the flavoprotein subunit of E. coli succinate dehydrogenase. This report represents the first nt sequence analysis of a gene of known function from the obligate intracellular parasite, C. burnetii.

Amino Acid Sequence↗

PubMedDate not supplied

Effect of leader primary structure on the translational efficiency of phosphoglycerate kinase mRNA in yeast.

In order to determine the effect of the nucleotide composition of the 5'-untranslated (leader) region on the translational efficiency of mRNA in yeast, we replaced a large part of the leader region of the phosphoglycerate kinase (PGK) gene by various deoxyoligonucleotides of defined sequence. All mutations left the context of the transcription initiation site and AUG start codon intact. The mutant genes were introduced into yeast cells on a multicopy vector and the ratio of the steady-state levels of PGK mRNA and protein were determined. We found translational efficiency to be unaffected by the presence of either an 18 nucleotides (nt) long polyA or polyC tract or by sequences consisting of mixtures of A and C residues in any proportion. In contrast, a poly U tract, as well as mixtures of U and C residues, reduced translational efficiency by a factor of two to three, presumably by long-range base-pairing between the leader and sequences elsewhere in the coding or 3'-non-coding regions of the messenger. In agreement with this hypothesis, a five-fold reduction in translational efficiency was found for an mRNA carrying a polyC tract in the leader as well as a polyG tract in the trailer, neither of which had any effect on translational efficiency by itself. Therefore, we conclude that the leader and trailer regions (including the polyA tail) of PGK mRNA are sufficiently close to base-pair when containing complementary sequences. The resulting secondary structure evidently constitutes a barrier for incoming 40S subunits on their way to the AUG start codon. The presence of an 18 nt long polyG tract in the leader completely abolished translation of the PGK mRNA in accordance with earlier observations. However, we found that leaders containing up to 40% G residues interspersed with either A or U, still allow highly efficient translation. This value is about four times as high as the average G content of leader sequences in naturally occurring yeast mRNAs. Finally, neither deletion of about 40% of the trailer sequence of PGK mRNA, nor replacement of this sequence by homopolymer tracts had any effect on translational efficiency.

Amino Acid Sequence↗

PubMed1995

Human immunodeficiency virus type 1 Nef protein inhibits activation pathways in peripheral blood mononuclear cells and T-cell lines.

Human immunodeficiency virus type 1 (HIV-1) Nef protein causes the loss of cell surface CD4 and interleukin-2 (IL-2) receptor (Tac) from peripheral blood mononuclear cells (PBMC) and CD4+ T-cell lines. As both CD4 and the IL-2 receptor play crucial roles in antigen-driven helper T-cell signalling and T-cell proliferation, respectively, the role of Nef in the viral life cycle may be to perturb signalling pathways emanating from these receptors. However, the intracellular targets for Nef that result in receptor down-regulation are unknown. Using a recombinant glutathione S-transferase-full-length 27 kDa Nef (Nef27) fusion protein, produced in Escherichia coli by translation from the first start codon of HIV-1 nef clone pNL4-3, as an affinity reagent to probe cytoplasmic extracts of MT-2 cells and PBMC, we have shown interaction with at least seven host cell protein species ranging from 24 to 75 kDa. Immunoblotting identified four of these proteins as p56lck, CD4, p53, and p44mapk/erk1, all of which are intimately involved in intracellular signalling. To assess the relevance of these interactions and further define the biochemical activity of Nef in signal transduction pathways, highly purified Nef27 protein was introduced directly into PBMC by electroporation. Nef27-treated PBMC showed reduced proliferative responsiveness to exogenous recombinant IL-2. Normally, stimulation of T-cells by IL-2 or phorbol 12-myristate 13-acetate provokes both augmentation of p56lck activity and corresponding posttranslational modification of p56lck. These changes were also inhibited by treatment of PBMC with Nef, suggesting that Nef interferes with activation of p56lck and as a consequence of signalling via the IL-2 receptor. Further evidence for Nef interfering with cell proliferation was the decreased production of the proto-oncogene c-myb, which is required for cell cycle progression, in Nef-treated MT-2 cells. In contrast to the binding characteristics and biological effects of Nef27, the alternate 25-kDa isoform of Nef (Nef25) produced by translation from the second start codon of HIV nef pNL4-3 (57 nucleotide residues downstream) was shown to interact with only three cellular proteins of approximately 26, 28, and 56 kDa from PBMC and MT-2 cells, one of which was identified as p56lck. Also, proliferation and posttranslational modification of p56lck in response to IL-2 stimulation were not profoundly affected by treatment of PBMC with Nef25 compared with Nef27.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1986

Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase.

The Bacillus subtilis genes encoding threonine synthase (thrC) and homoserine kinase (thrB) have been cloned via complementation of Escherichia coli thr mutants. Determination of their nucleotide sequences indicates that the thrC stop codon overlaps the thrB start codon; this genetic organization suggests that the two genes belong to the same operon, as in E. coli. However, the gene order is thrC-thrB in B. subtilis whereas it is thrB-thrC in the thr operon of E. coli. This inversion of the thrC and thrB genes between E. coli and B. subtilis is indicative of a possible independent construction of the thr operon in these two organisms. In other respects, comparison of the predicted amino acid sequences of the B. subtilis and E. coli threonine synthases with that of Saccharomyces cerevisiae threonine dehydratase and that of E. coli D-serine dehydratase revealed extensive homologies between these pyridoxal phosphate-dependent enzymes. This sequence homology, which correlates with similarities in the catalytic mechanisms of these enzymes, indicates that these proteins, catalyzing different reactions in different metabolic pathways, may have evolved from a common ancestor.

Amino Acid Sequence↗

PubMed1988

Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene.

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.

Amino Acid Sequence↗

PubMed1994

Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system.

The lysostaphin gene of Staphylococcus simulans was cloned into Escherichia coli. The 5' end of the gene was modified to include a eukaryotic start codon, the Kozak expression start site consensus sequence, and an enzyme site to facilitate manipulation of the gene. Transcription of the modified gene in vitro yielded an RNA transcript which, when added to a rabbit reticulocyte cell-free translation system, directed the synthesis of several products. The largest product, migrating at approximately 93 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was probably preprolysostaphin, since it was cleaved in the presence of an S. simulans culture supernatant to yield a polypeptide of a size similar to that of mature lysostaphin. When canine pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. The gene was also expressed transiently from the human cytomegalovirus promoter in COS-7 cells. Active enzyme could be detected in the cell lysate, and the prokaryotic signal appeared to target secretion of active enzyme to the culture medium. The successful expression of the lysostaphin gene and processing of the precursor to produce active secreted enzyme open up the possibility of controlling staphylococcal mastitis by targeting expression of this gene to the mammary glands of transgenic animals.

Animals↗

PubMed1995

Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy.

In Pseudomonas aeruginosa, the transcriptional activator LasR and the Pseudomonas autoinducer PAI, are necessary for efficient transcriptional activation of the lasB gene, encoding elastase (L. Passador, J. M. Cook, M.J. Gambello, L. Rust, and B. H. Iglewski, Science 260:1127-1130, 1993). The transcriptional start points of lasI in Escherichia coli and P. aeruginosa were determined by S1 nuclease mapping. In the presence of both LasR and PAI, the start site, T1, is located at position -25 relative to the ATG translational start codon. A minor transcriptional start, T2, is found at position -13 when lasI is transcribed in the absence of either LasR or PAI in P. aeruginosa and E. coli, respectively. To begin to closely examine the regulation of lasI, whose product is involved in the synthesis of PAI, a lasI-lacZ fusion on a lambda phage was constructed to form monolysogens of E. coli MG4. Lysogens supplied only with either lasI or lasR via multicopy plasmids demonstrated no significant increase in beta-galactosidase expression compared with control levels. Lysogens in which both lasR and lasI were supplied in multicopy exhibited a 62-fold increase in expression, and a lysogen in which lasR was supplied in trans and which was grown in the presence of exogenous PAI exhibited a 60-fold increase. Thus, LasR and PAI are necessary for the full expression of lasI in E. coli. The interchangeability of the P. aeruginosa and Vibrio fischeri homologs LasR and LuxR and their respective autoinducers, PAI and VAI, as activators of lasI-lacZ was examined. Only the combination of LasR and PAI significantly increased the expression of lasI. The comparison of lasI-lacZ and lasB-lacZ expression lysogens grown in the presence of lasR and PAI revealed that half-maximal expression of lasI required 0.1 nM PAI, in contrast to the 1.0 nM PAI necessary for lasB half-maximal expression. These results suggest an autoinduction regulatory hierarchy in which LasR and low PAI concentrations primarily activate lasI expression in a regulatory loop. With the accumulation of PAI, secondary activation of virulence product genes such as lasB occurs.

Bacterial Proteins↗

PubMed1994

Control of cationic amino acid transport and retroviral receptor functions in a membrane protein family.

A partial cDNA sequence indicated that the T lymphocyte early-activation gene (Tea) encodes a protein related to the dual-function ecotropic retrovirus receptor/cationic amino acid transporter (ecoR/CAT1), and RNA blots suggested highest Tea expression in T lymphocytes and liver (MacLeod, C.L., Finley, K., Kakuda, D. Kozad, C.A., and Wilkinson, M.F. (1990) Mol. Cell. Biol. 7, 3663-3674). The sequence of full-length Tea cDNA from liver (3683 bases) predicts a 657-amino-acid protein (CAT2 alpha) with 12-14 transmembrane domains. A long (515 base) region with six initiation codons and termination codons precedes the translation start codon. The liver Tea cDNA is identical to Tea cDNA from T lymphocytes (encoding CAT2 beta) with the exception of an apparent alternatively spliced sequence encoding a hydrophilic loop of 43 amino acids. The liver-specific sequence contains unique consensus sites for phosphorylation by cyclic AMP-dependent protein kinase and by protein kinase C. Injection of Xenopus oocytes with CAT2 alpha or CAT2 beta messenger RNA resulted in expression of Na(+)-independent cationic amino acid transport that was detected by current measurements under voltage-clamp. Although the amino acid sequences of the isoforms differ in only 21 of 43 residues with the majority of substitutions being conservative, the apparent affinity of CAT2 beta for arginine uptake was 70-fold higher than the CAT2 alpha isoform (Km 38 microM versus 2.7 mM). Neither isoform functioned as a receptor for ecotropic or amphotropic murine retroviruses. However, CAT1-CAT2 chimeric proteins that contain the first three putative extracellular loops of ecoR/CAT1 functioned as ecotropic receptors despite a diminished capacity to bind the viral envelope glycoprotein. The chimeric proteins also functioned as basic amino acid transporters with substrate affinities corresponding to the CAT2 isoform constituting the carboxyl-terminal portion. These results demonstrate that domains of these transporters can function in chimeric combinations to control viral receptor and transport functions.

Amino Acid Sequence↗

PubMed1984

Yeast LEU1. Repression of mRNA levels by leucine and relationship of 5'-noncoding region to that of LEU2.

Yeast LEU1 encodes the second enzyme in leucine biosynthesis. A 3.5-kilobase pair (kb) yeast genomic DNA fragment which complements a leu1 auxotroph was isolated by yeast transformation. After recloning into an integrating vector, a subfragment (of the 3.5-kb fragment) directs a URA3 marker to integrate at the LEU1 locus. About 1.9 kb was sequenced from the 5'-end of the 3.5-kb insert, and a long open reading frame and potential ATG start codon were located. S1 nuclease mapping showed a major start for LEU1 transcripts at 79 nucleotides upstream of the ATG codon. Northern blots with a LEU1-specific probe showed the size of the LEU1 transcript (about 2.9 kb) is consistent with the size of the enzyme and steady state levels of the transcript are sharply reduced in cells grown in the presence of an elevated leucine concentration. The latter observation correlates with the repression by leucine of LEU1 gene product levels. Other work has shown that the level of the LEU2 gene product is also repressed by leucine. Sequence comparisons between LEU1 and LEU2 show that the LEU2 5'-sequences which are cognate to leucine are not found in LEU1; and three blocks of nucleotide sequence homology between LEU1 and LEU2 occur in the 330 nucleotides upstream of the respective start codons.

Amino Acid Sequence↗

PubMed1991

Rat urate oxidase: cloning and structural analysis of the gene and 5'-flanking region.

The structural gene (UOX) encoding rat urate oxidase (UOX) spans at least 23 kb and is composed of eight exons and seven introns. All of the exon-intron splice junction sequences conformed to the GT/AG consensus established for eukaryotic genes. The transcription start point (tsp) was determined using S1-type nuclease protection riboprobe, and assigned to an adenine 54 nucleotides (nt) upstream of the ATG start codon. A 456-bp 5'-terminal fragment, starting at the ATG codon, carries a putative TATA (ATAAAA) sequence at -32, and two putative 'CAAT box' sequences at -62 and -71 bp upstream from the tsp. No sequence resembling 'GC' box hexanucleotides (GGGCGG or CCGCCC) was found. The structural features of the 5'-flanking region of the UOX gene are distinct from the 5'-flanking sequences of peroxisomal beta-oxidation system genes which contain one or more 'GC' box elements but lack TATA- and CAAT-like features [Osumi et al., J. Biol. Chem. 262 (1987) 8138-8143; Ishii et al., J. Biol. Chem. 262 (1987) 8144-8150]. The 5'-flanking region of the UOX gene reveals a sequence, TTAGTAATT at nt -276 from the tsp, which appears to be complementary to the underlined part of the liver-specific LF-B1/HNF-1 consensus sequence, GTTAATNATTAAC (where N = A, C, T, G or no nt).

Amino Acid Sequence↗

PubMed1989

Effects of deletions in the N-terminal basic arm of brome mosaic virus coat protein on RNA packaging and systemic infection.

The first 25 amino acids of brome mosaic virus (BMV) coat protein include 8 basic and no acidic residues and are implicated in binding the encapsidated RNA. Using infectious transcripts from BMV RNA3 cDNA clones, we modified this region of the coat gene. A coat protein mutant with the first 25 amino acids deleted failed to direct either packaging of viral RNA in protoplasts or systemic infection of whole barley plants. Neither symptoms, virions, nor viral RNA was detectable in plants inoculated with this mutant or a mutant with a frameshift mutation in the coat gene. Mutants with the normal start codon changed to AAG or with the first eight codons deleted allowed translation to start at a downstream AUG, resulting in a deletion of the first 7 amino acids of the mature wild-type coat protein. These mutants not only packaged viral RNA in protoplasts but directed symptomatic, systemic infections that developed with normal speed and degree of spread within the host. The AUG-to-AAG point substitution did not revert to the wild type after long-term culture in planta. Wild-type BMV virions were also found to contain small amounts of a protein that coelectrophoresed with the truncated coat protein produced by the viable AAG and eight-codon-deletion mutants. This minor coat protein species presumably arose by infrequent translation initiation at the second AUG in the wild-type coat protein gene. Absence of encapsidation-competent coat protein appeared to stimulate production of nonstructural proteins in protoplast infections.

Amino Acid Sequence↗