We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput ...reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Using the catechol dehydrogenase gene as a reporter, we isolated random mutations in the plJ101 korB gene operator/promoter (OP) region that affect korB expression and regulation. We mapped these ...mutations to inverted repeat sequences within the promoter and studied their effects on binding of the KorB repressor protein to the OP, on expression of the korB gene, and on plasmid transmission during mating. Additionally, we investigated the biological effects of KorB binding to a locus (sti, for strong incompatibility) adjacent to the korB OP and implicated in plJ101 replication. Our results identify sites that influence the synthesis and autoregulation of KorB; they also show that interaction of KorB with sti affects repression of korB and transmission of plasmids to spores of recipients.
The autoregulated kil-kor system of pIJ101 represents an intricately balanced gene control mechanism that accomplishes regulated plasmid transmission, presumably, in response to developmentally ...determined signals. The two autorepressors of this system, KorA and KorB, are known to regulate the corresponding kil genes at the transcriptional level. In this work, the molecular nature of the interaction between the repressors and their target sequences were characterized and their biological roles in plasmid transmission were studied. The two repressor proteins were expressed in E. coli and it was found that two forms of the KorB protein were expressed in cells harboring the korB gene. One has the M.W. of 10 kD, the M.W. predicted from the gene sequence, while the other is a 6 kD peptide. In S. lividans carrying pIJ10I derivatives, only the 6 kD protein was found. It is proposed that in Streptomyces, KorB protein is synthesized as a 10 kD protein, which is processed into a 6 kD peptide that binds differentially to the two operator/promoter regions it regulates showing 50 fold greater affinity for the sequences controlling kilB expression. Results from the binding analysis indicate that KorB interacts with the sti locus, which controls pIJ101 replication and incompatibility. The significance of this interaction was demonstrated by an insertion that separates the sti locus from the KorB binding site and consequently reduces the rate of plasmid transmission to 30% of the wild type level. These results are consistent with the notion that KorB may be involved in the coordination of plasmid transmission with conjugative DNA replication. Studies on KorA binding to the divergent korA-tra promoter showed that two DNA binding units of the KorA protein interact with its operator sequence targets. This suggests that korA-tra regulation may be analogous to the bacteriophage $\lambda$ cro-cI system and is tightly correlated with the process of plasmid transmission. Collectively, these data indicate that the KorA and KorB repressors play multiple roles in maintaining the balance of the kil-kor system and in coordinating the expression of these genes with the onset of plasmid transmission.
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