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At least 505 records · Page 28Linked to original sources
PubMed1980

Nucleotide sequence of human preproinsulin complementary DNA.

Recombinant bacterial plasmids that contain DNA complementary to human preproinsulin messenger RNA have been constructed. One clone contains the entire preproinsulin coding region, as well as the 3' untranslated region of the messenger RNA and eight nucleotides of the 5' untranslated region. Additional sequence information for the 5' untranslated region was obtained with the use of insulinoma messenger RNA in conjunction with specific primers from the cloned DNA for enzymatic chain termination sequence analysis. The results confirm the amino acid sequence of human proinsulin previously determined, and predict the amino acid sequence of the human preproinsulin signal peptide.

Amino Acid Sequence↗
PubMed1994

Characterization of a tobacco gene encoding a pollen-specific polygalacturonase.

We report here the isolation and characterization of a gene which is specifically expressed during late pollen development in Nicotiana tabacum L. cv. Havana and which exhibits homology to bacterial, fungal and plant polygalacturonases. This gene is ca. 4.3 kb, from the transcription start-site to the 3' polyadenylation-site sequences. It contains three introns of 620, 706 and 1400 bp and encodes a 1.5 kb message that contains an A-rich 5'-untranslated-leader sequence of 81 bases and a variable-length 3'-untranslated sequence of between 180 and 320 bases. Located within intron 3 is a 414 bp sequence which exhibits 79% homology to a sequence within the endochitinase gene; both sequences share the same internal repeat structure and exhibit features consistent with them being defective transposable elements. The predicted protein sequence coded for by Npg1 shows, in addition to a number of highly conserved cysteines, four conserved domains with the bacterial and fungal polygalacturonase genes. The pollen-specific polygalacturonases as a group can be distinguished from the fruit-ripening polygalacturonases by a number of criteria. It is suggested that these differences reflect the functional differences between plant endo- and exo-polygalacturonases. Npg1 is one of a two-member gene family expressed predominantly in the male gametophyte upon first microspore mitosis. From expression studies of promoter::GUS transgenes it is clear that the -744 bp to +74/+85 bp of Npg1 sequence (with respect to the transcription start site) is sufficient to drive the expression of the GUS reporter gene in a manner that reflects the spatial and temporal expression of Npg1 as determined by dot-blot and northern analysis.

Amino Acid Sequence↗
PubMed1997

Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins: addition of a hemagglutinin epitope does not affect calmodulin-dependent activation of smooth muscle myosin light chain kinase.

An epitope-tagged calmodulin (CaM), capable of interacting with CaM-binding proteins in cellular extracts, would be a valuable tool for identifying proteins in signal transduction pathways involving calcium. A bacterial overexpression vector for epitope-tagged CaM has been constructed by inserting the coding sequence for a nine amino acid portion of the influenza virus hemagglutinin (HA) protein into the initiation site of an overexpression vector for chicken CaM. The HA-CaM fusion produced in bacteria was compared to native CaM for its ability to activate smooth muscle myosin light chain kinase (MLCK), one of the best understood CaM-dependent enzymes. MLCK activity was tested in both a purified system and a CaM-depleted "native actomyosin" preparation maintaining many of the regulatory properties of the intact smooth muscle. HA-CaM behaves identically to unmodified CaM in both systems, indicating that the HA epitope does not adversely affect CaM function. The recombinant HA-CaM was used to sensitively detect CaM interactions with smooth muscle proteins in a modified gel overlay assay, using a monoclonal antibody against the HA epitope as the secondary reagent. Enzymatically active complexes of HA-CaM and MLCK could be immunoprecipitated from actomyosin preparations using the same monoclonal antibody and protein G-Sepharose beads.

Actomyosin↗
PubMed1993

On concerted origin of transfer RNAs with complementary anticodons.

Pairs of antiparallely oriented consensus tRNAs with complementary anticodons show surprisingly small numbers of mispairings within the 17-bp- long anticodon stem and loop region. Even smaller such complementary distances are shown by illegitimately complementary anticodons, i.e. those with allowed pairing between G and U bases. Accordingly, we suppose that transfer RNAs have emerged concertedly as complementary strands of primordial double helix-like RNA molecules. Replication of such molecules with illegitimately complementary anticodons might generate new synonymous codons for the same pair of amino acids. Logically, the idea of tRNA concerted origin dictates very ancient establishment of direct links between anticodons and the type of amino acids with which pre-tRNAs were to be charged. More specifically, anticodons (first of all, the 2nd base) could selectively target 'their' amino acids, reaction of acylating itself being performed by another non-specific site of pre-tRNA or even by another ribozyme. In all, the above findings and speculations are consistent to the hypercyclic concept (Eigen and Schuster, 1979), and throw new light on the genetic code origin and associated problems. Also favoring this idea are data on complementary codon usage patterns in different genomes.

Amino Acids↗
PubMed1987

Translation framing code and frame-monitoring mechanism as suggested by the analysis of mRNA and 16 S rRNA nucleotide sequences.

Protein coding sequences carry an additional message in the form of a universal three-base periodical pattern (G-non-G-N)n, which is expressed as a strong preference for guanines in the first positions of the codons in mRNA and lack of guanines in the second positions. This periodicity appears immediately after the initiation codon and is maintained along the mRNA as far as the termination triplet, where it disappears abruptly. Known cases of ribosome slippage during translation (leaky frameshifts, out-of-frame gene fusion) are analyzed. At the sites of the slippage the G-periodical pattern is found to be interrupted. It reappears downstream from the slippage sites, in a new frame that corresponds to the new translation frame. This suggests that the (G-non-G-N)n pattern in the mRNA may be responsible for monitoring the correct reading frame during translation. Several sites with complementary C-periodical structure are found in the Escherichia coli 16 S rRNA sequence. Only three of them are exposed to various interactions at the surface of the small ribosomal subunit: (517)gcCagCagCegC, (1395)caCacCgcC and (1531)auCacCucC. A model of a frame-monitoring mechanism is suggested based on the weak complementarity of G-periodical mRNA to the C-periodical sites in the ribosomal RNA. The model is strongly supported by the fact that the hypothetical frame-monitoring sites in the 16 S rRNA that are derived from the nucleotide sequence analysis are also the only sites known to be actually involved or implicated in rRNA-mRNA interactions.

Base Sequence↗
PubMed1985

Expression in bacteria of gB-glycoprotein-coding sequences of Herpes simplex virus type 2.

A plasmid with an insert that encodes the glycoprotein B(gB) gene of Herpes simplex virus type 2 (HSV-2) has been isolated. DNA sequences coding for a portion of the HSV-2 gB peptide were cloned into a bacterial lacZ alpha expression vector and used to transform Escherichia coli. Upon induction of lacZpo-promoted transcription, some of the bacteria became filamentous and produced inclusion bodies containing a large amount of a 65-kDal peptide that was shown to be precipitated by broad-spectrum antibodies to HSV-2 and HSV-1. The HSV-2 insert of one of these clones specifies amino acid residues corresponding to 135 through 629 of the gB of HSV-1 [Bzik et al., Virology 133 (1984) 301-314].

Antibodies, Viral↗
PubMed2005

Transcription start sites in the promoter region of the Escherichia coli pcnB (plasmid copy number) gene coding for poly(A) polymerase I.

Escherichia coli pcnB gene was discovered as a regulator of ColE1 plasmid copy number about 20 years ago. Further studies indicated that this gene codes for poly(A) polymerase I (PAP I). However, although importance of RNA polyadenylation has been demonstrated and biochemical properties of PAP I have been studied extensively, little is known about regulation of the pcnB gene transcription. Here, we demonstrate that there are three transcription start sites upstream of the pcnB coding sequence. Activities of at least two of these promoters were in inverse correlation to bacterial growth rates, which agrees with recently published results indicating increased abundance of PAP I and higher levels of RNA polyadenylation in slowly growing bacteria.

Base Sequence↗
PubMed2002

Expression of human vasopressin and oxytocin receptors in Escherichia coli.

In order to produce large amounts of human vasopressin and oxytocin receptors compatible with direct structural biology approaches such as X-ray crystallography, NMR or mass spectrometry, we have expressed these neurohypophysial hormone receptors in Escherichia coli. To facilitate the level of expression, the coding sequence for the V1a vasopressin receptor and the oxytocin receptor were first optimized for bacterial expression. The resulting 'bacterial receptor cDNAs' were then subcloned into pET/T7-driven prokaryotic expression vectors. Different constructs have been prepared: each cDNA was incorporated alone or in fusion with a T7 tag sequence or a glutathione-S-transferase tag sequence at the N-terminus end. Moreover, a 6 x His tag sequence has been added at the C-terminus end for one-step purification of the receptors. Screening of BL21(DE3) and BL21(DE3)pLysS bacterial strains transformed with the different constructions was achieved by Coomassie blue-stained SDS-polyacrylamide gels and by 6 x His antibody Western blotting. Several clones were selected for purification of the receptors. Expression levels of the receptors are now encouraging and will be optimized for further structural and functional studies. Moreover, at the same time, the construction of the bacterial-optimized sequence of the V2 vasopressin receptor and its expression will be performed.

Amino Acid Sequence↗
PubMed1980

Predicting coding function from nucleotide sequence or survival of "fitness" of tRNA.

The sequence of a nucleotide region of f1 bacteriophage was determined on a bonded ultrathin acrylamide gel with a discontinuous buffer system by using the dideoxy-DNA sequencing method. This sequence and one other were analyzed for maximal base pairing with tRNAs. The results allow a prediction of the direction and phase of possible coding functions. The implication of sequence constraints on mRNA codon frequency, tRNA structure, the origin of protein synthesis, and triplet reading are discussed in terms of neutral, Darwinian, and genotypic selectionist perspectives of evolution. The model of F. H. C. Crick, S. Brenner, A. Klug, and G. Pieczenik [(1976) Origins of Life 7, 389-397] for the origin of the genetic code is used to interpet contemporary adaptive and functional nucleic acid sequences.

Anticodon↗
PubMed1982

The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection.

We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both S1 nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.

Acetyltransferases↗
PubMed2001

Alternate paradigm for intrinsic transcription termination in eubacteria.

Intrinsic transcription terminators are functionally defined as sites that bring about termination in vitro with purified RNA polymerase alone. Based on studies in Escherichia coli, intrinsic termination requires a palindromic stretch followed by a trail of T (or U) residues in the coding strand. We have developed a highly efficient algorithm to identify hairpin potential sequences in bacterial genomes in order to build a general model for intrinsic transcription termination. The algorithm was applied to analyze the Mycobacterium tuberculosis genome. We find that hairpin potential sequences are concentrated in the immediate downstream of stop codons. However, most of these structures either lack the U trail entirely or have a mixed A/U trail reflecting an evolutionarily relaxed requirement for the U trail in the mycobacterial genome. Predicted atypical structures were shown to work efficiently as terminators both inside the mycobacterial cell and in vitro with purified RNA polymerase. The results are discussed in light of the kinetic competition models for transcription termination. The algorithm identifies >90% of experimentally tested terminators in bacteria and is an invaluable tool in identifying transcription units in whole genomes.

Algorithms↗
PubMed1996

Accumulation of a mRNA decay intermediate by ribosomal pausing at a stop codon.

A RNA fragment which is protected from degradation by ribosome pausing at a stop codon has been identified in growing Escherichia coli. The fragment is 261 nt long and corresponds to the 3'-end of the mRNA expressed from a semi-synthetic model gene. The 5'-end of the RNA fragment, denoted rpRNA (ribosomal pause RNA), is located 13 bases upstream of the stop codon. In vivo decay of the complete mRNA and accumulation of rpRNA are dependent on the nature of the stop codon and its codon context. The data indicate that the rpRNA fragment arises from interrupted decay of the S3A'mRNA in the 5'-->m3'direction, in connection with a ribosomal pause at the stop codon. RF-2 decoding of UGA is less efficient than RF-1 decoding of UAG in identical codon contexts, as judged from rpRNA steady-state levels. The half-life of UGA-containing rpRNAs is at least 5 min, indicating that ribosomal pausing can be a major factor in stabilising downstream regions of messenger RNAs.

Base Sequence↗