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PubMed1985

A bidirectionally active signal for termination of transcription is located between tetA and orfL on transposon Tn10.

A terminator of transcription with bidirectional activity has been located between the translation termination codons of the genes tetA and orfL on Tn10. These genes are transcribed towards each other. Each orientation of the intervening sequence is shown to reduce the expression of the lacZ and galK genes when cloned between the respective structural gene and its promotor. The 3'ends of the respective mRNAs were determined by S1 mapping. The results confirm that the same sequence capable of forming a stem-loop structure with a GC rich stem is the termination signal for both orientations. In the more efficient tetA orientation (99%-96% reduction of expression) this sequence is followed by a run of six thymines. In the less efficient orfL orientation (96%-78% reduction of expression) it is followed by an AT rich sequence with seven thymines out of eleven base pairs.

Bacterial Proteins↗

PubMed1987

Nucleotide sequence and in vitro expression of rubella virus 24S subgenomic messenger RNA encoding the structural proteins E1, E2 and C.

The complete nucleotide sequence of the 24S subgenomic mRNA of wild-type M33 strain rubella virus has been determined. This RNA is 3,383 nucleotides in length excluding the 3'-terminal poly(A) tract. After the three multiple in-phase termination codons clustered in the 5' terminus of this RNA, there are 81 nucleotides of nontranslated nucleic acid followed by a reading frame of 2,978 nucleotides that encodes the 110 kD precursor of the structural proteins. The 3'-untranslated region is 263 nucleotides. The 110 kD polyprotein is processed to produce nucleocapsid C, the glycoproteins E2 and E1 in that order. Sites of post-translational cleavage to produce E2 and E1 were located using available N-terminal amino acid sequences. RNAs synthesized by transcription in vitro are effective messengers in the rabbit reticulocyte cell-free translation system. Post-translational processing of the structural proteins was observed in the cell-free system supplemented with microsomes from dog pancreas.

Amino Acid Sequence↗

PubMed1991

The location of the 5' end of the potato leafroll luteovirus subgenomic coat protein mRNA.

Northern blot analysis of nucleic acid from potato plant tissues and tobacco protoplasts infected with a Scottish isolate of potato leafroll luteovirus (PLRV) detected the 6 kb genomic RNA and one subgenomic RNA species of about 2.7 kb; RNA extracted from virus particles contained only the genomic species. Blotting with small defined probes suggested that the location of the 5' end of the subgenomic RNA was between 2380 and 2510 nucleotides from the 3' end of the PLRV genome (between 3370 and 3500 nucleotides from the 5' end of PLRV Dutch isolate RNA). When RNA extracted from PLRV-infected or mock-inoculated protoplasts was used as the template for primer extension using primers complementary to the sequence at, or upstream of, the initiation codon of the coat protein gene, a single major infection-specific product was detected. A primer complementary to the sequence between 162 and 179 nucleotides upstream of the coat protein AUG yielded a product of 56 nucleotides. Thus, the subgenomic RNA has a leader sequence of 212 nucleotides, is 2505 nucleotides in length and starts at a position equivalent to 3376 nucleotides from the 5' end of the PLRV-Dutch genome, 11 nucleotides upstream of the termination codon of the putative polymerase gene. The nucleotide sequence immediately downstream of this position closely resembles that of the 5' end of the PLRV genomic RNA.

Base Sequence↗

PubMed1991

Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis.

Bordetella pertussis produces a porin protein which is a prominent outer membrane component found in both virulent and avirulent strains. N-terminal amino acid analysis of purified B. pertussis porin was performed and this amino acid sequence was used to design an oligonucleotide that was then utilized to screen a lambda gt11 library containing randomly sheared fragments of DNA from B. pertussis strain 347. One clone, lambda BpPor, was identified and subcloned into pUC18. A portion of the DNA insert in this subclone, pBpPor1, was sequenced and shown to contain the N-terminal region of the structural porin gene. This truncated gene sequence was used to design an additional oligonucleotide that was used to identify a clone, pBpPor2, which overlapped with pBpPor1 and contained a termination codon. The structural gene deduced from this sequence would encode a 365-amino-acid polypeptide with a predicted mass of 39,103 daltons. The predicted product also contains a signal sequence of 20 residues that is similar to that found in other porin genes. The predicted B. pertussis porin protein sequence contains regions that are homologous to regions found in porins expressed by Neisseria species and Escherichia coli, including the presence of phenylalanine as the carboxy-terminal amino acid. DNA hybridization studies indicated that both virulent and avirulent strains of B. pertussis contain only one copy of this gene and that Bordetella bronchiseptica and Bordetella parapertussis contain a similar gene.

Amino Acid Sequence↗

PubMed1988

Molecular cloning and characterization of the human dbl proto-oncogene: evidence that its overexpression is sufficient to transform NIH/3T3 cells.

We isolated cDNA clones representing the human dbl proto-oncogene transcript. Nucleotide sequence analysis revealed an open reading frame encoding a predicted protein of 925 amino acids. Using peptide antisera directed against specific proto-dbl peptides, a 115-kd protein was detected in COS cells transfected with an expression vector containing the entire coding region of proto-dbl. This mol. wt is consistent with that predicted from the open reading frame. We have previously shown that the dbl oncogene was generated by substitution of the 5' portion of proto-dbl with an unrelated human sequence. In this study we show that this rearrangement resulted in the loss of the 497 amino-terminal codons of the dbl proto-oncogene. Under the influence of a strong promoter proto-dbl could readily transform NIH/3T3 cells but its transforming activity was less than that of the dbl oncogene driven by the same promoter. Proto-dbl overexpression is, therefore, sufficient to transform NIH/3T3 cells, but specific structural alterations of its coding region significantly enhance its transforming activity. No apparent similarity was detected between the predicted proto-dbl product and other known proto-oncogenes. However, a stretch of 300 amino acids within the N-terminal half of proto-dbl showed structural similarity to the intermediate filament vimentin. This region in proto-dbl contains a heptad repeat motif characteristic of an alpha-helical coiled-coil structure. Taken together, these findings indicate that the human proto-dbl represents a new class of cellular oncogenes that may be related to cytoskeletal elements of the cell.

Amino Acid Sequence↗

PubMed1989

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis.

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗

PubMed1998

An efficient system for site-directed mutagenesis to make various mutants of the env gene of human immunodeficiency virus type 1.

We developed an efficient system of site-directed mutagenesis for the envelope (env) gene of human immunodeficiency virus type 1 (HIV-1). To make a template plasmid for mutagenesis, pS+B/MluI, two independent selection markers, i.e. a unique restriction site, MluI, and an in-frame termination codon, were introduced into the region encoding the V3 domain of the env gene of an HIV-1 strain, NL4-3, which had been cloned in the pUC118 plasmid. When the env gene of the pS+B/MluI plasmid was mutated successfully using mutagenic primers such as synthetic oligonucleotides or PCR-amplified DNA fragments longer than 1.5 kbp, the plasmids became resistant to digestion with MluI and competent env genes were formed by suppression of the in-frame termination. Various site-directed mutants of the env gene of HIV-1 were accurately constructed in a short time even in the absence of proper restriction sites by this system. The system of site-directed mutagenesis we reported here will be a useful method to analyze the functions of variable genes like the env gene of HIV-1 precisely and rapidly.

Amino Acid Sequence↗

PubMed1988

Cloning, sequence, and expression of the Drosophila cAMP-dependent protein kinase catalytic subunit gene.

Genomic DNA containing the protein coding region for Drosophila cAMP-dependent protein kinase catalytic subunit has been cloned and sequenced. The probe used to detect and isolate the gene fragment was constructed from two partially complementary synthetic oligonucleotides and contains 60 base pairs that encode (using Drosophila codon preferences) amino acids 195-214 of the beef heart catalytic subunit. In reduced stringency hybridization conditions, the probe recognizes two target sites in fly genomic DNA with 85% homology. One of these sites is in the cAMP-dependent protein kinase catalytic subunit gene, which was isolated as a 3959-base pair HindIII fragment. This fragment contains all of the protein coding portion, 900 base pairs upstream of the initiator ATG, and 2000 base pairs downstream of the termination codon (TAG). The coding portion of the gene contains no introns and yields a protein of 352 amino acids. There is a 2-amino acid insertion near the N terminus of the fly protein relative to the beef and mouse enzymes. Of the remaining 350 amino acids, 273 are invariant in the three species. A probe derived from the coding sequence of the HindIII clone hybridizes strongly to a 5100-base poly(A)+ RNA and weakly to 4100- and 3400-base poly(A)+ RNAs expressed in adult flies. A 2100-base pair EcoRI genomic fragment containing the second site recognized by the 60-base pair probe has also been cloned. DNA sequence analysis demonstrates that this fragment is part of the cGMP-dependent protein kinase gene or a close homolog. The catalytic subunit gene and the cGMP-dependent protein kinase gene have been located in regions 30C and 21D, respectively, of chromosome 2.

Amino Acid Sequence↗

PubMed1986

Characterization of four constant region genes of rabbit immunoglobulin-lambda chains.

A cDNA plasmid insert encoding the constant (C) region of a rabbit immunoglobulin-lambda light chain was used as a probe for screening a rabbit liver genomic DNA cosmid library. This allowed the isolation and identification of four distinct C lambda genes, designated C lambda 1, C lambda 2, C lambda 3, and C lambda 4, which were shown to be widely separated from each other along chromosomal DNA. Their nucleotide sequences have been determined. No in-frame termination codons were found within the coding regions. The C lambda 1, C lambda 2, and C lambda 3 sequences are quite similar to each other, but share less homology with the C lambda 4 gene or the cDNA-C lambda sequence used as a probe. The C lambda gene coding for the cDNA sequence was not isolated. Translation of the C lambda 1, C lambda 2, and C lambda 3 sequences predicts a Cys-Pro carboxy-terminal amino acid sequence, as found so far only for horse lambda-chains. Compared to the other rabbit C lambda genes, the C lambda 3 sequence exhibits two deletions, one of 9 bp, the other of 3 bp. The latter occurs at the same position as in the mouse C lambda 2 and C lambda 3 genes. These two deletions are located in the loops between anti-parallel beta-pleated sheets of the C lambda domain. When the C lambda nucleotide sequences from man, mouse, and rabbit are compared, there is less divergence within the same species than for interspecies comparisons. Possible genetic implications of this finding are discussed.

Amino Acid Sequence↗

PubMed1981

The primary structures of two yeast enolase genes. Homology between the 5' noncoding flanking regions of yeast enolase and glyceraldehyde-3-phosphate dehydrogenase genes.

Segments of yeast genomic DNA containing two enolase structural genes have been isolated by subculture cloning procedures using a cDNA hybridization probe synthesized from purified yeast enolase mRNA. Based on restriction endonuclease and transcriptional maps of these two segments of yeast DNA, each hybrid plasmid contains a region of extensive nucleotide sequence homology which forms hybrids with the cDNA probe. The DNA sequences which flank this homologous region in the two hybrid plasmids are nonhomologous indicating that these sequences are nontandemly repeated in the yeast genome. The complete nucleotide sequence of the coding as well as the flanking noncoding regions of these genes has been determined. The amino acid sequence predicted from one reading frame of both structural genes is extremely similar to that determined for yeast enolase (Chin, C. C. Q., Brewer, J. M., Eckard, E., and Wold, F. (1981) J. Biol. Chem. 256, 1370-1376), confirming that these isolated structural genes encode yeast enolase. The nucleotide sequences of the coding regions of the genes are approximately 95% homologous, and neither gene contains an intervening sequence. Codon utilization in the enolase genes follows the same biased pattern previously described for two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes (Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596-2605). DNA blotting analysis confirmed that the isolated segments of yeast DNA are colinear with yeast genomic DNA and that there are two nontandemly repeated enolase genes per haploid yeast genome. The noncoding portions of the two enolase genes adjacent to the initiation and termination codons are approximately 70% homologous and contain sequences thought to be involved in the synthesis and processing messenger RNA. Finally there are regions of extensive homology between the two enolase structural genes and two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes within the 5- noncoding portions of these glycolytic genes.

Base Sequence↗

PubMed1987

Sequence elements essential for rho-dependent transcription termination at lambda tR1.

To determine the location of DNA sequences required for the utilization of rho factor in transcription termination at the tR1 terminator of phage lambda, we constructed and analyzed a series of deletion mutants affecting several distinct regions of the cro-gene sequence. Two distinct sequence blocks, rutA and rutB, are shown to be particularly important for rho action at tR1 during in vitro transcription. They are located in the region between the sequence encoding the translation termination codon of cro mRNA and the start of tR1. Although other sequences contribute to rho action, their function can be replaced by unrelated heterologous DNA sequences encoding highly structured RNA segments, while the function of the rut sequences cannot. The lambda tR1 rut sequences encode RNA segments that contain a high proportion of cytidylate residues and that lack extensive intramolecular base pairing. Thus, the rut transcript segments are likely to be parts of the site on the nascent cro RNA that binds specifically with rho factor as an essential step in the termination process. This view is supported further by the findings (Chen, C.-Y. A., Galluppi, G. R., and Richardson, J. P. (1986) Cell 46, 1023-1028) that rho action at tR1 is specifically inhibited by DNA oligonucleotides that form hybrid helices with rut transcript segments. We also examined the effect of the auxiliary transcription factor NusA on transcription of our mutant templates and show that deletion of the boxA sequence of cro gene does not diminish the ability of NusA to reduce rho action at tR1.

Bacterial Proteins↗

PubMed1988

Rat testis P-450(17)alpha cDNA: the deduced amino acid sequence, expression and secondary structural configuration.

A complete amino acid sequence for rat testis P-450(17)alpha was deduced from nucleotide analysis of a cDNA clone isolated from a rat Leydig cell cDNA library. This DNA clone, containing initiation and termination codons and a polyA tail, translated a polypeptide in COS-1 cells that expressed both 17 alpha-hydroxylase and 17,20 lyase activities. It exhibited significant similarity to the nucleotide and deduced amino acid sequences of the bovine and human cytochrome P-450(17)alpha, particularly with respect to the highly conserved regions and secondary structure. The P-450(17)alpha appears to be anchored to the membrane of the endoplasmic reticulum through two transmembrane regions, specifically the N terminal insertion peptide and the stop-transfer sequence. Hydropathic analysis indicates that the remainder of the C terminus is associated with the membrane through four hydrophobic clefts, including the putative steroid binding site.

Amino Acid Sequence↗

PubMed2002

Multiple mechanisms for degradation of bacteriophage T4 soc mRNA.

The dmd gene of bacteriophage T4 is required for regulation of mRNA stability in a stage-dependent manner during infection. When this gene is mutated, late genes are globally silenced because of rapid degradation of mRNAs. To investigate the mechanism of such mRNA degradation, we analyzed the late gene soc transcripts. The degradation of soc mRNA was remarkably stabilized when its ability to be translated was impaired; either disruption of translation initiation signals or elimination of termination codons was effective in stabilization of soc mRNA and removal of elongation modestly stabilized it. Even in the absence of translation, however, the residual activity was still significant. These results suggested that the degradation of soc transcripts was promoted by two different mechanisms; one is dependent on translation and the other independent of translation. We found several cleavages introduced into soc RNA specifically when the dmd gene was mutated; some of them could be linked to polypeptide chain elongation and termination, suggesting the correlation with ribosomal action, and the others were independent of translation.

Bacteriophage T4↗

PubMed1991

Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant.

A heterologous system expressing functional Sindbis virus nonstructural proteins (nsPs) has several possible uses for studying Sindbis virus-specific RNA replication and transcription in vivo and in vitro. Of the many possible approaches, vaccinia virus offers an attractive transient expression system given that Sindbis virus replication can occur in cells which have been previously infected by vaccinia virus. In this report, a vaccinia virus recombinant (called vSINNS), which contains the cDNA encoding the Sindbis virus nsPs under the control of either the vaccinia virus 7.5K promoter or the bacteriophage T7 promoter, has been constructed and characterized. Upon infection of several cell types with vSINNS, Sindbis virus nsP precursors and processed forms, including nsP1, nsP2, and both phosphorylated and nonphosphorylated forms of nsP3, were synthesized. Proteins containing the putative RNA-dependent RNA polymerase domain (nsP4 and nsP34), which are normally produced in small amounts by readthrough of an opal termination codon, were not detected in vSINNS-infected cells. However, all nsP functions necessary for Sindbis virus-specific RNA synthesis must have been expressed, since both replication and subgenomic mRNA transcription of an engineered Sindbis virus defective interfering RNA in cells infected with vSINNS was observed. Furthermore, vSINNS could be used as a helper virus to amplify, to relatively high titers, a replication-defective Sindbis virus mutant containing an in-frame deletion in the conserved N-terminal domain of nsP3. These data, as well as the observation that normal yields of parental Sindbis virus are produced in cells which have been previously infected with vSINNS, indicate that expression of Sindbis virus nsPs, in the absence of Sindbis virus-specific RNA replication, is not sufficient to block the formation of active RNA replication complexes by superinfecting Sindbis virus.

Animals↗

PubMed1984

Characterization of viable mutants of polyomavirus cold sensitive for maintenance of cell transformation.

We mutagenized a cloned fragment of polyoma DNA encoding portions of the middle size (MT) and large T antigens. We regenerated infectious viral genomes containing the mutagenized DNA and tested their transforming ability at 32 and 39 degrees C. We isolated three nontransforming mutants and two mutants which were cold sensitive for the maintenance of cell transformation. The nontransforming mutants contained amber termination codons in the reading frame for the MT antigen. They synthesized truncated MT antigens which lacked MT-associated protein kinase activity. The cold-sensitive mutants synthesized MT antigens indistinguishable from wild type with regard to size, stability at 32 and 39 degrees C, intracellular location, and associated protein kinase activity. One of the mutants was shown by nucleotide sequence analysis to contain a single amino acid change in the MT antigen, located two residues upstream from the C-terminal hydrophobic region, and no changes in the large T antigen. The other mutant contained two amino acid changes in the MT antigen and two amino acid changes in the large T antigen.

Animals↗

PubMed2000

Relative quantitation of mRNA in beta-thalassemia/Hb E using real-time polymerase chain reaction.

beta-Thalassemia and Hb E patients, with seemingly identical genotypes, have a remarkable variability in severity. Reduction in red cell survival in beta-thalassemia is correlated with the amount of intracellular unmatched alpha-globin chains. However, it was only recently realized that mRNA, whose translation is prematurely terminated, is also unstable. No systematic attempts have been made to investigate mRNA stability in beta-thalassemia arising from nonsense mutations located upstream from the normal termination codon. In this study, one-step real-time polymerase chain reaction has been employed to compare the levels of alpha- and beta-globin mRNA in reticulocytes from beta-thalassemia/Hb E subjects. The results showed the highest alpha/beta-globin mRNA ratio (median = 5.70, n = 13) in frameshift codons 41/42 (-TTCT)/Hb E individuals compared to normal subjects (median = 1.02, n = 6), or those with Hb E trait (median = 2.15, n = 8). In addition, there was a concomitant increase in the alpha/beta-globin mRNA ratio with decrease in hemoglobin level, i.e., increase in severity. The difference in the ratio among beta-thalassemia/Hb E patients with the same genotype may be attributed to individual variations of efficiency in betaE-globin mRNA splicing and in the destruction of prematurely terminated mRNA.

Adult↗

PubMed1985

Deletion of C-terminal amino acid codons of PhiX174 gene E: effect on its lysis inducing properties.

The lysis gene E of bacteriophage PhiX174 has been subjected to deletion and fusion analysis. Deletions of 11 to 90% of gene E specific nucleotides coding for to C-terminal region of the gene product were cloned under transcriptional control of lambda pL. For this purpose plasmid pSU1 was constructed which carries an extended polylinker region downstream of pL. Depending on the number of nucleotides after the last gene E specific codon, various C-terminal segments of protein E were replaced by 4, 5, 53 or 314 unrelated amino acids. Functional analysis for lysis inducing properties of the various gene E mutants revealed that the final 9 codons of the gene could be deleted without loss of function. However, replacements of 19 or more C-terminal codons eliminated gene E activity. Although the functional site of the gene E product is located within the N-terminal half of the polypeptide, the C-terminal part of the protein appears to exhibit severe influence on conformation and/or regulation of the functional site.

Amino Acid Sequence↗

PubMed1989

A nonsense mutation in the apolipoprotein C-IIPadova gene in a patient with apolipoprotein C-II deficiency.

The apo C-II gene from a patient with apo C-II deficiency has been sequenced after amplification by the polymerase chain reaction. A substitution of an adenosine for a guanosine at position 3002 in exon 3 of the patient's gene was identified by sequence analysis. This mutation leads to the introduction of a premature termination codon (TAA) at a position corresponding to amino acid 37 of mature apo C-II and to the formation of a new Rsa I restriction enzyme site not present in the normal apo C-II gene. Amplification of DNA from family members by the polymerase chain reaction and digestion with Rsa I established that the patient is a true homozygote for the mutation. Analysis of the patient's plasma by two-dimensional gel electrophoresis and immunoblotting detected an apo C-II that exhibited abnormal electrophoretic mobility. We propose that the C to A substitution in the apo C-IIPadova gene is the primary genetic defect that leads to premature termination and the synthesis of a truncated 36 amino acid apo C-II that is unable to activate lipoprotein lipase.

Adenosine↗