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J T Lis

Publications and source records attributed to J T Lis.

At least 73 records · Page 4Linked to original sources
PubMed1988

The RNA polymerase II molecule at the 5' end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged.

Protein-DNA cross-linking of cultured Drosophila cells has shown that, in vivo, prior to the induction of heat shock, there is approximately one molecule of RNA polymerase II associated with the promoter region of the major heat shock gene, hsp70. Here, we show that this promoter-associated RNA polymerase II molecule is transcriptionally engaged and has formed a nascent RNA chain of approximately 25 nucleotides in length, but is apparently arrested at that point and unable to penetrate further into the hsp70 gene without heat induction. The detection of a post-initiation RNA polymerase complex on the promoter region of the inactive gene suggests that there is a transcriptional control mechanism that acts at a step early in transcript elongation.

Animals↗
PubMed1988

Germline transformation used to define key features of heat-shock response elements.

The heat-shock consensus element (HSE), CTNGAANNTTCNAG, is found in multiple copies upstream of all heat-shock genes. Here, the sequence requirements for heat-shock induction are tested by Drosophila germline transformation with an hsp70-lacZ gene fused to a pair of synthetic HSEs. Certain single-base substitutions in either HSE cause a dramatic reduction (forty-fold) in expression. Surprisingly, variations in sequences immediately flanking the HSEs also reduced levels of induction. One such variant that contains two perfect 14-base pair HSEs, which are correctly spaced relative to each other and the TATA box, retained only 7% of wild type-induced expression. These and additional analyses indicate that the heat-shock regulatory element includes sequences beyond the 14-base pair HSE and may be better described as a dimer of a 10-base pair sequence, NTTCNNGAAN.

Animals↗
PubMed1988

Localized heat-shock induction in Drosophila melanogaster.

We describe a technique for inducing localized expression of genes fused to heat-shock gene promoters. We demonstrate that a localized heat-shock response can be induced in Drosophila melanogaster at any developmental stage after formation of the cellular blastoderm by contacting a region of the animal with a heated needle. The size of the induced region can be altered by varying parameters such as the temperature and size of the needle tip. The test system utilized here is a D. melanogaster strain transformed with a fusion of the Drosophila hsp26 gene and the E. coli lacZ gene; the activity of this hybrid gene is monitored in whole animals by staining for beta-galactosidase activity. Induced beta-galactosidase activity is confined to the cells in the region of heating; the beta-galactosidase activity can still be detected 48 hr after the heat shock. Given the heat inducibility of Drosophila heat-shock promoters in heterologous systems, we suggest that this technique will be useful for allowing spatially controlled induction of a gene of interest in any organism into which fusion genes can be introduced. Additional uses of the technique for following cell movements during development are discussed.

Animals↗
PubMed1988

The positive and negative transcriptional regulation of the Drosophila Gapdh-2 gene.

One of the genes encoding glyceraldehyde 3-phosphate dehydrogenase of Drosophila melanogaster, Gapdh-2, is expressed in all cell types examined, but its level of expression is regulated developmentally. Here we report the analysis of the regulatory sequences for the transcription of Gapdh-2. We have generated Gapdh-2-LacZ fusion genes in which the 5'-flanking sequence of Gapdh-2 has been mutated. Examination of the expression of these fusion genes, which have been introduced by transfection into the Schneider II cell line and by germ-line transformation into flies, led to the identification of two distinct regulatory regions, URS-1 and URS-2, within the first 145 bp of the 5'-flanking sequence of Gapdh-2. URS-1 activates transcription throughout the development of Drosophila. However, URS-2 exhibits a dual function during the development. It clearly represses transcription in Schneider II cells, and perhaps also in mid-stage embryos. In contrast, it activates transcription in larvae and adult flies. Thus, URS-2 plays a key role in the developmental regulation of Gapdh-2. Additionally, the efficient transcription of Gapdh-2 in larval and adult stages appears to depend on a synergistic function of URS-1 and URS-2.

Animals↗
PubMed1988

Structure and expression of ubiquitin genes of Drosophila melanogaster.

We isolated and characterized two related ubiquitin genes from Drosophila melanogaster, polyubiquitin and UB3-D. The polyubiquitin gene contained 18 repeats of the 228-base-pair monomeric ubiquitin-encoding unit arranged in tandem. This gene was localized to a minor heat shock puff site, 63F, and it encoded a constitutively expressed 4.4-kilobase polyubiquitin-encoding mRNA, whose level was induced threefold by heat shock. To investigate the pattern of expression of the polyubiquitin gene in developing animals, a polyubiquitin-lacZ fusion gene was introduced into the Drosophila genome by germ line transformation. The fusion gene was expressed at high levels in a tissue-general manner at all life stages assayed. The ubiquitin-encoding gene, UB3-D, consisted of one ubiquitin-encoding unit directly fused, in frame, to a nonhomologous tail sequence. The amino acid sequence of the tail portion of the protein had 65% positional identity with that of yeast UBI3 protein, including a region that contained a potential nucleic acid-binding motif. The Drosophila UB3-D gene hybridized to a 0.9-kilobase mRNA that was constitutively expressed, and in contrast to the polyubiquitin gene, it was not inducible by heat shock.

Amino Acid Sequence↗
PubMed1988

Sex-specific control of Drosophila melanogaster yolk protein 1 gene expression is limited to transcription.

The sex of Drosophila melanogaster is determined by a hierarchy of genes. The ultimate targets of this regulatory hierarchy are the genes encoding terminal differentiation products of one sex. For one of the best-characterized target genes, that encoding female-specific yolk protein 1 (YP1), sex-specific transcriptional controls have been clearly demonstrated. In addition, sex-specific posttranscriptional controls were suggested from experiments in which YP1 RNA was induced in males with hormones. To determine whether males can efficiently process and translate a transcript which is normally found only in females, we used a non-sex-specific promoter, the hsp70 gene promoter, to drive YP1 gene transcription in germ line transformed males. The efficiency of expression of the YP1 gene at levels of RNA splicing, translation, and protein secretion in these males was compared with that in wild-type females. These experiments show that there are no sex-specific posttranscriptional controls operating to limit the production of secreted YP1 in males. Promoters containing different numbers of heat shock elements were tested for their ability to drive YP1 gene transcription in males. These results show that incompatibility between the hsp70 gene heat shock elements and the YP1 gene promoter can be overcome by increasing the amount of hsp70 gene sequence up or downstream of the TATA box. In the course of this study, two vectors useful for placing genes under heat shock regulation were constructed. One of these vectors is designed so that the heat-induced transcript produced is the "authentic" primary transcript; it should be useful for studies of posttranscriptional regulation.

Animals↗
PubMed1987

A germline transformation analysis reveals flexibility in the organization of heat shock consensus elements.

Maximal expression of the Drosophila heat shock gene hsp70 can be activated by a pair of heat shock consensus elements (HSE's) positioned close to the transcription start site. In contrast, required HSE's of other heat shock genes (i.e., hsp26, 27, 23) are located several hundred base pairs (bp) farther upstream of their start sites. Using germline transformation, we analyzed the requirements for HSE organization in the hsp70 and hsp26 regulatory regions. A 51 bp fragment containing the two proximal hsp70 HSE's was sufficient to rescue the heat shock response of an hsp26-lacZ gene devoid of its HSE's. Heat inducibility was restored with either orientation of the fragment relative to the hsp26 transcription start. In hsp70 gene constructions, relocation of hsp70 HSE's to more remote positions by inserting 127 or 331 bp into the regulatory region failed to substantially reduce expression. Thus, in contrast to their native configurations, the hsp26 promoter can be activated by HSE's solely in a proximal position and the hsp70 promoter can be activated by remote HSE's. In addition, a simple and sensitive assay for quantitative measurement of beta-galactosidase activity in crude fly extracts is described.

Animals↗
PubMed1987

Protein-DNA cross-linking reveals dramatic variation in RNA polymerase II density on different histone repeats of Drosophila melanogaster.

In Drosophila melanogaster the five histone genes are within a 5-kilobase region which is repeated 100 times at a single chromosomal site. These 5-kilobase repeats are of two distinct classes, short and long, that differ by approximately 200 base pairs of DNA in the spacer region between the H1 and H3 genes. Since the mRNA-homologous regions of the repeats are highly conserved, one cannot examine differential expression of the repeats by classical hybridization methods. In this study, we assessed their transcriptional activity by measuring in vivo the relative amounts of RNA polymerase II that were cross-linked by UV irradiation to the two different histone repeats. The RNA polymerase II density on the long repeat in Schneider line 2 cells was strikingly lower (10-fold) than the density on the short repeat. The magnitude of this difference cannot be accounted for by reduced transcription of only one or two genes of the repeat. The density of topoisomerase I, an indicator of transcriptional activity, was also much higher on the short repeat than on the long repeat of line 2 cells. In contrast, the RNA polymerase II density was slightly higher on the long repeat than on the short repeat in a second cell line, KcH. The major difference between active (KcH) and inactive (S2) long repeats resides in the H1-H3 nontranscribed spacer. This portion of the spacer may contain a component necessary for expression that can act over a moderate distance and affect multiple genes of the repeat.

Animals↗
PubMed1986

Expression of the major heat shock gene of Drosophila melanogaster in Saccharomyces cerevisiae.

A copy of the gene which encodes the major heat shock protein (hsp70) of D. melanogaster was integrated in both orientations into the genome of S. cerevisiae at the leu2 locus. The level of transcript from the D. melanogaster gene was measured under both normal conditions and conditions which are known to give rise to the heat shock response in S. cerevisiae. In both orientations the D. melanogaster gene gave rise to an abundant transcript in uninduced cells. The level of this transcript was increased transiently on heat shock, peaking after about 30 min at the elevated temperature. The average induction observed was around 5-fold. Although the D. melanogaster gene is heat inducible in S. cerevisiae, the transcripts are initiated at several sites which lie between 10 and 40 base pairs downstream of the initiation site in D. melanogaster. Thus, the transcriptional apparatus of S. cerevisiae appears to recognize the promoter and regulatory elements of the D. melanogaster major heat shock gene, although the manner in which transcription is initiated differs between the two species.

Animals↗
PubMed1986

Topoisomerase I interacts with transcribed regions in Drosophila cells.

The in vivo distribution of topoisomerase I on specific DNA sequences is determined at high resolution in Drosophila cells using a photo-crosslinking method. Topoisomerase I-DNA adducts are generated by irradiation of intact cells with UV light and then purified by immunoprecipitation with antibody to topoisomerase I. Analyses of the DNA sequences crosslinked to topoisomerase I by blot-hybridization with appropriate DNA probes indicate that topoisomerase I is concentrated on transcribed regions and not on nontranscribed flanking sequences. Like RNA polymerase II, topoisomerase I is recruited to heat-shock genes during the heat-shock response. However, topoisomerase I and RNA polymerase II can interact independently with the transcribed region because different ratios of topoisomerase I and RNA polymerase II are crosslinked to the highly transcribed hsp70 gene and the moderately transcribed copia genes. We hypothesize that topoisomerase I allows topological changes in DNA that are required for transcription.

Animals↗
PubMed1986

Spatial and temporal pattern of hsp26 expression during normal development.

The tissue-specific patterns of developmental expression of hsp26-lacZ fusion genes inserted into Drosophila melanogaster by germline transformation were analyzed in several transformant lines utilizing a histochemical assay for beta-galactosidase activity on whole animals. We compared this pattern to the tissue-specific distribution of endogenous hsp26 RNA determined using hybridization of probes to RNA in situ in tissue sections. Both assays reveal that hsp26 is expressed in numerous tissues during development including spermatocytes, nurse cells, epithelium, imaginal discs, proventriculus and neurocytes. The ease and resolution of the whole-animal beta-galactosidase assay makes it particularly attractive for the elucidation of sequences involved in such complex regulation. The original hsp26-lacZ fusion gene contained 2 kb of sequence upstream of the transcription start. A construct containing only 278 bp upstream was still expressed in spermatocytes but no longer in nurse cells. In a few instances, the fusion genes were expressed in tissues for which there was no evidence for expression of the endogenous hsp26 gene. These novel patterns appear to be a result of chromosomal position since they were observed in only one or a subset of transformant lines containing identical inserts.

Animals↗
PubMed1986

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.

Animals↗
PubMed1986

A consensus sequence polymer inhibits in vivo expression of heat shock genes.

Promoter function for hsp70 gene expression in Drosophila melanogaster was studied with an in vivo competition assay. A polymer of 40 tandem copies of the pair of regulatory elements of the hsp70 gene was constructed and cloned into a plasmid vector. Various marked genes were cotransfected with the polymer plasmid into Schneider line 2 cells, and their expression was determined by enzyme activity measurements. The polymer dramatically inhibited expression of cotransfected hsp70, hsp26, and hsp83 genes, but not cotransfected copia and histone genes. Our results indicate that in vivo, a trans-acting, positive regulatory factor, which can be titrated by heat shock consensus sequences, is required for activation of heat shock genes and is specific for these genes; the coordinate induction of different heat shock genes appears to be mediated by similar, but not identical, interactions of the trans-acting induction factor and the cis-acting heat shock consensus sequences; and the uninduced or basal level expression of the transfected hsp70 gene is also due to interaction of the consensus sequence with a positively acting factor.

Acetyltransferases↗
PubMed1985

Localization and expression of transformed DNA sequences within heat shock puffs of Drosophila melanogaster.

In situ hybridization at high resolution with biotin-labeled DNA was used to locate specific transcriptional units within the chromosomal puffs of normal heat shock loci and of new heat shock loci generated by transformation. This method resolves copies of the hsp70 gene that are separated by 40 kb within the 87C heat shock locus. In the case of two new puff loci generated by transformation, the heat-activated transcript is encoded by sequences that reside entirely within the puff domain and the activated promoter is positioned asymmetrically within the puff. However, an adjacent promoter and its transcriptional unit which do not appear to be induced by heat shock are also within the puff. These results support the idea that a chromosomal puff is a structure that facilitates or results from vigorous transcription, but that the structural change alone is insufficient to induce transcription.

Animals↗
PubMed1985

Determinants of heat shock-induced chromosome puffing.

Modified Drosophila heat shock genes were introduced into the germ line by P element transformation. The genes were altered such that several factors could be tested for their influence upon chromosome puffing. Deletion of promoter sequences upstream of position -73 of an hsp70-IacZ hybrid gene was sufficient to abolish puffing. Analysis of progressive 5' deletions defines a 16 bp interval that contains sequences required for both heat-induced puffing and gene expression. An internal deletion of the hsp70-IacZ gene that reduces the transcript size from 9 kb to 0.8 kb results in a dramatic reduction in puff size. The chromosomal insertion sites of 26 variant hsp70 or hsp26 genes fail to influence puffing greatly with one marked exception. This transformant possesses an insert that fails to puff and exhibits a tissue-restricted pattern of expression. These results indicate that variation in either promoter strength or transcript length have profound effects on puffing.

Animals↗
PubMed1985

In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.

We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.

Animals↗
PubMed1984

Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.

We present an approach for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. To test this approach, we examine the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, lambda cI gene, and to the uninduced and isopropyl beta-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. After these initial tests, we examine the distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant, and promoter mutant strains. Our in vivo data are in complete agreement with the predictions of the attenuation model of regulation. From these and other experiments, we discuss the resolution, sensitivity, and generality of these methods.

DNA, Bacterial↗