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M A Krasnow

Publications and source records attributed to M A Krasnow.

At least 37 records · Page 2Linked to original sources
PubMed1996

The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system.

We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth.

Animals↗
PubMed1996

Development of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events.

The tracheal (respiratory) system of Drosophila melanogaster is a branched network of epithelial tubes that ramifies throughout the body and transports oxygen to the tissues. It forms by a series of sequential branching events in each hemisegment from T2 to A8. Here we present a cellular and initial genetic analysis of the branching process. We show that although branching is sequential it is not iterative. The three levels of branching that we distinguish involve different cellular mechanisms of tube formation. Primary branches are multicellular tubes that arise by cell migration and intercalation; secondary branches are unicellular tubes formed by individual tracheal cells; terminal branches are subcellular tubes formed within long cytoplasmic extensions. Each level of branching is accompanied by expression of a different set of enhancer trap markers. These sets of markers are sequentially activated in progressively restricted domains and ultimately individual tracheal cells that are actively forming new branches. A clonal analysis demonstrates that branching fates are not assigned to tracheal cells until after cell division ceases and branching begins. We further show that the breathless FGF receptor, a tracheal gene required for primary branching, is also required to activate expression of markers involved in secondary branching and that the pointed ETS-domain transcription factor is required for secondary branching and also to activate expression of terminal branch markers. The combined morphological, marker expression and genetic data support a model in which successive branching events are mechanistically and genetically distinct but coupled through the action of a tracheal gene regulatory hierarchy.

Animals↗
PubMed1996

The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered.

The adult Drosophila wing is formed by an epithelial sheet, which differentiates into two non-neural tissues, vein or intervein. A large number of genes, many of them encoding components of an EGF-receptor signaling pathway, have previously been shown to be required for differentiation of vein tissue. Much less is known about the molecular control of intervein differentiation. Here we report that the Drosophila homolog of the mammalian Serum Response Factor gene (DSRF), which encodes a MADS-box containing transcriptional regulator, is expressed in the future intervein tissue of wing imaginal discs. In adult flies carrying only one functional copy of the DSRF gene, additional vein tissue develops in the wing, indicating that DSRF is required to spatially restrict the formation of veins. In mitotic clones lacking DSRF, intervein tissue fails to differentiate and becomes vein-like in appearance. Genetic and molecular evidence demonstrates that DSRF is encoded by the blistered locus, which produces ectopic veins and blistered wings when mutant. Our results show that DSRF plays a dual role during wing differentiation. It acts in a dosage-dependent [correction of dosage-dependant] manner to suppress the formation of wing veins and is required cell-autonomously to promote the development of intervein cells. We propose that DSRF acts at a key step between regulatory genes that define the early positional values in the developing wing disc and the subsequent localized expression of intervein-specific structural genes.

Alleles↗
PubMed1995

Extent and character of circadian gene expression in Drosophila melanogaster: identification of twenty oscillating mRNAs in the fly head.

BACKGROUND: Although mRNAs expressed with a circadian rhythm have been isolated from many species, the extent and character of circadianly regulated gene expression is unknown for any animal. In Drosophila melanogaster, only the period (per) gene, an essential component of the circadian pacemaker, is known to show rhythmic mRNA expression. Recent work suggests that the encoded Per protein controls its own transcription by an autoregulatory feedback loop. Per might also control the rhythmic expression of other genes to generate circadian behavior and physiology. The goals of this work were to evaluate the extent and character of circadian control of gene expression in Drosophila, and to identify genes dependent on per for circadian expression. RESULTS: A large collection of anonymous, independent cDNA clones was used to screen for transcripts that are rhythmically expressed in the fly head. 20 of the 261 clones tested detected mRNAs with a greater than two-fold daily change in abundance. Three mRNAs were maximally expressed in the morning, whereas 17 mRNAs were most abundant in the evening--when per mRNA is also maximally expressed (but when the flies are inactive). Further analysis of the three 'morning' cDNAs showed that each has a unique dependence on the presence of a light-dark cycle, on timed feeding, and on the function of the per gene for its oscillation. These dependencies were different from those determined for per and for a novel 'evening' gene. Sequence analysis indicated that all but one of the 20 cDNAs identified previously uncloned genes. CONCLUSIONS: Diurnal control of gene expression is a significant but limited phenomenon in the fly head, which involves many uncharacterized genes. Diurnal control is mediated by multiple endogenous and exogenous mechanisms, even at the level of individual genes. A subset of circadianly expressed genes are predominantly or exclusively dependent on per for their rhythmic expression. The per gene can therefore influence the expression of genes other than itself, but for many rhythmically expressed genes, per functions in conjunction with external inputs to control their daily expression patterns.

Amino Acid Sequence↗
PubMed1995

Extradenticle protein is a selective cofactor for the Drosophila homeotics: role of the homeodomain and YPWM amino acid motif in the interaction.

The Drosophila homeotic selector (HOM) genes encode a family of DNA binding transcription factors that specify developmental fates of different body segments by differentially regulating the activity of downstream target genes. A central question is how the HOM proteins achieve their developmental specificity despite the very similar DNA binding specificities of isolated HOM proteins in vitro. Specificity could be achieved by differential interactions with protein cofactors. The extradenticle gene might encode such a cofactor since it interacts genetically in parallel with Ultrabithorax, abdominal-A, and perhaps other HOM genes. By using a yeast two-hybrid system, we demonstrate selective interaction of the extradenticle homeodomain protein with certain Ultrabithorax and abdominal-A proteins but not with an Antennapedia protein or a more distant homeodomain protein. Strong interaction with Ultrabithorax proteins requires only the Ultrabithorax homeodomain and a 15-residue N-terminal extension that includes Tyr-Pro-Trp-Met (YPWM), a tetrapeptide motif found near the homeodomain in most HOM proteins and their mammalian Hox counterparts. The size and sequence of the region between the YPWM element and the homeodomain differ among Ultrabithorax isoforms, and this variable region appears to affect the interaction detected in the assay.

Amino Acid Sequence↗
PubMed1994

A differential response element for the homeotics at the Antennapedia P1 promoter of Drosophila.

Homeotic genes encode DNA-binding transcription factors that specify the identity of a segment or segments in particular body regions of Drosophila. The developmental specificity of these proteins results from their differential regulation of various target genes. This specificity could be achieved by use of different regulatory elements by the homeoproteins or by use of the same elements in different ways. The Ultrabithorax (UBX), abdominal-A (ABD-A), and Antennapedia (ANTP) homeoproteins differentially regulate the Antennapedia P1 promoter in a cell culture cotransfection assay: UBX and ABD-A repress, whereas ANTP activates P1. Either of two regions of P1 can confer this pattern of differential regulation. One of the regions lies downstream and contains homeoprotein-binding sites flanking a 37-bp region called BetBS. ANTP protein activates transcription through the binding sites, whereas UBX and ABD-A both activate transcription through BetBS and use the flanking binding sites to prevent this effect. Thus, homeoproteins can use the same regulatory element but in very different ways. Chimeric UBX-ANTP proteins and UBX deletion derivatives demonstrate that functional specificity in P1 regulation is dictated mainly by sequences outside the homeodomain, with important determinants in the N-terminal region of the proteins.

Amino Acid Sequence↗
PubMed1993

Intercellular signalling in Drosophila segment formation reconstructed in vitro.

Genetic studies show that intercellular signalling is involved in key steps in Drosophila melanogaster development, but it has not previously been possible to investigate these processes in simplified in vitro systems. Analysis of engrailed (en) and wingless (wg) and other segment polarity genes suggests that two or more intercellular signalling processes may be involved in intrasegmental patterning. Expression of en and wg begins about three hours after egg laying, in adjacent rows of cells in the posterior half of each segmental primordium. In wg- embryos and in conditional mutants in which wg function is inactivated during a critical period between three and five hours after egg laying, early en expression begins normally but then disappears within several hours. The wg gene encodes a protein highly similar to the product of the mouse Wnt-1 proto-oncogene, a secreted glycoprotein; wg protein is proposed to function as an extracellular signal, maintaining en expression and activating other molecular and morphogenetic processes in nearby cells. Several lines of evidence support the model, including the secretion of wg protein in the embryo, genetic mosaic experiments and cell lineage studies. We tested this model using purified embryonic cells isolated by whole animal cell sorting, and validated three key predictions: (1) when en-expressing cells from early embryos are grown alone in culture, they rapidly and selectively lose en expression; (2) purified wg-expressing cells provide a locally active signal that prevents this loss; (3) heterologous cells engineered to express wg also show signalling activity, indicating that wg protein alone, or in conjunction with more generally expressed factors, is the signal.

Animals↗
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↗
PubMed1983

Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites.

We studied the dynamics of site-specific recombination by the resolvase encoded by the Escherichia coli transposon Tn3. The pure enzyme recombined supercoiled plasmids containing two directly repeated recombination sites, called res sites. Resolvase is the first strictly site-specific topoisomerase. It relaxed only plasmids containing directly repeated res sites; substrates with zero, one or two inverted sites were inert. Even when the proximity of res sites was ensured by catenation of plasmids with a single site, neither relaxation nor recombination occurred. The two circular products of recombination were catenanes interlinked only once. These properties of resolvase require that the path of the DNA between res sites be clearly defined and that strand exchange occur with a unique geometry. A model in which one subunit of a dimeric resolvase is bound at one res site, while the other searches along adjacent DNA until it encounters the second site, would account for the ability of resolvase to distinguish intramolecular from intermolecular sites, to sense the relative orientation of sites and to produce singly interlinked catenanes. Because resolvase is a type 1 topoisomerase, we infer that it makes the required duplex bDNA breaks of recombination one strand at a time.

DNA Topoisomerases, Type I↗