PubMed Health⌕ Search

Biomedical subjects

M A Krasnow

Publications and source records attributed to M A Krasnow.

7 recordsLinked to original sources
PubMed1992

Differential regulation of transcription preinitiation complex assembly by activator and repressor homeo domain proteins.

Different eukaryotic transcription factors can act through the same upstream binding site to differentially regulate target gene expression, but little is known of the underlying mechanisms. Here, we show that Ultrabithorax and even-skipped homeo domain proteins (UBX and EVE) of Drosophila melanogaster exert active and opposite effects on in vitro transcription when bound to a common site upstream of a core promoter. Both the activator UBX and the repressor EVE affect the extent but not the rate constant of preinitiation complex (preIC) formation. Both regulators act early in preIC assembly and are dispensable later. Assembling complexes become resistant to regulation by the bound proteins, but activation by UBX is restored upon ATP or dATP addition, and regulation by both proteins is restored after the addition of all four nucleoside triphosphates and transcription initiation. The results establish that upstream activators and repressors can function by fundamentally similar mechanisms, by differentially regulating an early step in preIC assembly, leading to formation of functionally distinct transcription complexes. A subsequent step renders mature complexes transiently refractory to activation and repression. Implications for the mechanism of transcription complex assembly and turnover and its regulation are discussed, including a new role for ATP in turnover.

Adenosine Triphosphate↗
PubMed1991

Whole animal cell sorting of Drosophila embryos.

Use of primary culture cells has been limited by the inability to purify most types of cells, particularly cells from early developmental stages. In whole animal cell sorting (WACS), live cells derived from animals harboring a lacZ transgene are purified according to their level of beta-galactosidase expression with a fluorogenic beta-galactosidase substrate and fluorescence-activated cell sorting. With WACS, incipient posterior compartment cells that express the engrailed gene were purified from early Drosophila embryos. Neuronal precursor cells were also purified, and they differentiated into neurons with high efficiency in culture. Because there are many lacZ strains, it may be possible to purify most types of Drosophila cells. The same approach is also applicable to other organisms for which germ-line transformation is possible.

Animals↗
PubMed1990

Stimulation of transcription by an Ultrabithorax protein in vitro.

The Ultrabithorax (Ubx) gene of Drosophila melanogaster encodes a family of UBX proteins that are thought to specify the developmental fates of segments in the posterior thorax and anterior abdomen by controlling the expression of a set of target genes. UBX proteins bind DNA in vitro, and they activate or repress different natural and synthetic target promoters in cultured cells. Here it is shown that a purified UBX protein can stimulate transcription of a synthetic target gene in extracts of cultured D. melanogaster cells. Stimulation is dependent on the presence of upstream, promoter-region binding sites but is independent of binding site orientation. A naturally occurring binding site cluster and a binding site consensus sequence consisting of TAA trinucleotide repeats can mediate this activation. A minimal promoter fused to such sites is activated by UBX, suggesting that transcriptional stimulation could result from an interaction between the promoter-bound protein and the general transcriptional machinery.

Animals↗
PubMed1989

Transcriptional activation and repression by Ultrabithorax proteins in cultured Drosophila cells.

Homeotic genes of Drosophila melanogaster such as Ultrabithorax (Ubx) and Antennapedia (Antp) have long been thought to select metameric identity during development by controlling the expression of various target genes. Here we describe a cotransfection assay in cultured D. melanogaster cells that is used to demonstrate that Ubx proteins (UBX) can repress an Antp promoter fusion and activate a Ubx promoter fusion, activities predicted from genetic studies. We show (a) that UBX proteins regulated the level of accurately initiated Antp P1 and Ubx transcripts, (b) that activation of the Ubx promoter required a downstream cluster of UBX binding sites, and (c) that binding site sequences were sufficient to confer regulation on a heterologous promoter, regardless of their orientation or precise position. We conclude that UBX proteins are transcriptional repressors and activators, and that their actions are mediated by binding to promoter region sequences. Each member of the UBX protein family has similar regulatory abilities, but the properties of synthetic mutant forms suggest that UBX proteins may have a modular design similar to other transcriptional regulators.

Animals↗
PubMed1988

An Ultrabithorax protein binds sequences near its own and the Antennapedia P1 promoters.

The homeotic gene Ultrabithorax (Ubx), located in the bithorax complex of Drosophila, encodes a family of closely related proteins that direct the developmental fates of posterior thoracic and anterior abdominal metameres. We have purified a member of the Ubx protein family from an overproducing E. coli strain and have shown that it is sequence-specific DNA binding protein. The protein binds tightly to sequences near its own promoter and near the P1 promoter of Antenna-pedia (Antp), a homeotic gene Ubx is known to repress from genetic studies. The binding sites occur in clusters downstream of the transcription start sites, and far upstream at Antp P1. They range in size from 40 to 90 bp, and contain tandem repeats of the trinucleotide TAA or the related hexanucleotide TAA-TCG. These results suggest that the regulatory activities of Ubx are direct and are mediated by binding of Ubx proteins to promoter region sequences.

Animals↗
PubMed1985

Recombination site selection by Tn3 resolvase: topological tests of a tracking mechanism.

In vitro recombination by Tn3 resolvase of plasmids containing two directly repeated recombination (res) sites generates two singly interlinked catenated rings. This simple product catenane structure was maintained over a wide range of substrate supercoil densities and in a reaction mixture in which phage lambda Int-mediated recombination generated its characteristic multiply interlinked forms. Using substrates containing four res sites, we found that resolvase recombined neighboring res sites with high preference. This position effect implies that resolvase searches systematically along the DNA for a partner site. Intervening res sites in the opposite orientation did not prevent translocation. We analyzed the geometric arrangement of the interlocked rings after multiple recombination events in a four-site substrate and the pattern of segregation of nonspecific reporter rings catenated to the standard substrate. The results of these novel topological tests imply that the translocating enzyme may not make continuous contact with the DNA.

DNA Topoisomerases, Type II↗