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S C Elgin

Publications and source records attributed to S C Elgin.

At least 73 records · Page 4Linked to original sources
PubMed1986

Multiple forms and cellular localization of Drosophila DNA topoisomerase II.

Purified type II topoisomerase from Drosophila melanogaster embryos was reported earlier to contain a major polypeptide of 166,000 daltons and several smaller peptides between 132,000 and 145,000 daltons (Shelton, E. R., Osheroff, N. and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9530-9535). Using purified topoisomerase II we have raised antibodies against the 132,000-166,000-dalton cluster of polypeptides. In this paper we demonstrate that at least three of these polypeptides are also present in embryos immediately upon lysis. Using antigen-affinity purified antibody from the cluster of purified topoisomerase II antigens, we have also discovered several smaller polypeptides in the molecular size range of 30,000-40,000 daltons in embryo extracts. These observations suggest the presence of multiple forms of DNA topoisomerases in the cell. In addition, we demonstrate that purified Drosophila topoisomerase II antibody recognizes yeast topoisomerase II antigens expressed by lambda gt 11-yeast topoisomerase II recombinants (Goto, T. and Wang, J. C. (1984) Cell 36, 1073-1080) establishing a structural homology between yeast and Drosophila enzymes. Antibody preparations were also used to localize the distribution of topoisomerase II in polytene nuclei. In contrast with the distribution of topoisomerase I which is located primarily at puffs, the Drosophila topoisomerase II is distributed generally along the chromosomes paralleling the distribution of DNA itself.

Animals↗
PubMed1986

Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.

Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.

Amino Acid Sequence↗
PubMed1986

Chromatin structure of a P-element-transduced hsp-28 gene in Drosophila melanogaster.

The Drosophila hsp-28 gene was heat inducible when transduced to novel chromosomal sites even when no direct selection for transduced gene expression was imposed. The pattern of DNase I-hypersensitive sites 5' to the wild type and transduced copy of hsp-28 was similar. In addition, DNase I-hypersensitive sites occurred within the P-element sequences flanking transduced loci.

Animals↗
PubMed1986

Nucleosomal instability and induction of new upstream protein-DNA associations accompany activation of four small heat shock protein genes in Drosophila melanogaster.

We investigated in detail the structural changes that occur in nuclear chromatin upon activation of the four small heat shock protein genes in D. melanogaster. Both the chemical cleavage reagent methidiumpropyl-EDTA X iron(II) [MPE X Fe(II)] and the nuclease DNase I revealed a complex pattern of four or five hypersensitive sites upstream of each gene before activation. In addition, MPE X Fe(II) detected a short positioned array of nucleosomes located on each coding region. Upon heat shock activation a number of changes in the patterns occurred. For each gene, at least one of the upstream hypersensitive regions was eliminated or substantially shifted in position. Regions were established which became highly refractile to digestion by either MPE X Fe(II) or DNase I and, as such, appeared as small "footprints" in the pattern. The location of these refractile regions relative to the cap site varied for each gene examined. The coding regions themselves became highly accessible to DNase I. The nucleosomal arrays detected by MPE X Fe(II) were characterized by a considerable loss of detail and significantly enhanced accessibility, the extent of which probably reflected the relative transcription rate of each gene. Careful mapping of the location and extent of each upstream footprint and comparison with the DNA sequence revealed the presence at each location of two (or more) contiguous or overlapping segments that bear high homology to the heat shock consensus sequence C-T-N-G-A-A-N-N-T-T-C-N-A-G. A specific protein factor (or factors) is most likely bound at or near these sequence in heat-shocked Drosophila cells.

Animals↗
PubMed1984

Chemical footprinting of 5S RNA chromatin in embryos of Drosophila melanogaster.

We have used the footprinting reagent (methidiumpropyl-EDTA) iron(II) [MPE X Fe(II)] to investigate the chromatin structure of the tandemly repeated 5S RNA genes of Drosophila melanogaster embryos. Indirect end-labelling analysis of the products of mild MPE X Fe(II) digestion of nuclei reveals one extended region of accessibility of the DNA-protein complex per 375-bp gene-spacer unit. Within this region, which spans the 135-bp gene itself, a segment of particularly high sensitivity (covering 40-60 bp) is located in the distal (3') portion of each gene. The majority of the nontranscribed spacer between genes is in a highly inaccessible chromatin conformation. This pattern is repeated for all copies of the gene-spacer unit examined, approximately 15 at each end of the cluster. Salt extraction experiments show a diminution of pattern intensity after treatment with 0.5 M KCl, but the pattern is not lost under conditions which permit nucleosome sliding. The results indicate a specific, periodic chromatin structure across these transcriptionally competent 5S genes, but one which is generated and maintained by factors other than simple nucleosome arrays. Presumably protein elements of the transcription complex play a dominant role in the structure observed.

Animals↗
PubMed1984

Chromatin structure at the 44D larval cuticle gene locus in Drosophila: the effect of a transposable element insertion.

The chromatin structure of the larval cuticle gene cluster at 44D was characterized in embryos from wild-type (Oregon R) and a variant line (2/3) of Drosophila melanogaster. A major DNase I hypersensitive (DH) site was found between genes II and III in the chromatin, in a position 5' to the transcriptional start of the genes in the cluster. The introduction of a 7.3 kilobase transposable element into the cluster in the 2/3 variant enhanced the sensitivity of the major site in 2/3 chromatin but had no other effect upon the pattern of DH sites associated with the wild-type sequences. The wild-type sequences were packaged into an ordered nucleosome-like array in embryos, as revealed by digestion with the chemical cleavage reagent (methidiumpropyl-EDTA) iron (II) [MPE . Fe(II)]. Nucleolytic cleavage within the transposable element chromatin shows it to be organized in an ordered array punctuated by several DH sites. While the patterns of DNase I hypersensitivity are similar in the vicinity of the direct terminal repeats, the patterns revealed by micrococcal nuclease and MPE . Fe(II) are not, indicating a different chromatin organization of these two identical sequences.

Animals↗
PubMed1984

Supercoil-dependent features of DNA structure at Drosophila locus 67B1.

We have analyzed the pattern of supercoil-dependent, single strand-specific nuclease cleavage sites across 11.6 kb (11.6 X 10(3) base-pairs) of cloned Drosophila melanogaster DNA from locus 67B1. This region contains coding sequences for the heat shock proteins hsp23, hsp26 and hsp28 as well as for a 1.6 kb developmentally regulated transcript (R). Two major sites are detected on digestion with S1 nuclease or mung bean nuclease. The most prominent site maps 100 base-pairs upstream of hsp26 in a very pyrimidine-rich region adjacent to a known region of chromatin DNAase I hypersensitivity. The other site is located approximately 800 base-pairs upstream of hsp28 in an area devoid of such chromatin-specific features. BAL31 nuclease produces a different array, with three to six strong cleavages located in the spacer DNA approx. 0.1 to 1.0 kb upstream of the DNAase I hypersensitive sites of hsp28, hsp23 and R. Thus, for each gene in the cluster a localized sequence sensitive to the winding state of the DNA is observed 5' to the gene. However, there is no precise coincidence of any of the major sites sensitive to BAL31 nuclease in the supercoiled plasmid with the sequences sensitive to DNAase I in chromatin. While all of the enzymes utilized in this study have prominent single strand-specific endonucleolytic activity, it is clear that they recognize different variants in the DNA structure induced by supercoiling. At least two classes of DNA perturbation have been detected.

Animals↗
PubMed1984

Patterns of DNA structural polymorphism and their evolutionary implications.

The pattern of sites within purified DNA that are highly susceptible to double-stranded cleavage by micrococcal nuclease has been analyzed in the vicinity of over 20 genes from widely separated loci in Drosophila. These genes have uniformly exhibited a distinctive organization of cleavage sites such that at early times of digestion major sites are observed in the spacer regions surrounding the genes, but not within the protein coding regions themselves. Examples examined include Drosophila genes for heat-shock proteins, cytoplasmic actin, ribosomal protein 49, alcohol dehydrogenase, Sgs 4 glue protein, and other developmentally regulated transcripts, a human beta-globin gene, and mouse alpha 3-globin pseudogene. It seems probable that this gene/spacer pattern will be a general one in the genomes of eucaryotes, but not in the genomes of procaryotes, since neither pBR322 nor phage lambda DNA display such a pattern. One observes a nonrandom spacing of strong cleavage sites in Drosophila DNA, with the most frequent intervals being 195 bp and 411 bp. Such a pattern of variation in DNA structure may have evolved to facilitate the packaging of eucaryotic DNA into chromatin.

Animals↗
PubMed1984

Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome.

The distribution of DNA topoisomerase I within Drosophila polytene chromosomes was observed by immunofluorescent staining with affinity-purified antibodies. The enzyme is preferentially associated with active loci, as shown by prominent staining of puffs. The heat shock loci 87A-87C are stained after, but not before, heat shock induction. A detailed comparison of the distribution of topoisomerase I with that of RNA polymerase II reveals a similar, although not identical, pattern of association. Topoisomerase I is also found in association with the nucleolus, the site of transcription by RNA polymerase I.

Animals↗
PubMed1984

A cytological approach to the ordering of events in gene activation using the Sgs-4 locus of Drosophila melanogaster.

The polytene chromosomes of Drosophila strains that differ in the synthesis of the major salivary gland glue protein sgs-4 were examined by indirect immunofluorescence using antisera to several nonhistone chromosomal proteins. The Oregon-R X chromosome, which produces sgs-4 messenger RNA, showed a strong fluorescent band at locus 3C11-12 when stained with anti-RNA polymerase II, whereas the null mutant Berkeley 1 failed to exhibit fluorescence at that locus. The presence of another antigen (Band 2), normally associated with developmentally active loci, was clearly evident at locus 3C11-12 of both transcriptionally competent and null strains, indicating that the association of Band 2 antigen with the chromatin is an event independent of RNA polymerase II binding. Antibodies directed against Drosophila topoisomerase I stained 3C11-12 in the Sgs-4+ (wild-type) strain brightly, but gave significantly less staining in the null strain. This indicates that the high concentrations of topoisomerase I seen at active loci are closely associated with the transcriptional event. In some of these analyses, we have made use of flies heterozygous for the wild-type and null alleles in order to make internally controlled comparisons. The results suggest that this type of analysis will enable conclusions to be drawn concerning the interdependence and order of action of chromosomal proteins involved in developmental gene activation.

Alleles↗
PubMed1983

Chromatin structure in pre- and postblastula embryos of Drosophila.

Early in embryogenesis of Drosophila melanogaster, DNA synthesis is extremely rapid while RNA synthesis is virtually undetectable. We have examined the chromatin structure of nuclei from preblastula embryos to determine whether these unusual rates of replication and transcription correlate with any alteration in the chromatin. DNase I-hypersensitive sites at the 5' end of genes have been postulated to be necessary but not sufficient for activity of the associated gene and have been shown to be established prior to the onset of transcription. In order to ascertain whether the apparent transcriptional incompetence of the early embryos is the result of the absence of such chromatin structure, we have examined nuclei from cleavage-stage embryos to determine whether the DNase I-hypersensitive sites have been established. A variety of genes, including inducible heat-shock genes, a constitutively expressed ribosomal protein gene, and two developmentally regulated genes, have been examined. In every case the pattern of DNase I-hypersensitive sites in preblastula embryos duplicates that in the later (6-18 hr after oviposition) embryos. In addition, two extra sites are observed in the early embryos, one at the 5' end of the hsp 70 gene and one in a gene at chromosomal locus 67B1. These sites do not correlate with any known function; however, neither can functional significance be ruled out. In a further investigation of the chromatin structure of early embryos, a nucleosomal array was generated. The pattern produced from nuclei of early embryos is extremely similar to that from 6- to 18-hr embryos, but somewhat less distinct. Both nucleosomal arrays and DNase I-hypersensitive sites must therefore be established very rapidly following DNA replication. The chromatin structure of cleavage-stage embryos detected by these tests appears to be essentially the same as that of older embryos, both in general, and at specific loci.

Animals↗
PubMed1983

Cleavage of chromatin with methidiumpropyl-EDTA . iron(II).

Methidiumpropyl-EDTA . iron(II) [MPE . Fe (II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage. MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.

Animals↗
PubMed1983

Analysis of DNA structural patterns and sequence organization at the larval cuticle locus in Drosophila melanogaster.

We examined the pattern of DNA organization at the larval cuticle gene complex 44D of Drosophila melanogaster, using micrococcal nuclease and the 1,10-phenanthroline-cuprous complex. The initial cleavage patterns obtained with both reagents exhibited "gaps" at the positions of each of the genes examined, as well as at a pseudogene sequence contained within the complex. An additional gap for which no gene exists was observed for both patterns. The cleavage pattern obtained with micrococcal nuclease was unaltered, at a level of resolution of +/- 50 base pairs, in a mutant containing a transposable element. Analysis of the sequence data from this 5.5-kilobase gene cluster indicated that the sequence per se, and not the general base composition, is a dominant factor in determining the patterns observed.

Animals↗
PubMed1982

Analysis of chromatin structure and DNA sequence organization: use of the 1,10-phenanthroline-cuprous complex.

Limited treatment of Drosophila nuclei with the 1,10-phenanthroline-cuprous complex leads to rapid production of nucleosomal ladders indistinguishable from those obtained by micrococcal nuclease digestion. An investigation of the preferential sites of cleavage of protein-free DNA at locus 67B1 surprisingly indicated that both reagents recognized very similar features. Thus, a virtually identical pattern of preferential cleavages was generated over a 12 kb fragment encoding four transcripts at this locus. The distribution of cleavage sites was highly non-random, with major sites falling in the spacers between the genes. Both reagents cleaved certain chromatin-specific sites near the 5' ends of the genes. However, an analysis of preferential cleavages at the sequence level did not reveal the same close correspondence. We suggest that both reagents can recognize some localized secondary structural features of the DNA and that the particular distribution of sequences present at this locus results in a distinctive pattern of cleavage sites that delineates gene and spacer segments.

Animals↗