The in vitro DNA binding properties of wild‐type and mutant fushi tarazu homeodomains (ftz HD) have been analysed. The DNA binding properties of the ftz HD are very similar to those of the Antp HD. In interference experiments with mutant ftz HDs, close approaches between specific portions of the ftz HD peptide and specific regions of the binding site DNA were mapped. A methylation interference, G7 on the beta strand of BS2, is absent from the interference pattern with a mutant ftz HD [ftz (R43A) HD] in which the Arg43 at the second position of helix III (the recognition helix) is replaced by an Ala. This indicated that Arg43 of the ftz HD is in close proximity to the N7 of G7 of the beta strand of BS2 in the major groove. The methylation and ethylation interference patterns with the ftz (NTD) HD, in which the first six amino acids of the homeodomain were deleted, were extensively altered relative to the ftz HD patterns. Methylation of A11 and G12 of the alpha strand and ethylation of the phosphate of nucleotide A12 of the alpha strand no longer interfere with binding. This indicated that the first six amino acids of the homeodomain of ftz interact with A11 of the alpha strand in the minor groove, the phosphate of the nucleotide A13 on the alpha strand and G12 of the alpha strand in the adjacent major groove of BS2. In a binding study using a change of specificity mutation [ftz (Q50K) HD], in which the Gln50 at the ninth position of the third helix is exchanged for a Lys (as in the bicoid HD), and variant binding sites, we concluded that position 50 of the ftz HD and the ftz (Q50K) HD peptides interacts with base pairs at positions 6 and 7 of BS2. These three points of contact allowed us to propose a crude orientation of the ftz HD within the protein‐DNA complex. We find that the ftz HD and the Antp HD peptides contact DNA in a similar way.
Nuclear hormone receptors and homeodomain proteins are two classes of transcription factor that regulate major developmental processes. Both depend on interactions with other proteins for specificity and activity. The Drosophila gene fushi tarazu (ftz), which encodes a homeodomain protein (Ftz), is required zygotically for the formation of alternate segments in the developing embryo. Here we show that the orphan nuclear receptor alphaFtz-F1 (ref. 3), which is deposited in the egg during oogenesis, is an obligatory cofactor for Ftz. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. This interaction suggests that other nuclear receptor/homeodomain protein interactions maybe important and common in developing organisms.
The in vitro DNA binding properties of a purified 68-amino acid Antennapedia homeodomain (Antp HD) peptide have been analyzed. Equilibrium and kinetic binding studies showed that stable DNA-protein complexes are formed with a Kd of 1.6 x 10(-9) M and 1.8 x 10(-10) M, respectively. Heterodimer analysis led to the conclusion that Antp HD interacts in vitro as a monomer with the DNA target sites used in our study. The results of methylation and ethylation interference studies indicated that the Antp HD closely approaches the target DNA primarily from one side in a region extending across three phosphate backbones. The DNA binding properties of the Antp HD and prokaryotic DNA binding domains that share a helix-turn-helix motif are compared.
A differential hybridization screen has been used to identify genes cloned from the yeast Saccharomyces cerevisiae that are expressed preferentially during sporulation. Duplicate copies of a partial Sau3A yeast DNA library prepared in the vector pBR322 were hybridized with radioactive cDNA probes representing the mRNA populations of sporulating aa cells and asporogenous aao cells at various times after transfer to sporulation medium. Thirty-eight clones showed an enhanced hybridization signal with the aa sporulation probe relative to the aa control cDNA probe. A comparison of the array of fragments produced by restriction endonuclease digestion of these plasmids suggested that 15 different sequences had been cloned. An RNA blot analysis using these cloned DNAs to probe RNAs purified from aa, aa, and aa cells harvested either during vegetative growth or at 10 h after transfer to sporulation medium indicated that 14 different sporulation-specific genes had been identified.
A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulationspecific genes, SPSI, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPSI gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATaIMATa strain homozygous for a disruption of the SPSI gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPSI-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.Sporulation in the yeast Saccharonvyces cerevisiae, initiated upon starvation of MATa/MATax diploid cells, represents a regulated program of differentiation which includes meiosis and the encapsulation of the four haploid nuclei into ascospores. The ease of performing a variety of genetic manipulations in S. cerei'isiae (29) makes sporulation in this organism an appealing eucaryotic system for studying developmentally regulated gene expression. The temporal program of genetic, biochemical, and morphological events occurring during sporulation has been described in detail (6), but little is known about the concomitant program of gene expression presumed to be essential for these events. Although a mutational analysis has identified several genes uniquely required for the successful completion of sporulation (6), the analysis of in vivo-labeled proteins revealed disappointingly few sporulation-specific proteins (11,13,24,30,33 (14,32).In the present study we have characterized a cluster of three sporulation-specific genes previously identified in a differential hybridization screen (23). Ultimately, the elucidation of the manner in which these genes are activated will provide insights into the mechanisms involved in controlling development-specific gene expression. In this report, we have demonstrated by a mutational analysis that a gene which had been deemed sporulation specific on the basis of its expression pattern is indeed functionally required for the * Corresponding author. completion of sporulation, supporting the suggestion that its activation is directly dependent on sporulation-specific regulatory events. MATERIALS AND METHODSStrains and culture conditions. Escherichia coli HB101 was used for the propagation of plasmids. The S. cerevisiae strains used were the AP3 MATotIMATot, MATa/MATa, and MA TaMA Tot diploids (10) previously employed (23), SC252 (MA Tot adel leu2-3 leu2-112 ura3-52) (provided by J. Friesen), SR25-1A (MATa his4-912 ura3-52) (pr...
Observation of how cells divide, grow, migrate and form different parts of a developing organism is crucial for understanding developmental programs. Here, we describe a multicolor imaging tool named Raeppli (after the colorful confetti used at the carnival in Basel). Raeppli allows whole-tissue labeling such that the descendants of the majority of cells in a single organ are labeled and can be followed simultaneously relative to one another. We tested the use of Raeppli in the Drosophila melanogaster wing imaginal disc. Induction of Raeppli during larval stages irreversibly labels >90% of the cells with one of four spectrally separable, bright fluorescent proteins with low bias of selection. To understand the global growth characteristics of imaginal discs better, we induced Raeppli at various stages of development, imaged multiple fixed discs at the end of their larval development and estimated the size of their pouch primordium at those developmental stages. We also imaged the same wing disc through the larval cuticle at different stages of its development; the clones marked by Raeppli provide landmarks that can be correlated between multiple time points. Finally, we used Raeppli for continuous live imaging of prepupal eversion of the wing disc.
The Drosophila segmentation gene fushi tarazu (ftz) encodes a homeodomain-containing protein, ftz, that can act as a DNA-binding activator of transcription. In the developing embryo, ftz is expressed in seven stripes which correspond to the even-numbered parasegments. These parasegments are missing in ftz- embryos. When ftz is expressed throughout blastoderm embryos under the control of a heat-shock promoter, the odd-numbered parasegments are lost. This 'anti-ftz' phenotype has been attributed to autoactivation of the endogenous ftz gene by the ectopically expressed protein. Here we show that the same phenotype is induced by ectopic expression of a ftz polypeptide containing a deletion in the homeodomain. Thus, ftz can alter gene expression without binding directly to DNA.
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