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Interactions among regulators of RNA abundance characterized using RNA fingerprinting by arbitrarily primed PCR.

ABSTRACT: Using RNA fingerprinting by arbitrarily primed PCR it is possible to infer convergent transcript regulatory pathways from the coordinate behavior of subsets of anonymous transcripts without cloning any genes. The number of transcripts in each response category can be estimated. The same may be true for differential display. We demonstrate these claims by treating a cell line with two known modulators of RNA abundance, transforming growth factor-beta (TGF beta) and cycloheximide (CX), used together and alone. The responses of over 1700 anonymous transcripts were monitored under these three conditions and in an untreated control. Eight of the twenty-seven [3(3)] possible transcript response categories were observed among 86 differentially expressed transcripts. For example, CX stabilizes or induces as many as 2.7% of transcripts of which about one third do not accumulate when TGF beta is also present. This intersection may reflect CX stabilization or induction of an important class of RNAs that otherwise usually have short half-lives. We predict that RNAs in this class constitute the majority of transcripts targeted for rapid down regulation in response to TGF beta and perhaps most other natural transcriptional modulators.

SUBMITTER: McClelland M

PROVIDER: S-EPMC308475 | biostudies-other | 1994 Oct

REPOSITORIES: biostudies-other

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Interactions among regulators of RNA abundance characterized using RNA fingerprinting by arbitrarily primed PCR.

McClelland M M Ralph D D Cheng R R Welsh J J

Nucleic acids research 19941001 21

Using RNA fingerprinting by arbitrarily primed PCR it is possible to infer convergent transcript regulatory pathways from the coordinate behavior of subsets of anonymous transcripts without cloning any genes. The number of transcripts in each response category can be estimated. The same may be true for differential display. We demonstrate these claims by treating a cell line with two known modulators of RNA abundance, transforming growth factor-beta (TGF beta) and cycloheximide (CX), used togeth ...[more]

PMID: 7526341

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Project description:We report an analysis of a sample of the SOS response of Salmonella enterica serovar Typhimurium using the differential display of RNA fingerprinting gels of arbitrarily primed PCR products. The SOS response was induced by the addition of mitomycin C to an exponentially growing culture of serovar Typhimurium, and the RNA population was sampled during the following 2 h. These experiments revealed 21 differentially expressed PCR fragments representing mRNA transcripts. These 21 fragments correspond to 20 distinct genes. All of these transcripts were positively regulated, with the observed induction starting 10 to 120 min after addition of mitomycin C. Fifteen of the 21 transcripts have no homologue in the public sequence data banks and are therefore classified as novel. The remaining six transcripts corresponded to the recE, stpA, sulA, and umuC genes, and to a gene encoding a hypothetical protein in the Escherichia coli lysU-cadA intergenic region; the recE gene was represented twice by nonoverlapping fragments. In order to determine if the induction of these 20 transcripts constitutes part of a classical SOS regulon, we assessed the induction of these genes in a recA mutant. With one exception, the increased expression of these genes in response to mitomycin C was dependent on the presence of a functional recA allele. The exception was fivefold induced in the absence of a functional RecA protein, suggesting another layer of regulation in response to mitomycin C, in addition to the RecA-LexA pathway of SOS induction. Our data reveal several genes belonging to operons known to be directly involved in pathogenesis. In addition, we have found several phage-like sequences, some of which may be landmarks of pathogenicity determinants. On the basis of these observations, we propose that the general use of DNA-damaging agents coupled with differential gene expression analysis may be a useful and easy method for identifying pathogenicity determinants in diverse organisms.

Project description:Evidence suggests that a repertoire of Vibrio cholerae genes are differentially expressed in vivo, and regulation of virulence factors in vivo may follow a different pathway. Our work was aimed at characterization of in vivo-grown bacteria and identification of genes that are differentially expressed following infection by RNA arbitrarily primed (RAP)-PCR fingerprinting. The ligated rabbit ileal loop model was used. The motility of in vivo-grown bacteria increased by 350% over that of in vitro-grown bacteria. Also, the in vivo-grown cells were more resistant to killing by human serum. By using the RAP-PCR strategy, five differentially expressed transcripts were identified. Two in vitro-induced transcripts encoded polypeptides for the leucine tRNA synthatase and the 50S ribosomal protein, and the three in vivo-induced transcripts encoded the SucA and MurE proteins and a polypeptide of unknown function. MurE is a protein involved in the peptidoglycan biosynthetic pathway. The lytic profiles of in vivo- and in vitro-grown cells suspended in distilled water were compared; the former was found to be slightly less sensitive to lysis. Ultrathin sections of both cells observed under the transmission electron microscope revealed that in contrast to the usual wavy discontinuous membrane structure of the in vitro-grown cells, in vivo-grown cells had a more rigid, clearly visible double-layered structure. The V. cholerae murE gene was cloned and sequenced. The sequence contained an open reading frame of 1,488 nucleotides with its own ribosome-binding site. A plasmid containing the murE gene of V. cholerae was transformed into V. cholerae 569B, and a transformed strain, 569BME, containing the plasmid was obtained. Ultrathin sections of 569BME viewed under a transmission electron microscope revealed a slightly more rigid cell wall than that of wild-type 569B. When V. cholerae 569B and 569BME cells were injected separately into ligated rabbit ileal loops, the transformed cells had a preference for growth in the ileal loops versus laboratory conditions.

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Interactions among regulators of RNA abundance characterized using RNA fingerprinting by arbitrarily primed PCR.

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Interactions among regulators of RNA abundance characterized using RNA fingerprinting by arbitrarily primed PCR.

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