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At least 253 records · Page 14Linked to original sources
PubMed1993

A cytoplasmic actin gene from the silkworm Bombyx mori is expressed in tissues of endodermal origin and previtellogenic germ cells of transgenic Drosophila.

A cytoplasmic actin gene from Bombyx mori introduced into Drosophila melanogaster by P-element mediated transformation, is efficiently transcribed in larvae, pupae and adults of the host. The exogenous mRNA has the same size as the one observed in the Bombyx cells and the intron located within the coding region is properly excised, indicating a correct recognition of the exogenous sequences by the Drosophila transcriptional and splicing machineries. The expression of the Bombyx gene in Drosophila tissues was determined by transforming flies with a hybrid gene in which a large part of the Bombyx actin coding sequences was replaced by those of the bacterial lac Z gene. This chimaeric gene is specifically and highly expressed, from the embryo to the adult of the transgenic lines, in tissues of endodermal origin, the midgut and its derivatives, i.e. gastric caeca, the outer layer of the proventriculus, and in the Malpighian tubules. This gene is also expressed, at a lower level, in germ cells but restricted to the sixteen cell cysts during previtellogenesis. The expression of the Bombyx gene during development of transgenic flies was compared to that of the two Drosophila endogenous cytoplasmic actin genes and the results are discussed.

Actins↗
PubMed1994

Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116.

Caedibacter taeniospiralis, an obligate bacterial endosymbiont of Paramecium tetraurelia, confers a killing trait upon its host paramecium. Type 51 R bodies (refractile inclusion bodies) are synthesized by these endosymbionts and are required for expression of the killing trait. The nucleotide sequence of the genetic determinants for type 51 R body synthesis and assembly was determined for C. taeniospiralis 47 and 116. Three independently transcribed genes (rebA, rebB, and rebC) were characterized. To date these are the only genes from C. taeniospiralis to be sequenced and characterized. DNA regulatory regions are recognized by Escherichia coli, and codon usage appears similar to that in E. coli. A fourth open reading frame with appropriate regulatory sequences was found within the reb locus, but no evidence was obtained to suggest that this putative gene is expressed in E. coli. The R body-encoding sequences from both strains are identical. Two-dimensional gel electrophoresis of deletion derivatives shows that two polymerization events are involved in R body assembly. One polymerization event requires only RebB and RebC; the other requires all three proteins. Expression of RebC is necessary for the posttranslational modification of RebA and RebB into species with three and two different molecular weights, respectively. In the presence of RebC, each species of RebB with a different molecular weight has six different isoelectric points.

Amino Acid Sequence↗
PubMed1984

Expression of genes introduced into cells by retroviral infection is more efficient than that of genes introduced into cells by DNA transfection.

Calcium phosphate-mediated DNA transfection and retroviral infection are two alternative gene transfer techniques designed to introduce specific DNA fragments into the chromosomes of recipient cells. To compare the efficiency of expression of genes introduced into cells by either of these two techniques, a retrovirus-derived vector was constructed from the genome of Moloney murine leukemia virus by replacing the coding sequences of the envelope gene with the bacterial Neor gene derived from Tn5, termed rEnv-Neor. Expression of the hybrid Neor gene in NIH 3T3 cells after DNA transfection or retroviral infection was determined by measuring the steady-state levels of the corresponding cytoplasmic polyadeylated RNA species. Cells containing one copy of the integrated rEnv-Neor DNA introduced into cells by retroviral infection expressed 10- to 50-fold-higher levels of vector-specific RNA compared with cells harboring one copy of the same DNA derived by DNA transfection. Analysis of the integrated rEnv-Neor DNA with the methylation-sensitive restriction enzyme SmaI has shown that DNA integrated after DNA transfection but not after viral infection is partially methylated, predominantly in the 5' long terminal repeat, the region involved in initiation of transcription.

Animals↗
PubMedDate not supplied

[Cloning preprolactin cDNA from Pacific salmon in Escherichia coli].

Prolactin coding mRNA was shown to be a prevalent part of chum salmon (Oncorhynchus keta) pituitary poly(A)-RNA during the spawning period. Clone lambda gtPrk12 was selected from the pituitary cDNA library by means of hybridization with the prolactin probe, and a nucleotide sequence of the insertion was determined and compared to the prolactin coding sequences from rainbow trout and Pacific chinook salmon, which had been published earlier. The sequences compared exhibited a significant homology. The deduced amino acid sequence of the chum salmon prolactin differed from a sequence determined directly in a single position. The prolactin-coding sequence can be used for constructing the bacterial strain producing prolactin.

Amino Acid Sequence↗
PubMed2005

Genomic peculiarity of coding sequences and metabolic potential of probiotic Escherichia coli strain Nissle 1917 inferred from raw genome data.

Probiotic Escherichia coli strain Nissle 1917 (O6:K5:H1) is a commensal E. coli isolate that has a long tradition in medicine for the treatment of various intestinal disorders in humans. To elucidate the molecular basis of its probiotic nature, we started sequencing the genome of this organism with a whole-genome shotgun approach. A 7.8-fold coverage of the genomic sequence has been generated and is now in the finishing stage. To exploit the genome data as early as possible and to generate hypotheses for functional studies, the unfinished sequencing data were analyzed in this work using a new method [Sun, J., Zeng, A.P., 2004. IdentiCS--identification of coding sequence and in silico reconstruction of the metabolic network directly from unannotated low-coverage bacterial genome sequence. BMC Bioinformatics 5, 112] which is particularly suitable for the prediction of coding sequences (CDSs) from unannotated genome sequence. The CDSs predicted for E. coli Nissle 1917 were compared with those of all five other sequenced E. coli strains (E. coli K-12 MG1655, E. coli K-12 W3110, E. coli CFT073, EHEC O157:H7 EDL933 and EHEC O157:H7 Sakai) published to date. Five thousand one hundred and ninety-two CDSs were predicted for E. coli Nissle 1917, of which 1065 were assigned with enzyme EC numbers. The comparison of all predicted CDSs of E. coli Nissle 1917 to the other E. coli strains revealed 108 CDSs specific for this isolate. They are organized as four big genome islands and many other smaller gene clusters. Based on CDSs with EC numbers for enzymes, the potential metabolic network of Nissle 1917 was reconstructed and compared to those of the other five E. coli strains. Overall, the comparative genomic analysis sheds light on the genomic peculiarity of the probiotic E. coli strain Nissle 1917 and is helpful for designing further functional studies long before the sequencing project is completely finished.

Chromosome Mapping↗
PubMed1990

Coding and noncoding sequences at the 3' end of yeast histone H2B mRNA confer cell cycle regulation.

Yeast (Saccharomyces cerevisiae) histone mRNA synthesis is tightly regulated to the S phase of the cell division cycle as a result of both transcriptional and posttranscriptional regulation. We focused on the role of posttranscriptional control in histone H2B1 gene (HTB1) regulation and studied a portion of the HTB1 message required for cell-cycle-specific accumulation. The 3' end of the HTB1 gene containing a 17-amino-acid coding sequence and entire noncoding sequence was fused to the bacterial neomycin phosphotransferase II gene (neo) under control of the GAL1 promoter. The expression of the endogenous and chimeric HTB1 genes was analyzed during the yeast cell cycle. As yeast cells entered a synchronous cell cycle following release from alpha-factor arrest, the level of GAL1-promoter-controlled neo-HTB1 message increased approximately 12-fold during S phase and dropped to basal level when the cells left S phase. This indicates that the 3' end of the HTB1 mRNA is capable of conferring cycle-specific regulation on a heterologous message. Deletion analysis of the 3' end showed that the signal for cell cycle control of HTB1 mRNA includes contiguous coding and noncoding sequences surrounding the stop codon. This differs from the situation in mammalian cells, whose posttranscriptional regulation of histone genes is mediated through a short sequence containing a stem-loop structure near the very terminus of the untranslated 3' end.

Base Sequence↗
PubMed1994

Targeting of promoters for trans activation by a carboxy-terminal domain of the NS-1 protein of the parvovirus minute virus of mice.

The NS-1 gene of the parvovirus minute virus of mice (MVM) (prototype strain, MVMp) was fused in phase with the sequence coding for the DNA-binding domain of the bacterial LexA repressor. The resulting chimeric protein, LexNS-1, was tested for its transcriptional activity by using various target promoters in which multiple LexA operator sequences had been introduced. Under these conditions, NS-1 was shown to stimulate gene expression driven by the modified long terminal repeat promoters (from the retroviruses mouse mammary tumor virus and Rous sarcoma virus) and P38 promoter (from MVMp), indicating that the NS-1 protein is a potent transcriptional activator. It is noteworthy that in the absence of LexA operator-mediated targeting, the genuine mouse mammary tumor virus and Rous sarcoma virus promoters were inhibited by NS-1. Together these data strongly suggest that NS-1 contains an activating region able to induce promoters with which this protein interacts but also to repress transcription from nonrecognized promoters by a squelching mechanism similar to that described for other activators. Deletion mutant analysis led to the identification of an NS-1 domain that exhibited an activating potential comparable to that of the whole polypeptide when fused to the DNA-binding region of LexA. This domain is localized in the carboxy-terminal part of NS-1 and corresponds to one of the two regions previously found to be responsible for toxicity. These results argue for the involvement of the regulatory functions of NS-1 in the cytopathic effect of this parvovirus product.

Animals↗
PubMed1995

Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus.

The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined. The sequence was found to contain an open reading frame (ORF) with a G+C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension. The ORF encodes a protein with an M(r) of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site. The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G+C content in the third position. The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence. Neither ATG nor GTG initiator codons were identified for the protein. However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon. The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension. The -10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 region was less similar. Comparison of the primary amino acid sequence of PHS of S. antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain. A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843. This phsA promoter-dependent xylE expression could be repressed by glucose in S. antibioticus when the organism was grown on glucose or galactose plus glucose. Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence analysis of 2''-aminoglycoside nucleotidyl-transferase ANT(2'') from Tn4000: its relationship with AAD(3'') and impact on Tn21 evolution.

Aminoglycoside 2''-O-nucleotidyltransferase (AAD(2'')) mediates bacterial resistance to dibekacin, gentamicin, kanamycin, sisomicin and tobramycin. Its coding sequence, aadB, is part of Tn21-related transposon, Tn4000. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 177 amino acids with a calculated molecular weight of 21,240. The predicted amino acid sequence revealed up to 27% homology to that of three nucleotidyltransferases of type AAD(3''), which are widely distributed among Gram-negatives, and to the AAD(9) from Staphylococcus aureus transposon Tn554. The regions flanking aadB suggest that its insertion into Tn21 arose from a site-specific recombination event adjacent to the aadA gene.

Amino Acid Sequence↗
PubMed2006

Predicting antisense RNAs in the genomes of Escherichia coli and Salmonella typhimurium using promoter-search algorithm PlatProm.

A pattern recognition software PlatProm, which takes into consideration both sequence-specific and structure-specific features in the genetic environment of the promoter sites and identifies transcription start points with a very high accuracy was used to reveal potentially transcribed regions in the genomes of two bacterial species. Along with the expected promoters located upstream from coding sequences PlatProm identified several hundred of very similar signals in other intergenic regions and within coding sequences. Homologous genes of Escherichia coli and Salmonella typhimurium, containing potential promoters on the template strand are suggested as putative targets for regulations by antisense RNA-products (aRNAs).

Algorithms↗
PubMed1993

Bacterially expressed Fabs of monoclonal antibodies neutralizing tumour necrosis factor alpha in vitro retain full binding and biological activity.

Antibody fragments specific for the human tumour necrosis factor alpha (TNF alpha) have been cloned from lambda combinatorial expression libraries using total RNA obtained from three different hybridoma cell lines of therapeutic interest. The previously described bacteriophage lambda vectors, lambda HC2 and lambda LC1, were modified to create unique antibody cloning sites in the combinatorial construct and a novel tag peptide was inserted at the C-terminal end of the expressed Fd chain. Sequence analysis of the cloned Fabs indicated that two of them were derived from a single B cell. Expression in E. coli showed that the amount of recovered Fab in the bacterial culture medium was related to the sequences of the variable coding regions. Hybrid Fabs created by chain exchange of similar antibodies were as active as the originally paired Fabs in binding assays. The relative affinities and the capacities of the bacterially expressed Fabs to neutralize TNF alpha cytotoxicity in vitro were identical to those of the parental antibodies. The results demonstrate that, using an in vitro approach, it is possible to generate from existing hybridoma cell lines high affinity Fabs which retain antigen specificity. The cloning sites incorporated into the C-terminal parts of these Fabs will now permit their further modification to include additional functional characteristics not possible with the original hybridoma antibodies.

Amino Acid Sequence↗
PubMed1995

Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111.

The O antigens found in Salmonella enterica (Se) and Escherichia coli (Ec) show a great deal of diversity, and only three structures are known to be common to both genera. Two of them contain the 3,6-dideoxyheoxse colitose, not found in other serogroups of the two species. The first of these is common to Ec O111 and Se O:35 (sv Adelaide); the other is found in both Ec O55 and Se O:50 (sv Greenside). The genes specific for the synthesis of O antigen are generally located in the rfb gene cluster at map position 45 min in Ec and 42 min in Se. The rfb (O antigen) gene cluster of an Ec O111 strain M92 had been cloned earlier and hybridisation analysis suggested that the rfb clusters of Ec M92 and a Se sv Adelaide strain had been acquired separately by the two species since their divergence. We have now sequenced part of the rfb cluster from Ec M92. We identify two genes of the GDP-colitose pathway, rfbM and rfbK, and show that several other ORFs have similarity to the rfb and cps (capsular polysaccharide) genes. Downstream of this block of genes is an ORF which encodes a protein with predicted transmembrane segments which is presumed to correspond to the rfbX gene. The % G+C values of the Ec M92 rfb sequence are extremely low, indicating that the rfb evolved in a low % G+C species of bacteria before transfer into Ec.

Amino Acid Sequence↗
PubMed1980

The influence of codon context on genetic code translation.

A class of mutations that increase the deficiency of a suppressor tRNA in translating a particular amber codon has been characterized. The increased efficiency is due to a mutation resulting in a change in the mRNA that affects the nucleotide adjacent to the 3' side of the UAG triplet. Thus the interaction of tRNA with mRNA is influenced by mRNA sequences outside the triplet codon.

Base Sequence↗
PubMed1995

Transcription of the archaeal trkA homolog in Methanosarcina mazei S-6.

Transcription of the archaeal trkA gene homolog in Methanosarcina mazei S-6 was studied at the optimal growth temperature of 37 degrees C and after heat shock at 45 degrees C. Northern (RNA) blotting results (transcript size) and data from primer extension experiments to map the transcription initiation site indicate that trkA is cotranscribed with another gene. The latter, orf11, encodes a protein of 94 amino acids (10,611 Da) and is located upstream of trkA, with which it overlaps: the translation stop codon of orf11, TGA, shares the bases T and G with the translation start codon of trkA, ATG. These genes' transcription was decreased by heat shock to the point of making the transcript undetectable by Northern or dot blotting procedures. orf11 and trkA differ in codon usage patterns, and the proteins coded by them, i.e., Orf11 and TrkA, are dissimilar in amino acid sequence and composition.

Amino Acid Sequence↗
PubMed1996

A Tn1545-like transposon carries the tet(M) gene in tetracycline resistant strains of Bacteroides ureolyticus as well as Ureaplasma urealyticum but not Neisseria gonorrhoeae.

The presence of the tet(M) determinant and conjugative transposons related to Tn1545 in urogenital pathogens, Bacteroides ureolyticus (10 strains), Neisseria gonorrhoeae (37 strains) and Ureaplasma urealyticum (81 strains), was studied by PCR analysis and hybridization assay. All tetracycline-resistant strains that hybridized with a probe specific for tet(M) gave the expected fragment when their DNA's were subject to PCR. The tet(M) gene and int-Tn, the gene encoding the protein required for the movement of Tn1545-like transposons, were always found together in tetracycline resistant strains of B. ureolyticus and U. urealyticum. In contrast, int-Tn was not detected in tet(M)-mediated tetracycline-resistant N. gonorrhoeae strains. These data suggest carriage of tet(M) by a Tn1545-like transposon in some tetracycline-resistant B. ureolyticus.

Bacteroides↗
PubMed1994

A frame-specific symmetry of complementary strands of DNA suggests the existence of genes on the antisense strand.

The bacterial DNA sequence in GenBank database were divided into coding and noncoding regions and examined for the base-trimer distribution in every triplet frame on the sense and antisense strands. The results revealed that for the noncoding region, both strands have very similar base-trimer distributions and have no frame specificity; that is, DNA is symmetric in the noncoding region. For the coding region, on the other hand, the symmetry is broken only in the triplet framework, and we found a special triplet-frame-specific symmetry which appears when the two complementary strands of the coding region are read from their 5' ends. In addition, the following frame specificity was also observed in the distribution of stop codons on the antisense strand of the coding region. When the antisense sequences of the open reading frames (ORFs) in the database are read in the three reading frames, the same reading frame as the corresponding ORF contains a significantly larger amount of long open frames without stop codons (i.e., nonstop frames [NSFs]) than expected, while the number of NSFs in the other two reading frames is similar to that of the expected one. That is, NSFs as well as ORFs are maintained in a frame-specific manner, and in this sense, DNA becomes symmetrical even in the coding region. These two kinds of frame-specific symmetries indicate that only an ORF and its complementary triplets are specifically recognized and maintained in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Antisense↗
PubMed1998

Combining diverse evidence for gene recognition in completely sequenced bacterial genomes.

Analysis of a newly sequenced bacterial genome starts with identification of protein-coding genes. Functional assignment of proteins requires the exact knowledge of protein N-termini. We present a new program ORPHEUS that identifies candidate genes and accurately predicts gene starts. The analysis starts with a database similarity search and identification of reliable gene fragments. The latter are used to derive statistical characteristics of protein-coding regions and ribosome-binding sites and to predict the complete set of genes in the analyzed genome. In a test on Bacillus subtilis and Escherichia coli genomes, the program correctly identified 93.3% (resp. 96.3%) of experimentally annotated genes longer than 100 codons described in the PIR-International database, and for these genes 96.3% (83.9%) of starts were predicted exactly. Furthermore, 98.9% (99.1%) of genes longer than 100 codons annotated in GenBank were found, and 92.9% (75.7%) of predicted starts coincided with the feature table description. Finally, for the complete gene complements of B.subtilis and E.coli , including genes shorter than 100 codons, gene prediction accuracy was 88.9 and 87.1%, respectively, with 94.2 and 76.7% starts coinciding with the existing annotation.

Algorithms↗