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PubMed1989

DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus.

The cloning of the gene(s) encoding a 36-kilodalton (kDa) cell envelope protein of Brucella abortus has been previously described (T. A. Ficht, S. W. Bearden, B. A. Sowa, and L. G. Adams, Infect, Immun. 56:2036-2046, 1988). In an attempt to define the nature of the previously described duplication at this locus we have sequenced 3,500 base pairs of genomic DNA encompassing this region. The duplication represented two similar open reading frames which shared more than 85% homology at the nucleotide level but differed primarily because of the absence of 108 nucleotides from one of the two gene copies. These two genes were read from opposite strands and potentially encoded proteins which are 96% homologous. The predicted gene products were identical over the first 100 amino acids, including 22-amino-acid-long signal sequences. The amino acid composition of the predicted proteins was similar to that obtained for the Brucella porin isolated by Verstreate et al. (D. R. Verstreate, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982) and presumably represented two copies of the porin gene, tentatively identified as omp 2a (silent) and omp 2b (expressed). The homology between the two genes extended to and included Shine-Dalgarno sequences 7 base pairs upstream from the ATG start codons. Homology at the 3' ends extended only as far as the termination codon, but both genes had putative rho-independent transcription termination sites. Localization of the promoters proved more difficult, since the canonical procaryotic sequences could not be identified in the region upstream of either gene. Promoter activity was demonstrated by ligation to a promoterless lacZ gene in pMC1871. However, only one active promoter could be identified by using this system. A 36-kDa protein was synthesized in E. coli with the promoter in the native orientation and was identical in size to the protein produced in laboratory-grown B. abortus. When the promoter-containing fragment was inverted, a 33-kDa protein was expressed. These results were consistent with the predicted sizes based on the nucleotide sequences of the open reading frames in omp 2b and omp 2a. Whether this locus contains one active and one silent or cryptic porin gene, or two active Brucella porin genes expressed under different environmental conditions, is discussed.

Amino Acid Sequence↗

PubMed2004

Evidence against a direct role for the Upf proteins in frameshifting or nonsense codon readthrough.

The Upf proteins are essential for nonsense-mediated mRNA decay (NMD). They have also been implicated in the modulation of translational fidelity at viral frameshift signals and premature termination codons. How these factors function in both mRNA turnover and translational control remains unclear. In this study, mono- and bicistronic reporter systems were used in the yeast Saccharomyces cerevisae to differentiate between effects at the levels of mRNA turnover and those at the level of translation. We confirm that upfDelta mutants do not affect programmed frameshifting, and show that this is also true for mutant forms of eIF1/Sui1p. Further, bicistronic reporters did not detect defects in translational readthrough due to deletion of the UPF genes, suggesting that their function in termination is not as general a phenomenon as was previously believed. The demonstration that upf sui1 double mutants are synthetically lethal demonstrates an important functional interaction between the NMD and translation initiation pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1985

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. I. Primary structure of a pyrI gene encoding a modified regulatory subunit.

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrI encoding the regulatory polypeptide chain of the enzyme. We now report the sequence of the mutated pyrI and show that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Escherichia coli chromosome. An accompanying paper emphasizes the importance of the carboxy-terminal end of the regulatory chain for the homotropic and heterotropic interactions of aspartate carbamoyltransferase.

Allosteric Site↗

PubMed1985

The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase.

The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.

Aldose-Ketose Isomerases↗

PubMed1980

DNA sequence of the Serratia marcescens lipoprotein gene.

The Serratia marcescens gene for the outer membrane lipoprotein (lpp) was cloned in lambda phage vector Charon 14. The recombinant phage was very unstable, and the lpp gene with a 300-base-pair deletion at the transcription termination site was further cloned in pBR322. The DNA sequence of 834 base pairs encompassing the lpp gene was determined and compared with that of the Escherichia coli lpp gene. The sequence comparisons exhibit several unique features. (i) The promoter region is highly conserved (84% homology) and has an extremely high A+T content (78%) as in E. coli (80%). (ii) The 5' nontranslated region of the lipoprotein mRNA is also highly conserved (95% homology). (iii) In the DNA sequence corresponding to the signal peptide of this secretory protein, there are three drastic changes, including addition of one base pair and deletion of four base pairs in S. marcescens as compared to E. coli. The resultant alterations in the amino acid sequence, however, do not change the basic properties of the signal peptide, which are assumed to be essential for its function in the secretory mechanism. (iv) The DNA sequence from the amino terminus to the 51st residue of the mature lipoprotein is highly conserved (95% homology) and there is no amino acid substitution. (v) The DNA sequence corresponding to the seven amino acid residues at the carboxyl terminus has only 42% homology, resulting in four amino acid substitutions. (vi) Within the section of 40 base pairs beginning with the termination codon (UAA) and ending immediately before the oligo(T) transcription termination site in the E. coli lpp gene, there is about 60% homology. However, after this section, there is no obvious homology between the two sequences, probably because of a deletion of 300 base pairs at this region. (vii) Seven stable stem-and-loop structures could be formed in the mRNA region. (viii) Alterations in the third position of codons used in the lpp gene suggest that the gene has evolved somewhat differently from other genes in S. marcescens.

Amino Acid Sequence↗

PubMed1991

Mutations in 16S rRNA that affect UGA (stop codon)-directed translation termination.

Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Escherichia coli 16S rRNA shown previously to be important for peptide chain termination. Analysis of stop codon suppression by the various mutants showed an exclusive response to UGA stop signals, which was correlated directly with the continuity of one or the other of two tandem complementary UCA sequences (bases 1199-1204). Since no other structural features of the mutated ribosomes were hampered and the translation initiation and elongation events functioned properly, we propose that a direct interaction occurs between the UGA stop codon on the mRNA and the 16S rRNA UCA motif as one of the initial events of UGA-dependent peptide chain termination. These results provide evidence that base pairing between rRNA and mRNA plays a direct role in termination, as it has already been shown to do for initiation and elongation.

Base Sequence↗

PubMed1980

Gene organization of the transforming region of weakly oncogenic adenovirus type 7: the E1a region.

The structures of adenovirus type 7 (Ad7) cytoplasmic RNAs transcribed from the leftmost 4.5% of the viral genome during lytic infection of KB cells have been determined. The E1a region was found to specify three differently spliced mRNAs (I, II and III) which have common 5' and 3' termini. mRNAs I and II are transcribed between identical initiation and termination codons and code for polypeptides of 28 kd and 24 kd, whose only difference is an internal sequence of 32 amino acids present in the 28-kd protein. Translation of mRNA III initiates at the same AUG codon as in mRNA I and II, but uses a different reading frame beyond the splice point; consequently, it terminates at an earlier stop codon and yields a 6.3-kd polypeptide. Cytoplasmic E1a RNA was used as a template for in vitro protein synthesis in a cell-free system and found to encode polypeptides with apparent molecular weights of 42 kd, 40 kd, and 11 kd.

Adenoviruses, Human↗

PubMedDate not supplied

[Context organization of mRNA 5'-untranslated regions of higher plants].

Computer analysis of nucleotide sequences of 5'-untranslated regions (5'-UTR) of higher plants mRNA adopted from the EMBL nucleotide sequence databank was carried out. It was demonstrated that the average nucleotide frequencies of the leader sequences and adjacent regions of basal promoters are similar, whereas introns and 3'-UTR have a higher content of T and a lower content of C. A particular 5'-UTR contextual feature is a misbalance in the content of complementary nucleotides; probably a stable secondary structure adversely affects the translation properties of the leader sequence. About 20% of 5'-UTR contain AUG triplets, which is twice the earlier estimate. Considered are the properties of leader open reading frames (uORF), the possible causes of their high content in 5'-UTRs of eukaryotic mTNAs, and correlations between the features of uORFs and of the protein-encoding sequence of the gene. It is demonstrated that in effectively translated mRNAs the leader AUG triplets are more frequently located in a nonoptimal context, whereas terminating codons of uORFs more frequently exist in the optimal one. A hypothesis is put forward that the efficiency of termination at the uORF stop codon might substantially interfere with the mRNA translation activity.

5' Untranslated Regions↗

PubMed2000

Upf1p control of nonsense mRNA translation is regulated by Nmd2p and Upf3p.

Upf1p, Nmd2p, and Upf3p regulate the degradation of yeast mRNAs that contain premature translation termination codons. These proteins also appear to regulate the fidelity of termination, allowing translational suppression in their absence. Here, we have devised a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100), and used it to determine the regulatory roles of the UPF/NMD gene products. Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression. Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy-number can1-100 expression in wild-type cells caused an increase in mRNA abundance similar to that obtained in upf/nmd cells but did not result in comparable suppression. can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA. Our findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay. Consistent with these relationships, Upf1p, Nmd2p, and Upf3p were shown to be present at 1, 600, 160, and 80 molecules per cell, levels that underscored the importance of Upf1p but minimized the likelihood that these proteins were associated with all ribosomes or that they functioned as a stoichiometric complex.

Adaptor Proteins, Signal Transducing↗

PubMed1987

Extracellular release of colicin A is non-specific.

The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase.

Base Sequence↗

PubMed1999

RB1 gene mutations in retinoblastoma.

Mutations in both alleles of the RB1 gene are causal for the development of retinoblastoma, a childhood tumor of the eye. The spectrum of somatic and germline mutations in this gene is dominated by small mutations. Data on small mutations are listed in a locus specific database available at http://www.d-lohmann.de/Rb/mutations.html. Analysis of 368 reported small mutations reveals considerable heterogeneity. A notable recurrence of transitions is observed at 13 CpG-dinucleotides that are part of CGA codons or splice donor sites. Most mutations create a premature termination codon. With few exceptions, patients heterozygous for mutations of this kind develop bilateral retinoblastoma. Missense mutations and inframe deletions are rare. Some of these mutations are associated with a distinct phenotype marked by incomplete penetrance and reduced expressivity.

Chromosome Mapping↗

PubMed1985

Verification of the DNA predicted amino acid sequence of bacteriophage P22 tail protein by mass spectrometry.

Mass spectrometry was used to verify portions of a proposed amino acid sequence of the bacteriophage P22 tail protein which had been inferred from the DNA base sequence. The exopeptidases dipeptidyl aminopeptidase I and IV and dipeptidyl carboxypeptidase were used to hydrolyse intact protein and fragments generated by cyanogen bromide treatment of the tail protein. After partial purification by high performance liquid chromatography, peptides were identified by gas chromatography/mass spectrometry and fast atom bombardment mass spectrometry. The results indicate that the initiation amino acid, N-formylmethionine, has been removed from the N-terminal of the protein and that the protein ends at the termination codon which is 667 amino acids from the N-terminal residue. Nine regions of the protein from 13 to 42 residues in length were verified. All of the sequences checked were in the same DNA reading frame and corresponded to the proposed sequence.

Amino Acid Sequence↗

PubMed1982

Construction of versatile expression cloning vehicles using the lipoprotein gene of Escherichia coli.

The gene for the outer membrane lipoprotein (lpp), the most abundant protein of Escherichia coli, was used to construct multi-purpose expression cloning vehicles. These vehicles consist of two types in terms of gene expression, one for constitutive (pIN-I type; three vehicles) and the other for inducible (pIN-II type; three vehicles) gene expression, and have the following features: (a) The lpp gene was inserted into a multicopy plasmid, pBR322, and the tet gene was removed to keep the size of the vehicles minimal (approximately 5 kb). (b) A nucleotide sequence of 22 bp which contains EcoRI, HindIII, and BamHI sites was inserted at the position of the third amino acid of the prolipoprotein. (c) The same nucleotide sequence was also inserted in two other reading frames at the same position. There are no other EcoRI, HindIII, and BamHI sites in the vehicles. Therefore, six different types of restriction fragments (EcoRI-EcoRI, HindIII-HindIII, BamHI-BamHI, EcoRI-HindIII, EcoRI-BamHI, and HindIII-BamHI) can be cloned at this position in any of the three different reading frames. (d) The nucleotide sequence from position 46 to 168 of the lpp gene was deleted. However, the 3' end position of the lpp gene of 154 bp was retained, which contains not only translation termination codons in three different reading frames but also the transcription termination signal of the lpp gene. Thus, this sequence is assumed to prevent unnecessary translation as well as transcriptional read-through of a cloned gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

The structure and expression of a novel gene activated in early mouse embryogenesis.

We report the cloning and sequence determination of the mouse H19 gene. This gene is under the genetic control of two trans-acting loci in the mouse, termed raf and Rif. These loci determine the adult basal and inducible levels, respectively, of H19 mRNA, as well as the mRNA for alpha-fetoprotein. By elucidating the sequence and structure of the H19 gene we show that it is unrelated to the alpha-fetoprotein gene, and therefore must have acquired its regulation by raf and Rif independently. The sequence also indicates that the H19 gene has a very unusual structure. It is composed of five exons, 1307, 135, 119, 127 and 560 bp in size, along with four very small introns whose combined lengths are 270 bases. The largest open reading frame of the gene, sufficient to encode a protein of approximately 14 kd, is contained entirely within the first large exon, 680 bases downstream of the cap site of the mRNA. Preceding the translation initiation codon are four ATG codons, each of which is followed shortly thereafter by translation terminator codons. The rest of the gene, which encompasses all five exons, is presumed to be untranslated. That the long 5' untranslated region may be used to regulate the translation of the mRNA is suggested from in vitro translation studies. Experiments which utilized tissue culture cell lines of the mesodermal lineage suggest that the gene is activated very early during muscle cell differentiation.

Animals↗

PubMed1992

Escherichia coli umuDC mutants: DNA sequence alterations and UmuD cleavage.

The products of the chromosomally encoded umuDC genes are directly required for mutagenesis in Escherichia coli. Strains with either umuD or umuC mutations are rendered phenotypically non-mutable. To ascertain the molecular basis of this non-mutability, we determined the DNA sequence alterations of seven chromosomal umuDC mutants. Six mutants (umuD1, umuD44, umuD77, umuC36, umuC25, and umuC104) were found to be single base-pair substitutions that resulted in missense mutations. The Tn5 transposon insertion mutation (umuC122) resulted in a missense mutation followed immediately by a termination codon, producing a truncated UmuC protein lacking 102 carboxyl-terminal amino acids. All of the mutations were found to reside in regions of the UmuD and UmuC proteins that share high homology with analogous proteins. Chemiluminescent immunoassays revealed that the umuD1, umuD44, and umuD77 mutations all resulted in a non-cleavable UmuD protein. Because UmuD cleavage is a prerequisite for mutagenesis, the lack of UmuD processing appears to be the molecular basis for the non-mutable phenotype in these strains. These studies re-emphasize the critical nature of the RecA-mediated cleavage of UmuD for inducible mutagenesis and provide insights into the functional domains of the UmuC protein.

Bacterial Proteins↗

PubMed2006

ErmK leader peptide : amino acid sequence critical for induction by erythromycin.

The ermK gene from Bacillus lichenformis encodes an inducible rRNA methylase that confers resistance to the macrolide-lincosamide-streptogramin B antibiotics. The ermK mRNA leader sequence has a total length of 357 nucleotides and encodes a 14-amino acid leader peptide together with its ribosome binding site. The secondary structure of ermK leader mRNA and a leader peptide sequence have been reported as the elements that control expression. In this study, the contribution of specific leader peptide amino acid residues to induction of ermK was studied using the PCR-based megaprimer mutation method. ermK methylases with altered leader peptide codons were translationally fused to E. coil beta-galactosidase reporter gene. The deletion of the codons for Thr-2 through Ser-4 reduced inducibility by erythromycin, whereas that for Thr-2 and His-3 was not. The replacement of the individual codons for Ser-4, Met-5 and Arg-6 with termination codon led to loss of inducibility, but stop mutation of codon Phe-9 restored inducibility by erythromycin. Collectively, these findings suggest that the codons for residue 4, 5 and 6 comprise the critical region for induction. The stop mutation at Leu-7 expressed constitutively ermK gene. Thus, ribosome stalling at codon 7 appears to be important for ermK induction.

Amino Acid Sequence↗

PubMed1984

An effect of codon context on the mistranslation of UGU codons in vitro.

Effects of codon context on nonsense codon suppression may act either through release factor recognition of termination codons or aminoacyl-tRNA selection by the ribosome. The latter hypothesis has been studied by comparing misreading by Escherichia coli UGA suppressor tryptophan tRNA of UGU (cysteine) codons in two synthetic polymers, poly(U-G) and poly( U5 , G), which differ in sequence around the UGU codons. In vitro translation of these polymers in a cell-free system from E. coli yielded selection errors of 4 X 10(-3) and 1.75 X 10(-2) for UGU codons in poly(U-G) and poly( U5 , G), respectively. This difference suggests that codon context may significantly affect misincorporation of amino acids into protein.

Codon↗

PubMed1975

Yeast super-suppressors are altered tRNAs capable of translating a nonsense codon in vitro.

tRNA isolated from two different yeast super-suppressor strains translates a known nonsense mutation in vitro, whereas tRNA from a closely related nonsuppressing strain does not. Suppression was assayed by translation of RNA isolated from an amber coat mutant of bacteriophage Qbeta (GB11) in a protein-synthesizing system derived from mouse tissue culture cells (L cells). Suppressed forms of Qbeta coat protein synthesized in vitro were quantitatively detected by a specific immunoprecipitation assay. The L-cell protein-synthesizing system also responds to E. coli suppressor tRNA. This indicates that the biochemical mechanism for nonsense suppression is very similar in yeast and E. coli. These findings also provide additional evidence that the amber codon (UAG) functions as one of the mammalian chain-terminating codons. Since the suppression assay utilizes protein-synthesizing components isolated from mammalian cells, it should prove useful in the search for mammalian nonsense suppressors.

Codon↗