J. Jose Bonner

The use of promoter fusions in Drosophila genetics: Isolation of mutations affecting the heat shock response

We have constructed a gene fusion using the promoter of Drosophila hsp70 and the structural gene for Drosophila alcohol dehydrogenase (Adh) and used this construct to transform Adh-deficient flies. In these transformants, Adh is expressed only after heat shock. Like hsp70 itself, this heat-shock-inducible Adh (Adhhs) is induced in a wide variety of tissues. It fails to be induced in primary spermatocytes. Although the tissue distribution of Adh activity is very different from wild type, this does not appear to be deleterious. Indeed, the induction of Adhhs allows flies to survive exposure to ethanol. We have used this latter characteristic to select dominant, trans-acting mutations that alter the response of flies to heat shock.

RNA polymerase II transcribes all of the heat shock induced genes of Drosophila melanogaster

Heat shock of Drosophila melanogaster induces the transcription of a small number of RNAs. Some of these encode protein products, but not all. We have investigated whether the several induced RNAs are transcribed by RNA polymerase II or by some other RNA polymerase. Immunochemical staining of polytene chromosomes indicates that, on heat shock, RNA polymerase II is relocalized; it “migrates” from previously-active transcription sites to the heat shock induced loci. All heat shock induced puffs show immunochemical staining. Such staining correlates with RNA polymerase II activity as judged by the sensitivity of RNA synthesis at these sites to low concentrations of a-amanitin. Thus the protein-coding and non-protein-coding heat shock-induced RNAs are transcribed by this polymerase specifically. We have also identified several non-puffed chromosomal sites at which RNA synthesis is induced by heat shock.

Mutations that induce the heat shock response of Drosophila

We have isolated a number of mutations in D. melanogaster that result in the constitutive expression of the heat shock response in a tissue-specific manner. These mutations induce alcohol dehydrogenase (ADH) when the ADH structural gene is fused to the promoter for the 70 kd heat shock protein (hsp70) gene. Flies carrying these mutations, the hsp70-Adh fusion, and a deletion in their endogenous Adh genes are ethanol tolerant and exhibit elevated ADH levels. Several of the tissue-specific mutations have also been shown to induce an hsp26-Adh fusion gene in trans. The mutation Act88FKM75, a G----A transition in the indirect flight muscle-specific actin gene, also exhibits this phenotype. Comparisons with the Act88FKM75 mutation suggest that the tissue-specific mutations induce the heat shock response by disrupting the physiology of the cells in which the variant gene product is expressed.

Induction of Drosophila heat-shock puffs in isolated polytene nuclei.

Abstract Procedures are given for the isolation of active nuclei from salivary glands of Drosophila melanogaster . Incubation of nuclei with extracts from “heat-shocked,” cultured Drosophila cells induced chromosomal puffing at the cytologically identified “heat-shock” loci. This procedure thus enables one to assay for the heat-shock transcriptional regulators. Partial fractionation of inducing extracts indicates that some puffs (63C, 93D, and 63F) can be induced in vitro under conditions which do not induce others (87A and 87C). This finding is consistent with the notion that these loci are differentially regulated in vivo , perhaps by different mechanisms.

Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisme

SummaryThe HSF1 gene of Saccharomyces cerevisiae directs the synthesis of the heat shock transcription factor, HSF. The gene is essential; disruption mutations are lethal. Using a plasmid shuffle screen, we isolated mutations in the HSF1 gene after in vitro mutagenesis of plasmid DNA with hydroxylamine. From a collection of both conditional (temperature-sensitive) and unconditional lethal mutations, we recovered mutations that map exclusively to the 5′ half of the gene. All are nonsense mutations, including conditional mutations that map 5′ to the portion of the HSF1 gene that encodes the DNA-binding domain of the transcription factor. For one such mutation, we demonstrated that the nonsense mutation is subject to translational readthrough, even though there are no known nonsense suppressors in the genetic background of our strain. Our results suggest that the HSF protein is highly tolerant of amino acid changes, a conclusion that is consistent with the very low degree of evolutionary conservation among HSF proteins. Our results also suggest that translational readthrough occurs with moderate efficiency in yeast, particularly when the terminator codon is followed immediately by an A or C residue. This result illustrates that the inference of gene function from mutant phenotype depends critically upon the analysis of a true null allele, and not merely an amber or ochre allele.

Vectors for the expression and analysis of DNA-binding proteins in yeast

A series of 13 vectors is described. All are yeast centromere plasmids with the LEU2 gene for selection in yeast, and pUC19 sequences for growth in Escherichia coli. All contain the GAL1 promoter directing transcription into a multiple cloning site (MCS). For twelve of the plasmids, synthetic oligodeoxyribonucleotides create an ATG start codon, in a productive context for yeast, prior to the MCS. Spacing between the ATG and the MCS is variable, to facilitate the cloning of gene fragments in the appropriate reading frame. Nine of the plasmids also contain the strong transcriptional activator from the herpes simplex virus VP16 gene, joined downstream from the MCS. In these nine vectors, all possible combinations of reading frames are available. The suitability of these plasmids for the expression and analysis of DNA-binding domains is tested by cloning into them fragments of the yeast HSF1 gene, encoding the heat shock transcription factor (HSF). The regulation of reporter gene expression by the chimeric HSF-VP16 fusions is described, as is the utility of these vectors for other applications.

Clonal analysis of two mutations in the large subunit of RNA polymerase II of Drosophila

SummaryTwo mutations in the gene, RpII215, were analyzed to determine their effects on cell differentiation and proliferation. The mutations differ in that one, RpII215ts(ts), only displays a conditional recessive lethality, while the other, RpII215Ubl (Ubl), is a recessive lethal mutation that also displays a dominant mutant phenotype similar to that caused by the mutation Ultrabithorax (Ubx). Ubl causes a partial transformation of the haltere into a wing; however, this transformation is more complete in flies carrying both Ubl and Ubx. The present study shows that patches of Ubl/- tissue in gynandromorphs are morphologically normal. Cuticle that has lost the wild-type copy of the RpII215 locus fails to show a haltere to wing transformation, nor does it show the synergistic enhancement of Ubx by Ubl. We conclude that an interaction between the two RpII215 alleles, Ubl and RpII215+, is responsible for the mutant phenotype. Gynandromorphs carrying the ts allele, when raised at permissive temperature, display larger patches of ts/- cuticle than expected, possibly indicating that the proliferation of ts/+ cells is reduced. This might result from an antagonistic interaction between different RpII215 alleles. Classical negative complementation does not appear to be the cause of the antagonistic interaction described above, as only one RpII215 subunit is thought to be present in an active multimeric polymerase enzyme. We have therefore coined the term ‘negative heterosis’ to describe the aforementioned interactions.We also observed that the effects of mutationally altered RNA polymerase II on somatic cells are different from its effects on germ cells. Mutant somatic cells (either Ubl/- or ts/-, the latter shifted to restrictive temperature) reduce cell proliferation, but otherwise do not appear to disrupt cell differentiation. However, mutant germ cells often differentiate into morphologically abnormal oocytes.