scholarly journals IS1414, an Escherichia coliInsertion Sequence with a Heat-Stable Enterotoxin Gene Embedded in a Transposase-Like Gene

2000 ◽  
Vol 68 (10) ◽  
pp. 5710-5715 ◽  
Author(s):  
Annette McVeigh ◽  
Alessio Fasano ◽  
Daniel A. Scott ◽  
Sandra Jelacic ◽  
Steve L. Moseley ◽  
...  
Keyword(s):  
Burkholderia Cepacia ◽  
Rhizobium Meliloti ◽  
Structural Features ◽  
Protein Product ◽  
Toxin Gene ◽  
Gene Probe ◽  
Coding Region ◽  
Reading Frame ◽  
Heat Stable

ABSTRACT Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed thatastA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the −1 reading frame in reference to the toxin gene. In vitro expression of the predicted M r-∼46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.

scholarly journals Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

2002 ◽  
Vol 76 (3) ◽  
pp. 1450-1460 ◽  
Author(s):  
S. Spaderna ◽  
H. Blessing ◽  
E. Bogner ◽  
W. Britt ◽  
M. Mach
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Component ◽  
Laboratory Strain ◽  
Immunoblot Analysis ◽  
Protein Product ◽  
Clinical Isolates ◽  
Coding Region ◽  
Reading Frame ◽  
Infected Cells ◽  
Strain Ad169

ABSTRACT Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.

A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

2007 ◽  
Vol 292 (4) ◽  
pp. R1649-R1656 ◽  
Author(s):  
John Yuh-Lin Yu ◽  
Chin-Hon Pon ◽  
Hui-Chen Ku ◽  
Chih-Ting Wang ◽  
Yung-Hsi Kao
Keyword(s):  
Mrna Expression ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Biological Effects ◽  
In Vitro Study ◽  
The Body ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle ( Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5′-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3′-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH2 terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly1 and Thr29 substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48–81% and 76–96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17β-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.

scholarly journals Primary sequence and developmental expression of a novel Drosophila melanogaster src gene.

1987 ◽  
Vol 7 (6) ◽  
pp. 2119-2127 ◽  
Author(s):  
R J Gregory ◽  
K L Kammermeyer ◽  
W S Vincent ◽  
S G Wadsworth
Keyword(s):  
Drosophila Melanogaster ◽  
Blot Hybridization ◽  
Structural Features ◽  
Developmental Expression ◽  
Reading Frame ◽  
Adult Females ◽  
Src Gene ◽  
Free Translation

We have sequenced a cDNA clone for the Drosophila melanogaster gene Dsrc28C, a homolog of the vertebrate gene c-src. The cDNA contains a single open reading frame encoding a protein of 66 kilodaltons which contains features highly conserved within the src family of tyrosine protein kinases. Novel structural features of the Dsrc28C protein include a basic pI and a polyglycine domain near the amino terminus. Cell-free translation of in vitro-transcribed RNA yielded a protein of the predicted size which could be immunoprecipitated by anti-v-src antisera. RNA blot hybridization revealed that the gene is expressed predominantly during embryogenesis, in imaginal disks of third-instar larvae, and in adult females. In situ hybridization showed that expression in adult females is largely confined to nurse cells and developing oocytes.

scholarly journals A Group II Intron-Type Open Reading Frame from the Thermophile Bacillus (Geobacillus) stearothermophilus Encodes a Heat-Stable Reverse Transcriptase

2004 ◽  
Vol 70 (12) ◽  
pp. 7140-7147 ◽  
Author(s):  
Jaishree Vellore ◽  
Samuel E. Moretz ◽  
Bert C. Lampson
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Protein Product ◽  
Thermophilic Bacterium ◽  
Full Length ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Group Ii

ABSTRACT The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75°C.

scholarly journals The Major Open Reading Frame of the β2.7 Transcript of Human Cytomegalovirus: In Vitro Expression of a Protein Posttranscriptionally Regulated by the 5′ Region

1998 ◽  
Vol 72 (10) ◽  
pp. 8425-8429 ◽  
Author(s):  
Giovanna Bergamini ◽  
Marko Reschke ◽  
Maria Concetta Battista ◽  
Maria Cristina Boccuni ◽  
Fabio Campanini ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Cytomegalovirus Infection ◽  
Intracellular Localization ◽  
Protein Product ◽  
Transient Transfection ◽  
Open Reading Frame ◽  
Major Open Reading Frame ◽  
Reading Frame ◽  
Human Cytomegalovirus Infection

ABSTRACT β2.7 is the major early transcript produced during human cytomegalovirus infection. This abundantly expressed RNA is polysome associated, but no protein product has ever been detected. In this study, a stable peptide of 24 kDa was produced in vitro from the major open reading frame (ORF), TRL4. Following transient transfection, the intracellular localization was nucleolar and the expression was posttranscriptionally inhibited by the 5′ sequence of the transcript, which harbors two short upstream ORFs.

Characterization of Escherichia coli Strains from Cases of Childhood Diarrhea in Provincial Southwestern Nigeria

2000 ◽  
Vol 38 (1) ◽  
pp. 7-12
Author(s):  
Iruka N. Okeke ◽  
Adebayo Lamikanra ◽  
Hartmut Steinrück ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Blot Hybridization ◽  
Toxin Gene ◽  
Gene Probe ◽  
Cytolethal Distending Toxin ◽  
Healthy Children ◽  
Southwestern Nigeria ◽  
E Coli ◽  
Heat Stable ◽  
Primary Health Care Centers

ABSTRACT In a study carried out in small-town and rural primary health care centers in southwestern Nigeria, 330 Escherichia coli strains isolated from 187 children with diarrhea and 144 apparently healthy controls were examined for virulence traits. Based on the results of colony blot hybridization, strains were categorized as enteropathogenic E. coli (1.8%), enterotoxigenic E. coli (2.4%), enteroinvasive E. coli (1.2%), enterohemorrhagic E. coli (0.6%), enteroaggregative E. coli (10.3%), diffusely adherent E. coli (7.9%), cell-detaching E. coli (6.9%), and cytolethal distending toxin-producing E. coli (0.9%). E. coli strains that hybridized with a Shiga toxin gene probe but lacked other characteristics usually present in enterohemorrhagic E. coli constituted 8.4% of the isolates. Ninety-seven E. coli isolates adhered to HEp-2 cells in an aggregative fashion but did not hybridize with any of the probes employed in the study. Overall the pathotypes, apart from cytolethal distending toxin-producing E. coli , were recovered both from children with diarrhea and from children without diarrhea, though to a lower extent from the healthy children. All diarrheagenic E. coli strains were associated with diarrhea ( P < 0.02). Heat-stable-enterotoxin-producing enterotoxigenic E. coli showed significant association with diarrhea ( P < 0.02), as did strains that demonstrated aggregative adherence to HEp-2 cells ( P < 0.04), but not those that hybridized with the CVD432 enteroaggregative probe.

scholarly journals Alternate RNA splicing of murine nfkb1 generates a nuclear isoform of the p50 precursor NF-kappa B1 that can function as a transactivator of NF-kappa B-regulated transcription.

1994 ◽  
Vol 14 (12) ◽  
pp. 8460-8470 ◽  
Author(s):  
R J Grumont ◽  
J Fecondo ◽  
S Gerondakis
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Rna Splicing ◽  
Coding Region ◽  
Nf Kappa B ◽  
Reading Frame ◽  
C Terminus ◽  
A Cell ◽  
Regulated Gene Expression

The NF-kappa B1 subunit of the transcription factor NF-kappa B is derived by proteolytic cleavage from the N terminus of a 105-kDa precursor protein. The C terminus of p105NF-kappa B1, like those of I kappa B proteins, contains ankyrin-related repeats that inhibit DNA binding and nuclear localization of the precursor and confer I kappa B-like properties upon p105NF-kappa B1. Here we report the characterization of two novel NF-kappa B1 precursor isoforms, p84NF-kappa B1 and p98NF-kappa B1, that arise by alternate splicing within the C-terminal coding region of murine nfkb1. p98NF-kappa B1, which lacks the 111 C-terminal amino acids (aa) of p105NF-kappa B1, has a novel 35-aa C terminus encoded by an alternate reading frame of the gene. p84NF-kappa B1 lacks the C-terminal 190 aa of p105NF-kappa B1, including part of ankyrin repeat 7. RNA and protein analyses indicated that the expression of p84NF-kappa B1 and p98NF-kappa B1 is restricted to certain tissues and that the phorbol myristate acetate-mediated induction of p84NF-kappa B1 and p105NF-kappa B1 differs in a cell-type-specific manner. Both p84NF-kappa B1 and p98NF-kappa B1 are found in the nuclei of transfected cells. Transient transfection analysis revealed that p98NF-kappa B1, but not p105NF-kappa B1 or p84NF-kappa B1, acts as a transactivator of NF-kappa B-regulated gene expression and that this is dependent on sequences in the Rel homology domain required for DNA binding and on the novel 35 C-terminal aa of this isoform. In contrast to previous findings, which indicated that p105NF-kappa B1 does not bind DNA, all of the NF-kappa B1 precursors were found to specifically bind with low affinity to a highly restricted set of NF-kappa B sites in vitro, thereby raising the possibility that certain of the NF-kappa B1 precursor isoforms may directly modulate gene expression.

scholarly journals The energy landscape of −1 ribosomal frameshifting

2020 ◽  
Vol 6 (1) ◽  
pp. eaax6969 ◽  
Author(s):  
Junhong Choi ◽  
Sinéad O’Loughlin ◽  
John F. Atkins ◽  
Joseph D. Puglisi
Keyword(s):  
Single Molecule ◽  
Messenger Rna ◽  
Information Transfer ◽  
Mechanistic Model ◽  
High Rate ◽  
Coding Region ◽  
Ribosomal Frameshifting ◽  
Reading Frame

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3′ messenger RNA (mRNA) structures, but how these mRNA structures enhance −1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal −1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

Primary sequence and developmental expression of a novel Drosophila melanogaster src gene

1987 ◽  
Vol 7 (6) ◽  
pp. 2119-2127
Author(s):  
R J Gregory ◽  
K L Kammermeyer ◽  
W S Vincent ◽  
S G Wadsworth
Keyword(s):  
Drosophila Melanogaster ◽  
Blot Hybridization ◽  
Structural Features ◽  
Developmental Expression ◽  
Reading Frame ◽  
Adult Females ◽  
Src Gene ◽  
Free Translation

We have sequenced a cDNA clone for the Drosophila melanogaster gene Dsrc28C, a homolog of the vertebrate gene c-src. The cDNA contains a single open reading frame encoding a protein of 66 kilodaltons which contains features highly conserved within the src family of tyrosine protein kinases. Novel structural features of the Dsrc28C protein include a basic pI and a polyglycine domain near the amino terminus. Cell-free translation of in vitro-transcribed RNA yielded a protein of the predicted size which could be immunoprecipitated by anti-v-src antisera. RNA blot hybridization revealed that the gene is expressed predominantly during embryogenesis, in imaginal disks of third-instar larvae, and in adult females. In situ hybridization showed that expression in adult females is largely confined to nurse cells and developing oocytes.

Nucleotide sequence and expression in vitro of cDNA derived from mRNA of int-1, a provirally activated mouse mammary oncogene

1985 ◽  
Vol 5 (12) ◽  
pp. 3337-3344
Author(s):  
Y K Fung ◽  
G M Shackleford ◽  
A M Brown ◽  
G S Sanders ◽  
H E Varmus
Keyword(s):  
Nucleotide Sequence ◽  
Mammary Tumor ◽  
Initiation Site ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Coding Region ◽  
Reading Frame ◽  
Donor And Acceptor

The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984) shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41-kilodalton protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of RNA transcribed from the cDNA clones with SP6 RNA polymerase.

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