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J T Lis

Publications and source records attributed to J T Lis.

87 records · Page 5Linked to original sources
PubMed1984

High-resolution mapping of DNase I-hypersensitive sites of Drosophila heat shock genes in Drosophila melanogaster and Saccharomyces cerevisiae.

High-resolution analysis of the chromatin structure of the promoter regions of five Drosophila heat shock genes showed a similar location for the hypersensitive sequences relative to the start of transcription. For each of the five genes examined--those coding for hsp27, hsp26, hsp23, hsp70, and hsp83--the DNase I-hypersensitive sites in Drosophila melanogaster nuclei mapped to two regions upstream of the coding region. These sites occurred on the average, 115 and 17 base pairs upstream from the start of transcription of the five heat shock genes examined. This latter site corresponded to sequences at or near the TATA consensus sequence. Sites even further upstream of the hsp27, hsp26, and hsp83 genes were also evident. Additionally, for the two genes examined--hsp70 and hsp83--the DNase I-hypersensitive sites were preserved, at least within this level of resolution (+/- 10 base pairs), when the Drosophila genes were integrated into the Saccharomyces cerevisiae genome. This result indicates that the signals responsible for generating these hypersensitive sites are inherent in the DNA sequences and, in this case, are not highly species specific.

Animals↗
PubMed1983

Localization of the hsp83 transcript within a 3292 nucleotide sequence from the 63B heat shock locus of D. melanogaster.

We have determined the complete nucleotide sequence of a 3292 bp cloned segment derived from the 63B heat shock cytogenetic locus of D. melanogaster. Within this segment we have positioned the start of transcription and RNA splice sites of the unique gene that encodes the 83,000 d heat shock polypeptide (hsp83 gene) by S1 mapping and synthesis of cDNA from restriction fragment primed mRNA. The sequence begins at a point 879 bp upstream from the transcription start and includes the 149 bp nontranslated first exon, the 1139 bp intron and extends 1125 bp into the protein coding region. These data identify a single open translation reading frame for the first 375 amino acids of the 83,000 d polypeptide, beginning with the first ATG codon located at the 3' intron-exon junction. We discuss and demonstrate the use of E. coli exonuclease III generated single-strand DNA probes as an alternative to strand separation for S1 mapping of mRNA. We also use homology search criteria based upon known protein-DNA binding sites to compare our hsp83 sequence with other sequenced Drosophila heat shock genes. These comparisons indicate that a large region of approximately 80 bp centered around the transcription initiation point of the hsp83 gene shares only a 31% homology with the corresponding region of the hsp70 gene, whereas the hsp22, 23, 26, and 27 genes share a 54% homology with hsp70 in this region. The lower homology of the hsp83 gene is consistent with the deviant nature of this heat shock gene.

Amino Acid Sequence↗
PubMed1983

New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene.

A hybrid gene that consists of the Drosophila heat shock gene, hsp70, fused to the E. coli beta-galactosidase gene has been introduced into the Drosophila germline by the P element microinjection method. This hybrid includes 194 bp of sequence upstream of the start of the hsp70 transcript. Three strains of transformed flies were isolated and characterized by DNA blotting experiments and by in situ hybridization to polytene chromosomes. Strain Bg61 has a single insert of the hybrid gene at the tip of chromosome 3L, site 61A, and the insert consists of a structure that is consistent with P-element-mediated transposition. Strain Bg9,61 has inserts at both 61A and 9E, while Bg64 has a single insert at 64D. Heat shock induces the formation of a large chromosomal puff at all three sites. These puffs appear and regress with kinetics indistinguishable from the puffing of the heat shock locus, 87C, from which the hsp70 gene, used in these studies, was isolated. The beta-galactosidase activity in the transformants is inducible by heat shock and shows a widespread distribution throughout the tissues of larvae and adults.

Animals↗
PubMed1981

Genomic organization and transcription of the alpha beta heat shock DNA in Drosophila melanogaster.

Previous studies have shown that (i) several RNAs induced by heat shock of Drosophila melanogaster cells are homologous to tandemly repeated alpha beta units found in cloned segments of D. melanogaster DNA, and (ii) the alpha beta sequences are present both at a major heat shock locus, 87Cl, and the chromocenter of polytene chromosomes (Lis, J.T., Prestidge, L. and Hogness, D.S. [1978] Cell 14, 901-919). We have used deficiencies that delete DNA from the 87C region to examine the arrangement of alpha beta sequences at this locus and in the centromeric heterochromatin that comprises the chromocenter, and also to determine the chromosomal location of the induced transcription. The tandemly repeated alpha beta units are restricted to the 87C locus. In contrast, the chromocentral alpha beta sequences do not form intact alpha beta units, and are dispersed at heterochromatic sites in some other form. Although only half of the alpha beta DNA is at the 87C locus, essentially all alpha beta transcripts (greater than 99.5%) are derived from this locus.

Animals↗
PubMed1981

Two closely linked transcription units within the 63B heat shock puff locus of D. melanogaster display strikingly different regulation.

We report the isolation and characterization of a cloned DNA of D. melanogaster, Dm4L, that is derived from the major heat shock puff site at 63B. This segment contains two closely linked genes that are each present once per Drosophila haploid genome. One of these, the hsp 83 gene, encodes an abundant heat shock mRNA that, unlike other major heat shock mRNAs, is also abundant in uninduced (23 degrees) kco cells. Although only a slight increase in the level of total hsp 83 RNA can be detected after heat shock in Kco cells, the level of hsp 83 poly(A)+ mRNA increases more than 6-fold and the level of pulse-labeled hsp 83 RNA in total cellular RNA increases 11-fold relative to uninduced cells. In contrast, the levels of total, poly(A)+, and pulse-labeled RNA homologous to the second gene, 63B-T2, are approximately the same in both induced and uninduced cells. Hence, even though these genes are separated by only one thousand base pairs, and, from in situ hybridization to polytene chromosomes, both lie within the heat shock puff, they display strikingly different regulatory properties, These results demonstrate that close linkage of a gene to a heat shock puff is not sufficient to render its expression heat inducible.

Animals↗
PubMed1981

Cloning and characterization of nine heat-shock-induced mRNAs of Drosophila melanogaster.

We have cloned cDNA segments representing nine different genes whose expression is activated by heat-shock treatment of Drosophila melanogaster cells. These nine were selected from a larger population based on the fact that the levels of the polysomal, poly(A)-containing RNA homologous to each of these cDNA segments is significantly greater in induced cells. Among these genes are four that hybridize in situ to major heat-shock puff sites on polytene chromosomes, and five previously uncharacterized genes that hybridize to other sites. Some of these additional sites have been previously implicated as responders to heat shock. Hybridization of in vivo pulse-labeled. total RNA from induced and uninduced cells to specific cDNA segments provided an estimate of both the relative level of different heat-shock transcripts and the inducibility of the nine genes. Although the most abundant RNA species are derived from genes at major heat-shock puff sites, all nine show significant induction. These results indicate that the repertoire of gene expression that is activated or amplified by heat shock is not confined to these major puff sites and that the number of genes involved in the heat-shock response is much larger than the seven commonly noted.

Animals↗
PubMed1981

DNA sequence analysis reveals extensive homologies of regions preceding hsp70 and alphabeta heat shock genes in Drosophila melanogaster.

Two kinds of RNA are synthesized at the 87C1 chromosomal locus of Drosophila melanogaster in response to heat shock. One of these codes for the major heat shock protein, hsp70; the other, alphabeta RNA, derives from tandemly repeated alphabeta units consisting of adjacent alpha and beta DNA elements and has no identified translation product. Another DNA element, gamma, flanks the 5' ends of some alphabeta units. Here we report the complete nucleotide sequence of the 617-base-pair alpha and the 733-base-pair gamma element as well as a portion of the longer beta element. Sequence comparisons between the gamma element and the two hsp70 genes at 87C1 reveal that the 406 base pairs of gamma immediately upstream from the 5' end of the alphabeta unit exhibit 97.5% homology with the sequences at and upstream from the 5' end of the hsp70 genes. A similar homology also exists between gamma and an hsp70 gene present at another heat shock locus, 87A7, which contains no alphabeta units. These results, in conjunction with previous observations, strongly suggest that the coordinate induction by heat shock of the hsp70 and alphabeta genes is a consequence of their homologous 5' flanking sequences. We propose that this extraordinary degree of sequence conservation stems from the recent transposition of alphabeta DNA to the 87C1 locus, an event that brought alphabeta sequences adjacent to, and under the regulation of, the hsp70 control element.

Journal Article↗
PubMed1978

A novel arrangement of tandemly repeated genes at a major heat shock site in D. melanogaster.

Three cloned segments of Drosophila melanogaster DNA have been isolated that derive from the major heat shock site at 87C in chromosome 3. Each of these segments contains sequences homologous to a class of polysomal poly(A)+ RNAs whose synthesis is induced by heat shock of cultured cells. A combination of R loop, heteroduplex and restriction fragment maps of these segments reveals that their RNA-homologous sequences are arranged in tandemly repeated units, each unit consisting of an alpha element (0.49 kb) joined to a beta element (1.10 kb). The polysomal RNAs homologous to these alphabeta units (1.59 kb) are distributed into three size classes exhibiting approximate lengths of 1, 2 and 3 kb. R loop mapping demonstrated that the sequence of the 2 kb RNA is alphabetaalpha, indicating that it, and presumably the 3 kb RNA, derive from transcripts covering more than one repeated unit. One of the cloned segments contains a variant repeat unit, alphagamma, located between two alphabeta units. This unit has the same alpha element, but the beta element has been replaced by a nonhomologous gamma element (0.87 kb). Analysis of the total D. melanogaster DNA indicates that the 87C locus contains at least 21 tandemly repeated units, twelve of which were identified as alphabeta units and six as alphagamma units. The 21 or more units are distributed among at least three different tandem arrays separated by different spacer regions, one of which is within a cloned segment. Sequences in the gamma element, but not those in the alpha or beta elements, are also found at 87A, which contains another heat shock site. The possible roles of the alphabeta-type RNAs and of the gamma sequences are discussed in the light of these results.

Animals↗
PubMed1975

Size fractionation of double-stranded DNA by precipitation with polyethylene glycol.

We show that DNA molecules of differing molecular mass are separable by selective precipitation with polyethylene glycol (PEG+.. Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA. Double-stranded DNA can be fractionated at least in the range of 3 times 10-7 to 1 times 10-5 daltons. The effects on PEG concentration, sodium chloride concentration, DNA concentration, pH, divalent ions, precipitation time, and centrifugal force have been determined. These studies show PEG precipitation offers a size fractionation method for DNA which is convenient, of high capacity, and applicable over a wide range of conditions. However, resolution is not high and separation of two species approaches 100% only if they differ in molecular mass by at least a factor of two.

Cations, Divalent↗
PubMedDate not supplied

A hypersensitive site in hsp70 chromatin requires adjacent not internal DNA sequence.

Nuclease-hypersensitive sites in chromatin exist at the 5' side of many eukaryotic genes. To gain some understanding of the molecular basis of these hypersensitive sites, we have now examined the pair of sites upstream of the Drosophila hsp70 gene in a series of plasmids that contain deletions in the hypersensitive region and have been transformed into yeast cells. Hypersensitive sites 5' to a Drosophila hsp70 gene are preserved when this gene is introduced into yeast by transformation. We find that a yeast strain containing a plasmid in which the deletion extends through the first hypersensitive site still displays the normal pair of hypersensitive sites, so DNA sequences over which the first hypersensitive site is centred are not required for hypersensitivity at this position and the site can form over a foreign DNA sequence juxtaposed against this deletion end point. Deletions progressing further into the region bracketed by the pair of 5' hypersensitive sites eliminate the first hypersensitive site and alter the downstream site. We propose that the hypersensitive sites are generated through the binding of a protein that renders flanking sequences more accessible to nucleases, perhaps by preventing normal chromatin packaging.

Animals↗