NORMAL GENOMIC

• Ghrist AC, Stauffer GV
• Promoter characterization and constitutive expression of the Escherichiacoli gcvR gene.
• J Bacteriol. 1998; 180: 1803-7
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• The Escherichia coli glycine cleavage repressor protein (GcvR) negativelyregulates expression of the glycine cleavage operon (gcv). In this study,the gcvR translational start site was determined by N-terminal amino acidsequence analysis of a GcvR-LacZ fusion protein. Primer extension analysisof the gcvR promoter region identified a primary transcription start site27 bp upstream of the UUG translation start site and a minor transcriptionstart site approximately 100 bp upstream of the translation start codon.The -10 and -35 promoter regions upstream of the primary transcriptionstart site were defined by mutational analysis. Expression of a gcvR-lacZfusion was unaltered in the presence of glycine or inosine, moleculesknown to induce or repress expression of gcv, respectively. In addition,it was shown that gcvR-lacZ expression is neither regulated by the glycinecleavage activator protein (GcvA) nor autogenously regulated by GcvR. FromDNA sequence analysis, it was predicted that the translation start codonof the downstream bcp gene overlaps the gcvR stop codon, suggesting thatthese genes may form an operon. However, a down mutation in the -10promoter region of gcvR had no effect on the expression of a downstreambcp-lacZ fusion, and primer extension analysis of the bcp promoter regiondemonstrated that bcp has its own promoter within the gcvR codingsequence. These results show that gcvR and bcp do not form an operon.Furthermore, the deletion of bcp from the chromosome had no effect ongcv-lacZ expression.

• Solenberg PJ, Burgett SG
• Method for selection of transposable DNA and characterization of a newinsertion sequence, IS493, from Streptomyces lividans.
• J Bacteriol. 1989; 171: 4807-13
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• A method to select for transposable elements from Streptomyces spp. byusing insertional inactivation of a repressor gene that functions inEscherichia coli was developed. Plasmid pCZA126, which can replicate inStreptomyces spp. or E. coli, contains a gene coding for the lambda cI857repressor and a gene, under repressor control, coding for apramycinresistance. E. coli cells containing the plasmid are apramycin sensitivebut become apramycin resistant if the cI857 repressor gene is disrupted.Plasmids propagated in Streptomyces spp. can be screened for transposableelements that have disrupted the cI857 gene by transforming E. coli cellsto apramycin resistance. This method was used to isolate a new1.6-kilobase insertion sequence, IS493, from Streptomyces lividans CT2.IS493 duplicated host DNA at the target site, had inverted repeats at itsends, and contained two tandem open reading frames on each strand. IS493was present in three copies in the same genomic locations in several S.lividans strains. Two of the copies appeared to be present in regions ofsimilar DNA context that extended at least 11.5 kilobases. Several otherStreptomyces spp. did not appear to contain copies of IS493.

• Hawley DK, McClure WR
• Compilation and analysis of Escherichia coli promoter DNA sequences.
• Nucleic Acids Res. 1983; 11: 2237-55
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• The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coliRNA polymerase were compiled. The complete listing was divided into twogroups depending upon whether or not the promoter had been defined bygenetic (promoter mutations) or biochemical (5' end determination)criteria. A consensus promoter sequence based on homologies among 112well-defined promoters was determined that was in substantial agreementwith previous compilations. In addition, we have tabulated 98 promotermutations. Nearly all of the altered base pairs in the mutants conform tothe following general rule: down-mutations decrease homology andup-mutations increase homology to the consensus sequence.

• Gold L, Pribnow D, Schneider T, Shinedling S, Singer BS, Stormo G
• Translational initiation in prokaryotes.
• Annu Rev Microbiol. 1981; 35: 365-403

• Valentin-Hansen P, Svenningsen BA, Munch-Petersen A, Hammer-Jespersen K
• Regulation of the deo operon in Escherichia coli: the double negativecontrol of the deo operon by the cytR and deoR repressors in a DNAdirected in vitro system.
• Mol Gen Genet. 1978; 159: 191-202
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• The synthesis of the four enzymes of the deo operon in Escherichia coli isknown from in vivo experiments to be subject to a double negative control,exerted by the products of the cytR and deoR genes. A DNA-directed invitro protein synthesizing system makes the deo enzymes (exemplified bythymidine phosphorylase) in agreement with in vivo results. Enzymesynthesis is stimulated by cyclic AMP and repressed by the cytR and deoRgene products. Repression by the cytR repressor is reversed by cytidine oradenosine in the presence of cyclic AMP, while repression by the deoRrepressor is reversed by deoxyribose-5-phosphate. Assays for the presenceof the cytR and deoR repressors were established by use of S-30 extractsprepared from the regulatory mutants. Dissociation constants forrepressor-operator binding as well as for repressor-inducer interactionshave been estimated from the results.