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J T Lis

Publications and source records attributed to J T Lis.

At least 55 records · Page 3Linked to original sources
PubMed1994

The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development.

B52 is a Drosophila melanogaster protein that plays a role in general and alternative splicing in vitro. It is homologous to the human splicing factor ASF/SF2 which is essential for an early step(s) in spliceosome assembly in vitro and also regulates 5' and 3' alternative splice site choice in a concentration-dependent manner. In vitro, B52 can function as both a general splicing factor and a regulator of 5' alternative splice site choice. Its activity in vivo, however, is largely uncharacterized. In this study, we have further characterized B52 in vivo. Using Western blot (immunoblot) analysis and whole-mount immunofluorescence, we demonstrate that B52 is widely expressed throughout development, although some developmental stages and tissues appear to have higher B52 levels than others do. In particular, B52 accumulates in ovaries, where it is packaged into the developing egg and is localized to nuclei by the late blastoderm stage of embryonic development. We also overexpressed this protein in transgenic flies in a variety of developmental and tissue-specific patterns to examine the effects of altering the concentration of this splicing factor in vivo. We show that, in many cell types, changing the concentration of B52 adversely affects the development of the organism. We discuss the significance of these observations with regard to previous in vitro results.

Animals↗
PubMed1993

Polymerase processivity and termination on Drosophila heat shock genes.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) limits RNA polymerase II transcription to a gene's 5'-end. Transcription of the uninduced Drosophila hsp70 gene is likewise restricted to the 5'-end, where the polymerase resides in a paused state. Furthermore, paused elongation complexes formed on the uninduced hsp70 gene and in DRB-inhibited reactions can both be restarted by Sarkosyl or high salt. These similarities prompted us to explore whether these complexes were generated by a block at the same polymerase modification step. In vivo UV cross-linking and KMnO4 hyperreactive site mapping show that while the naturally paused polymerase is restricted to the first approximately 42 base pairs of hsp70, DRB treatment of heat-induced cells allows the polymerase to transcribe past this site. Therefore, the DRB-sensitive step is probably not rate-limiting for hsp70 transcription under uninduced conditions. DRB treatment did, however, lead to the reduction of KMnO4 hyper-reactivity on hsp70 and hsp26 in a region correlating with open polymerase and/or early elongation complexes, suggesting a site for the DRB-sensitive polymerase modification step. Finally, we used the techniques of polymerase-DNA cross-linking and KMnO4 hyper-reactive site mapping to analyze the natural polymerase termination process at the 3'-end of the hsp26 gene. The data obtained are consistent with polymerases terminating at multiple sites downstream of the polyadenylation site.

Animals↗
PubMed1993

In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.

The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp70 gene of Drosophila melanogaster, for example, an RNA polymerase II complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk of capping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.

Animals↗
PubMed1993

DNA melting on yeast RNA polymerase II promoters.

Transcription-dependent DNA melting on the yeast GAL1 and GAL10 promoters was found to be more closely correlated with the TATA box than the transcription start site. On both these genes, melting begins about 20 base pairs downstream of the TATA box. Physical and genetic analyses suggest that RNA polymerase II associates with this region. Thus, the distance between promoter melting and the TATA box in yeast may be similar to that in higher eukaryotes, even though transcription initiates in a region about 10 to 90 base pairs farther downstream in yeast.

Base Sequence↗
PubMed1993

Rapid changes in Drosophila transcription after an instantaneous heat shock.

Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes provides a framework for evaluating the mechanisms of activation and repression of transcription. Here, using two methods, we examined the changes in RNA polymerase II association on a set of Drosophila genes at 30-s intervals following an instantaneous heat shock. In the first method, Drosophila Schneider line 2 cells were quickly frozen to halt transcription, and polymerase distribution was analyzed by a nuclear run-on assay. RNA polymerase transcription at the 5' end of the hsp70 gene could be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase could already be detected near the 3' end of the gene. A similar rapid induction was found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene was found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. Similar results were obtained by an in vivo UV cross-linking assay. In this second method, cell samples removed at 30-s intervals were irradiated with 40-microseconds bursts of UV light from a Xenon flash lamp, and the distribution of polymerase was examined by precipitating UV cross-linked protein-DNA complexes with an antibody to RNA polymerase II. Both approaches also showed the in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene to be approximately 1.2 kb/min.

Animals↗
PubMed1992

Promoter sequence containing (CT)n.(GA)n repeats is critical for the formation of the DNase I hypersensitive sites in the Drosophila hsp26 gene.

We have analyzed P-element-transformed lines carrying hsp26/lacZ transgenes with various deletions and substitutions within the Drosophila melanogaster hsp26 promoter region in order to identify the sequences required for the formation of the DNase I hypersensitive sites (DH sites). DH sites are generally found associated with promoters and enhancer elements of active and inducible eukaryotic genes, and are thought to be nucleosome-free regions of DNA that interact with regulatory proteins and the transcriptional machinery. There are two major DH sites located within the promoter region of the hsp26 gene, centered at -50 and at -350 (relative to the hsp26 transcription start site). The sequences from -135 to -85, which contain (CT)n.(GA)n repeats, contribute significantly to the formation of the DH sites in the hsp26 promoter region. Deletion or substitution of this (CT)n region drastically reduces the accessibility of the DNA at these sites to DNase I. This reduction in accessibility was quantified by measuring the susceptibility of the DNA within nuclei to cleavage at a restriction site within the DH site. In addition to the (CT)n region and the promoter at -85 to +11 (region P), one of two other regions must be present for effective creation of the DH sites: sequences between -351 and -135 (region A), or sequences between +11 and +632 (region D). Disruption of the wild-type chromatin structure, as assayed by the loss of accessibility to the DH sites, is correlated with a decrease in inducible transcriptional activity, even when the TATA box and heat shock regulatory elements are present in their normal positions.

Animals↗
PubMed1992

Promoter melting and TFIID complexes on Drosophila genes in vivo.

In vivo UV cross-linking and nuclear transcriptional run-on experiments have shown that a number of Drosophila genes possess an elongationally paused RNA polymerase on their 5' ends. Here, we examine in vivo promoters that do and do not possess paused polymerases using the single-stranded DNA-probing reagent KMnO4. Melted DNA helices are found associated with the pause site of the uninduced hsp70 and hsp26 heat shock genes and the constitutively expressed beta-1 tubulin gene. The histone H1 and H2B genes, which lack a paused polymerase, have no comparable region of melted DNA. Melting at the pause site persists upon heat shock induction of the hsp70 and hsp26 genes, indicating that pausing continues after gene activation. Interestingly, activation triggers additional melting, both at the start site (in the region where open complexes would be expected to form) and downstream of the uninduced pause site. In the course of our studies, we discovered that some T residues of the TATA box were protected from KMnO4 modification in both induced and uninduced cells. This protection appears to be a consequence of TFIID binding, as a similar protection pattern could be produced in vitro with purified protein.

Animals↗
PubMed1992

DNA sequence requirements for generating paused polymerase at the start of hsp70.

RNA polymerase II is transcriptionally engaged but paused approximately 25 nucleotides from the start site of the hsp70 gene of Drosophila melanogaster in uninduced (non-heat-shocked) flies. Here, we identify regions of the hsp70 promoter that are required for formation of this paused polymerase. Various hsp70 promoter sequences are substituted for promoter sequences of a yolk protein gene, yp1, which, in males, is normally not expressed and has no paused polymerase. Run-on assays with nuclei of male transgenic flies are used to measure the level of paused polymerase on the hybrid genes. Sequences that reside upstream of the hsp70 TATA element, when fused upstream of the yp1 TATA element, specify the formation of a paused polymerase on the 5' end of this hybrid gene. Within this region are multiple copies of the GAGA element, which is known to bind a constitutively expressed factor. This element appears to play a role in generating the pause. Also, in the absence of much of this upstream region, hsp70 sequences in the vicinity of the transcriptional start and pause site participate in specifying the pause. Deletions of the pause site reduce the level of paused polymerase but do not lead to constitutive transcription. However, a connection between transcription and pausing is seen. The level of paused polymerase on the various hybrid hsp70-yp1 promoters correlates with the promoter's potential to direct heat-induced transcription.

Animals↗
PubMed1991

Cooperative binding of Drosophila heat shock factor to arrays of a conserved 5 bp unit.

Drosophila heat shock factor (HSF) exists as a multimer in solution and when bound to its regulatory element (HSE). We have previously reported evidence that subunits of HSF associate to form homotrimers and that each subunit contacts a conserved 5 bp DNA sequence repeated within an HSE. Here we show that HSF binding is highly cooperative at two distinct levels: between subunits of the HSF multimer, and between multimers. The binding of HSF to one of a pair of adjacent trimeric binding sites facilitates HSF binding to the second by over 2000-fold. This cooperativity is particularly important in binding HSF at 37 degrees C, and could account for the requirement for multiple binding sites in vivo and, in part, for the differential expression of heat shock genes.

Allosteric Regulation↗
PubMed1991

Characterization of a Drosophila protein associated with boundaries of transcriptionally active chromatin.

We have used indirect immunofluorescence of polytene chromosomes to examine the chromatin distribution of a 52-kD Drosophila protein designated B52. B52 is localized to transcriptionally active loci and, at the highly decondensed heat shock loci, can be seen to bracket the RNA polymerase II fluorescence signals symmetrically. We have also examined the distribution of B52 on nonpolytene chromosomes in Drosophila cell cultures with an in vivo UV cross-linking method and find that, here too, B52 is associated with boundaries of transcriptionally active chromatin. The predicted primary amino acid sequence of B52 reveals two regions with similarities to a number of other proteins known to interact with nucleic acids.

Amino Acid Sequence↗
PubMed1991

RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene.

An RNA polymerase II molecule is associated with the 5' end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides (A. E. Rougvie and J. T. Lis, Cell 54:795-804, 1988). Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here we report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5' end of hsp70 in cells exposed to the intermediate heat shock temperatures of 27 and 30 degrees C. At 30 degrees C, each copy of hsp70 was transcribed approximately five times during the 25-min heat shock that we used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

Animals↗
PubMed1990

Optimal heat-induced expression of the Drosophila hsp26 gene requires a promoter sequence containing (CT)n.(GA)n repeats.

We report here the analysis of the sequence requirements for the heat-induced expression of the Drosophila melanogaster hsp26 gene using germline transformation. Heat-induced expression is augmented fivefold by a homopurine/homopyrimidine region from -85 to -134 that is devoid of heat-shock elements but contains numerous (dC-dT).(dG-dA) repeats. Sequences within this interval have been shown to assume a nuclease S1-hypersensitive structure in vitro. In this paper, we extend those in vitro observations, demonstrating that the S1-hypersensitive structure is triple-helical H-DNA formed by a symmetric (dC-dT).(dG-dA) sequence. Thus, the sequences that form H-DNA in vitro are also required in vivo for optimal hsp26 transcription. However, mutational analysis and diethylpyrocarbonate modification experiments in isolated nuclei suggest that the (dC-dT).(dG-dA) sequence does not form H-DNA in vivo and argue against a role for H-DNA in the heat-induced expression of hsp26.

Animals↗
PubMed1990

Multiple, compensatory regulatory elements specify spermatocyte-specific expression of the Drosophila melanogaster hsp26 gene.

The hsp26 gene of Drosophila melanogaster is expressed in six tissues during development and in a tissue-general response to heat shock. To be able to compare tissue-specific and heat-induced mechanisms of hsp26 expression, we have begun an analysis of the sequences involved in the spermatocyte-specific expression of the hsp26 gene by using germ line transformation. hsp26 mRNA synthesized in the spermatocytes has the same start site as sites previously demonstrated for nurse cell-specific and heat-induced mRNAs. Three regions of the hsp26 gene (nucleotides -351 to -135, -135 to -85, and +11 to +632) were able to stimulate spermatocyte-specific expression when fused with promoter sequences (nucleotides -85 to +11) that alone were insufficient to stimulate expression. These stimulatory regions appear to contain elements that provide redundant functions. While each region was able to stimulate expression independently, the deletion of any one region from a construct was without consequence as long as another compensatory region(s) was still present. There must reside, at a minimum, two independent spermatocyte-specifying elements within the sequences that encompass the three stimulatory regions and the promoter. At least one element is contained within sequences from -351 to -48. This region, in either orientation, can stimulate spermatocyte-specific expression from a heterologous promoter. A second element must reside in sequences from -52 to +632, since these sequences are also sufficient to direct spermatocyte-specific expression.

Animals↗
PubMed1990

Postinitiation transcriptional control in Drosophila melanogaster.

Drosophila hsp70 genes have an RNA polymerase II molecule paused at their 5' ends in uninduced cells. In this study we have shown that this pausing also occurs on other heat shock and constitutively expressed genes. We propose that a rate-limiting step in early elongation occurs in many Drosophila genes and may be a target for transcriptional regulation.

Animals↗
PubMed1990

Closely related DNA sequences specify distinct patterns of developmental expression in Drosophila melanogaster.

Three short synthetic DNA sequences, which are closely related to one another, confer three distinct patterns of developmental expression on the heat shock hsp70 gene in transgenic Drosophila melanogaster lines. These results show that small variations or even single base pair changes in a repeated element of a regulatory sequence can create promoters that display new specificities of tissue and developmental regulation. Interestingly, the three patterns of developmental expression conferred by the synthetic DNAs resemble in part those of the known developmental genes: glucose dehydrogenase (Gld), Dopa decarboxylase (Ddc), and salivary gland secretory proteins (Sgs), respectively. In each case, the defined regulatory region of the known developmental gene contains multiple sequences that are similar or identical to the synthetic sequence that confers a similar pattern of developmental expression on the hsp70 gene. Thus, these results are congruent with the view that short sequence elements in multiple copies can confer either simple or relatively complex patterns of developmental expression on a receptive promoter like that of hsp70. Furthermore, the fact that the three variants tested produced three distinct patterns of expression in transgenic animals suggests that the number of different elements is large.

Animals↗
PubMed1989

Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit.

The minimal DNA sequence required for the formation of a stable complex with Drosophila heat shock factor (HSF) in vitro is an inverted repeat of a 5 bp recognition unit, -GAA-. Surprisingly, both permutations of this 5 bp unit, head-to-head and tail-to-tail, bind to HSF with similar affinity and with striking 2-fold symmetry. HSF also binds to longer arrays of inverted 5 bp units, and the size of the HSF footprint increases with the addition of each 5 bp unit to these arrays. However, the electrophoretic mobility of the HSF-DNA complexes decreases most distinctly with the addition of every three 5 bp units. Cross-linking of purified HSF in the absence of DNA generates complexes with the sizes expected of HSF trimers. We propose that trimers of HSF bind to DNA and that the number of HSF subunits in direct contact with DNA is determined by the number of correctly positioned 5 bp recognition units.

Animals↗
PubMed1989

Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.

Animals↗