Biomedical subjects

R Schleif

Publications and source records attributed to R Schleif.

17 recordsLinked to original sources

PubMed1992

Repression of the araBAD promoter from araO1.

DNA looping between the araO2 and araI sites holds the uninduced or basal level of expression of the araBAD genes from the pBAD promoter at a low level. Despite the presence of another and closer site potentially capable of mediating looping to araI, no repression from this site, called araO1, is observed. Here we show, using both in vivo and in vitro experiments, that the araO1 site is not normally occupied by AraC protein under repressing conditions, but that if AraC protein is overproduced and the araO2 site is absent, araO1 is then occupied and repression of pBAD can be observed.

AraC Transcription Factor↗

PubMed1992

DNA looping.

DNA looping is widely used in nature. It is well documented in the regulation of prokaryotic and eukaryotic gene expression, DNA replication, and site-specific DNA recombination. Undoubtedly looping also functions in other protein-DNA transactions such as repair and chromosome segregation. While the underlying physical chemistry of DNA looping is common to all systems, the precise biochemical details of looping and the utilization of looping by different systems varies widely. Looping appears to have been chosen by nature in such a wide variety of contexts because it solves problems both of binding and of geometry. The cooperativity inherent in binding a protein to multiple sites on DNA facilitates high occupancy of DNA sites by low concentrations of proteins. DNA looping permits a sizeable number of DNA-binding proteins to interact with one of their number, for example RNA polymerase. Finally, DNA looping may simplify evolution by not requiring a precise spacing between a protein's binding site and a second site on the DNA.

Chemical Phenomena↗

PubMed1991

Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon.

We report the mapping, sequencing, and study of the physiological role of the fourth arabinose-inducible operon from Escherichia coli, araJ. It is located at 9 min on the chromosome and codes for a single 42-kDa protein that shows no significant homology to other known proteins. Destruction of the chromosomal araJ gene does not detectably affect either of the two arabinose transport systems, the ability of cells to grow on arabinose, or the induction kinetics of the araBAD operon, and thus the physiological role of AraJ, if any, remains unknown. We have also found a long open reading frame upstream of araJ. The sequence of this upstream open reading frame was found to be identical to the previously reported sequence of the sbcC gene (I. S. Naom, S. J. Morton, D. R. F. Leach, and R. G. Lloyd, Nucleic Acids Res. 17:8033-8044, 1989). The carboxyl region of SbcC has an amino acid sequence consistent with this region of SbcC forming an extended alpha-helical coiled-coil.

Amino Acid Sequence↗

PubMed1990

Characterization of the Escherichia coli araFGH and araJ promoters.

The identities of two cloned, arabinose-inducible promoters were tested by hybridizing promoter DNA fragments with restriction digests of chromosomal DNA containing Mudlac phage inserted in either araFGH or in araE transport operons. One promoter, thought to be araE, is within 10(3) base-pairs of a Mudlac insertion in the araE gene. The second promoter was not found within several thousand base-pairs of either of the known transport genes. This promoter is now named araPJ (araJ). The DNA sequence of the fragment containing the araFGH promoter was determined. The start site of transcription in vivo was located to within +/- 1 base-pair (bp) by S1 nuclease mapping. DNase 1 footprinting revealed that, in comparison with the araBAD and araE promoters, the locations of the AraC and cyclic AMP receptor protein (CRP) binding sites are reversed with CRP lying between AraC and RNA polymerase. The central location of the CRP binding site may explain why the araFGH promoter is more catabolite sensitive than the other ara promoters. AraC and CRP were both required for maximal transcription in vitro, although a low level of transcription was detected with CRP alone. S1 nuclease mapping of mRNA-DNA hybrids from the araJ promoter located the transcription start point to within #/- 3 bp, and demonstrates that the promoter is dependent upon AraC protein and CRP in vivo. DNase footprinting showed that the location of the AraC protein binding site on araJ is adjacent to the RNA polymerase site, as seen at the araBAD and araE promoters. Two CRP sites were observed; one is upstream from the AraC site and one is downstream from the transcription start site.

Amino Acid Sequence↗

PubMed1989

Determining residue-base interactions between AraC protein and araI DNA.

Depurination/depyrimidation binding-interference experiments (missing contact probing) identified specific candidate residue-base interactions lost by mutants of Escherichia coli L-arabinose operon regulatory protein, AraC, to one of its binding sites, araI. These candidates were then checked more rigorously by comparing the affinities of wild-type and alanine-substituted AraC protein to variants of araI with alterations in the candidate contacted positions. Residues 208 and 212 apparently contact DNA and support, but do not prove the existence of a helix-turn-helix structure in this region of AraC protein whereas contacts by mutants with alterations at positions 256, 257 and 261 which are within another potential helix-turn-helix region do not support the existence of such a structure there. The missing contacts displayed by three AraC mutants are found within two major groove regions of the DNA and are spaced 21 base-pairs apart in a pattern indicating a direct repeat orientation for the subunits of AraC.

Binding Sites↗

PubMed1989

Transvaginal hysterosalpingo-contrast-sonography (Hy-Co-Sy) compared with conventional tubal diagnostics.

Transvaginal hysterosalpingo-contrast-sonography (Hy-Co-Sy) is described as a new method for direct imaging of the tubal passage in tubal diagnostics. During one period of anaesthesia, a transvaginal Hy-Co-Sy was followed by either hysterosalpingography or chromolaparoscopy in 42 patients with sterility disorders. The contrast-enhanced sonography of the tubes was performed in eight cases with sterile saline solution (group 1) and 34 cases with a specially developed ultrasound contrast medium (SH U 454, group 2). The findings obtained by transvaginal Hy-Co-Sy were compared with those of the conventional method. By both methods, there was complete agreement with respect to the same evaluation of tubal passage for both sides, partial agreement when firstly the tubes were assessed as unilaterally or bilaterally open--without precise localization of the side--depending on the appearance of fluid in the pouch of Douglas, or secondly, only one side, when compared, showed agreement. In group 1 complete agreement was found once, partial agreement five times and non-agreement twice, while in group 2 complete agreement was found 22 times (65%), partial agreement 11 times and non-agreement once.

Adult↗

PubMed1978

Nucleotide sequence of the L-arabinose regulatory region of Escherichia coli K12.

The nucleotide sequence of a 250-base pair segment of L-arabinose operon DNA containing the 150-base pair regulatory region has been determined. This segment includes the promoter for rightward araBAD transcription, pBAD, the promoter for leftward araC transcription, pC, and sites responsible for repression.

Arabinose↗

PubMed1977

Overproducing araC protein with lambda-arabinose transducing phage.

Escherichia coli infected with bacteriophage lambda-arabinose transducing phage were tested as sources of araC protein. Infection of cells with such phage produces an intracellular concentration of araC protein up to 100 times that present in wild-type E. coli, apparently resulting from fusion of the araC gene to bacteriophage lambda promoters. Lysates from these phage-infected cells may be fractionated to yield another 100-fold enrichment in araC activity so that the total enrichment is 10,000-fold. A nonsense mutation in araC provided proof of the identification on gel electrophoresis of a band in the purified material. Biologically active araC protein is a dimer with 28,000 M.W. subunits. The araC gene in these phage replaces the int-xis genes but is oriented in the opposite direction. Nonetheless, it appears to be transcribed in this position by the phage promoter pr via transcription the long way around. Furthermore, because araC gene is in this position, we were able to isolate phage on which the araC gene was under phage late gene control by deletion of the late gene transcription stop signals in the b2 region.

Arabinose↗

PubMed1977

In vitro construction of plasmids which result in overproduction of the protein product of the araC gene of Escherichia coli.

Derivatives of the Escherichia coli drug resistance plasmid pMB-9 were constructed which contain the promoter from the lactose operon of E. coli fused to the araC gene of E. coli. E. coli possessing these plasmids contain about 50 times as much of the araC gene product as do cells with a wild-type araC gene and promotor.

Arabinose↗

PubMed1977

The araC promoter: transcription, mapping and interaction with the araBAD promoter.

The start sites of the araC and araBAD gene messenger of E. coli were located by transcription in vitro from short DNA fragments, by high magnification electron microscopy and by genetic mapping. Transcription for these messengers proceeds in opposite directions from the start sites that are 150 base pairs apart. Transcription from the araBAD promoter requires araC protein plus arabinose and CAP protein plus cyclic AMP. In the experiments performed in vitro, inducing the araBAD promoter represses activity of the araC promoter.

Arabinose↗

PubMed1976