Biomedical subjects

S S Zinkel

Publications and source records attributed to S S Zinkel.

5 recordsLinked to original sources

PubMed1992

Identification of a negative regulatory element that inhibits c-mos transcription in somatic cells.

We have used transient expression assays to identify a cis-acting region in the 5' flanking sequence of murine c-mos which, when deleted, allows expression from the c-mos promoter in NIH 3T3 cells. This negative regulatory sequence, located 400 to 500 nucleotides upstream of the c-mos ATG, also inhibited expression from a heterologous promoter. In addition to NIH 3T3 cells, the c-mos negative regulatory sequence was active in BALB/3T3 cells, PC12 rat pheochromocytoma cells, and A549 human lung carcinoma cells. Site-specific mutagenesis identified three possibly interacting regions that were involved in negative regulatory activity, located around -460, -425, and -405 with respect to the ATG. RNase protection analysis indicated that once the negative regulatory sequences were deleted, transcription in NIH 3T3 cells initiated from the same transcription initiation sites normally utilized in spermatocytes, approximately 280 nucleotides upstream of the ATG. Deletions beyond the spermatocyte promoter, however, allowed transcription initiation from progressively downstream c-mos sequences. Deletion or mutation of sequences surrounding the oocyte promoter at -53 also had little effect on expression of c-mos constructs in NIH 3T3 cells. Therefore, the major determinant of c-mos expression in NIH 3T3 cells was removal of the negative regulatory sequence rather than the utilization of a unique promoter. The c-mos negative regulatory sequences thus appear to play a significant role in tissue-specific c-mos expression by inhibiting transcription in somatic cells.

3T3 Cells↗

PubMed1991

Catabolite activator protein-induced DNA bending in transcription initiation.

We describe experiments that enable us to track the presence and direction of the DNA bend induced by Escherichia coli catabolite activator protein (CAP) through the intermediate stages of transcription initiation at the lac promoter. Transcriptional complexes examined were formed on superhelical templates to enhance specific complex formation, and detected by electrophoretic analysis after restriction digestion. We found that the bend is maintained and even increased upon formation of closed and open complexes. Our results exclude the hypothesis that the energy of the CAP-induced bend is used to promote open complex formation. We now suggest a new model, in which DNA wraps around the CAP-polymerase complex to form a writhing structure equivalent to that at the end of an interwound superhelical domain. Formation of this structure may facilitate open complex formation. We further propose that the stored bend energy may be used to help counteract strong protein-protein or protein-DNA interactions, thus assisting the process of RNA polymerase escape from the promoter.

Base Sequence↗

PubMed1991

c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site.

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.

Animals↗

PubMed1990

Comparative gel electrophoresis measurement of the DNA bend angle induced by the catabolite activator protein.

We describe a method to determine the magnitude of protein-induced DNA bends relative to a set of standard A tract bends using comparative gel electrophoresis. The DNA bend of interest was that induced by the catabolite activator protein (CAP), the transcriptional activator protein of the lac operon. The set of comparison molecules contained both bends of known magnitude and a bound CAP. The electrophoretic influence of the bound protein was accounted for by placing its binding site at the end of the molecule where its induced bend has little influence. Standard bends at the DNA center were introduced by incorporating 3-9 A6 tracts at approximately 10.5 base-pair phasing. The mobility of these control molecules was compared to the mobility of a test molecule of comparable length containing a central CAP-induced DNA bend. The CAP bend angle was found to be 5.6 +/- 0.3 A tract equivalents, or approximately 100 degrees, independent of the concentration of the gel used within the range tested. The dependence of gel retardation on DNA end-to-end distance was found to break down for A tract bend angles above 120 degrees, corresponding roughly to the angle beyond which the long axis of the molecule is no longer parallel to the end-to-end vector. We speculate that this may reflect a switch in the mode of migration of molecules through the gel.

Base Sequence↗

PubMedDate not supplied

DNA bend direction by phase sensitive detection.

Gel electrophoresis of DNA and protein-DNA complexes has been a key method used in studies of sequence-directed and protein-induced DNA bending. Natural DNA sequences can have protein binding sites adjacent to A-tract bending sites, resulting in the potential for the formation of topologically complex shapes in a localized DNA regulatory domain. An essential first step in deducing the structure and functional significance of such domains is elucidation of the relative direction of bending, which can be determined from the electrophoretic mobilities of isomers having varied helical phasing between the bends. Taking DNA bent around CAP protein as a standard, we conclude that the junction bending model correctly predicts the direction of bending at A tracts in kinetoplast DNA. The overall direction of the bend is towards the minor groove at the centre of the A tract.

Base Sequence↗