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S C Elgin

Publications and source records attributed to S C Elgin.

At least 91 records · Page 5Linked to original sources
PubMed1982

Nucleosome repeat structure is present in native salivary chromosomes of Drosophila melanogaster.

The regularly repeating periodic nucleosome organization is clearly resolved in the chromatin of the isolated salivary chromosomes of Drosophila melanogaster. A new microsurgical procedure of isolation in buffer A of Hewish and Burgoyne (1973, Biochem. Biophys. Res. Commun., 52:504-510) yielded native Drosophila salivary chromosomes. These chromosomes were then swollen and spread by a modified Miller procedure, stained or shadowed, and examined in the electron microscope. Individual nucleoprotein fibers were resolved with regularly repeated nucleosomes of approximately 10 nm diameter. Micrococcal nuclease digestion of isolated salivary nuclei gave a family of DNA fragments characteristic of nucleosomes for total chromatin, 5S gene, and simple satellite (rho = 1.688 g/cm3) sequences.

Animals↗
PubMed1981

DNase I hypersensitive sites of the chromatin for Drosophila melanogaster ribosomal protein 49 gene.

By using an indirect end-labelling technique for mapping, five DNase I hypersensitive sites have been located in Drosophila melanogaster chromatin at the 5'-end of the gene coding for ribosomal protein 49. These sites typically span about 100-600 base pairs and are approximately the length of a nucleosome apart (center to center distance ca 245 bp). This is the first analysis of the chromatin structure of a constitutive house-keeping gene. The results support the hypothesis that the presence of such a DNase 1 hypersensitive site in chromatin is necessary for transcription in vivo. The presence of such sites may reflect some local changes in the conformation of the chromatin in the presumptive regulatory region.

Animals↗
PubMed1981

Transcription of heat shock loci of Drosophila in a nuclear system.

We have investigated transcription in nucleic isolated from 6-18-h Drosophila melanogaster embryos. Kinetic aspects and specific products of the reaction have been analyzed; in particular, we have examined whether or not a faithful representation of an induced state, in this case the heat shock response, is maintained in the isolated nucleic. RNA polymerase II is active for at least 30 min. The combined RNA polymerase I and III activities are seen to continue synthesis for 60 min. Maximal synthesis by all enzymes is obtained in the absence of Mn2+. Transcripts from the 5S DNA have been characterized. These consist of two species, of 135 and 120 nucleotides, apparently the precursor and mature forms of 5S RNA. Extensive initiation by RNA polymerase III occurs on 5S DNA in this system. Transcription from the heat shock loci at 87A and 87Cl has been analyzed by hybridization of the newly synthesized RNA (selected by using 5'-mercuri-UTP) to plasmids containing the hsp 70 gene and adjacent regions. Only those segments of the DNA to which transcripts have been mapped in vivo hybridized with the in vitro synthesized RNA, and this transcription was observed only in nuclei isolated from heat-shocked embryos; no transcripts are detected in nuclei isolated from control embryos. These RNA species are synthesized by RNA polymerase II. Further analysis of transcription of the hsp68 gene (at locus 95D) has also been carried out. We conclude that in this system RNA polymerases II and II transcribe the chromatin template accurately and that the changes related to gene activation by heat shock are stable during nuclear isolation.

Animals↗
PubMed1981

Micrococcal nuclease as a probe of DNA sequence organization and chromatin structure.

We have investigated micrococcal nuclease digestion of chromatin and purified DNA at the heat-shock locus 67B in Drosophila melanogaster. At early stages of the reaction a distinct set of fragments is generated, indicating the presence of preferential cleavage sites. These sites are also observed when purified recombinant plasmid DNA is used as the substrate, demonstrating that the sites are specified by the DNA sequence. At Drosophila locus 67B, prominent sites occur frequently, spaced approximately 200 bp apart, within the nontranscribed portions of the locus, but are generally not observed within the regions that are transcribed. In contrast, such sites are randomly distributed along the procaryotic plasmid pBR322. The results indicate that specific patterns of digestion of eucaryotic chromatin by micrococcal nuclease cannot be simply interpreted as the consequence of the nucleosome array. However, it is possible that the organization of eucaryotic DNA sequences detected by micrococcal nuclease bears a functional relationship to the organization of DNA by nucleosomes and, in fact, was so selected through evolution.

Animals↗
PubMed1981

DNase I hypersensitive sites in Drosophila chromatin occur at the 5' ends of regions of transcription.

By using a map of the unique region of DNA encoding the fur small heat-shock proteins of Drosophila melanogaster (hsp 22, hsp 23, hsp 26, and hsp 28), and a simple mapping technique, the positions of the DNase I hypersensitive sites of chromatin in the vicinity of these genes have now been determined. The major chromatin-specific sites occur at the 5' ends of each of the four heat-shock protein genes in embryo nuclei. These genes are not active in the nuclei analyzed but can be quickly induced in these cells by the heat-shock stimulus. The chromatin structure indicated by DNase I hypersensitivity may be a necessary factor in the general mechanism of gene activation.

Animals↗
PubMed1981

Monoclonal antibodies against a specific nonhistone chromosomal protein of Drosophila associated with active genes.

Hybridomas secreting monoclonal antibodies have been produced by fusion of NS-1 mouse myeloma cells with the spleen cells of mice inoculated with a 60-65,000-mol wt fraction of proteins released from Drosophila embryo nuclei treated with DNase I. The antibodies secreted by the hybridomas were examined with polytene chromosomes of formaldehyde-fixed salivary gland squashes by an immunofluorescence assay. Most of the clonal antibodies obtained resulted in specific staining of the chromosomes relative to the cytoplasmic debris. In the case of clone 28, the antibodies showed a preferential association with sites of gene activity, both puffs and loci identified as puffing at some time during the third instar and prepupal period. In larvae that were heat shocked (exposed to 35 degrees C for 15 min before removal and fixation of the glands), the antibodies of clone 28 stained preferentially the induced heat-shock loci while continuing to stain most of the normal set of loci. The antigen for clone 28 was identified as a single protein of approximately 62,000 mol wt by using the antibodies followed by 125I-rabbit anti-mouse Ig to stain nitrocellulose replicas of SDS polyacrylamide gels of total chromosomal proteins. This study demonstrates that monoclonal antibodies can be used successfully in immunofluorescence staining of formaldehyde-fixed polytene chromosomes. The results verify the hypothesis that a specific nonhistone chromosomal protein is preferentially associated with the set of loci that includes both active sites and those scheduled to be active at some time in this developmental program. Such proteins may play a general role in the mechanisms of cell determination and gene activation.

Animals↗
PubMed1981

Distribution studies on polytene chromosomes using antibodies directed against hnRNP.

The distribution of nuclear ribonucleoprotein (hnRNP) particles in Drosophila polytene chromosomes has been investigated using anti-B-36 serum as a probe. The use of polytene chromosomes allows resolution at the level of the chromomere, and provides the opportunity to look for both positive and negative correlations with transcriptional activity. The antiserum was obtained using the nuclear protein B-36 from Physarum polycephalum as the immunogen. It has been shown to precipitate hnRNP particles from HeLa cells through a cross-reaction with the major 32,000- and 34,000-dalton hnRNP particle proteins. The antiserum cross-reacts with a Drosophila nuclear protein of approximately 34,000 daltons. By indirect immunofluorescence, we observed that the antiserum reacts preferentially with transcriptionally active loci of the polytene chromosomes, whereas loci previously or subsequently active do not show significant fluorescence. The overall pattern of fluorescence is very similar to that generated with anti-RNA polymerase B serum. The correlation of fluorescence and transcriptional activity observed suggests that the anti-B-36 serum is recognizing hnRNP proteins which have combined with nascent RNA molecules at the sites of transcription.

Animals↗
PubMed1980

Proteins of metaphase chromosomes and interphase chromatin.

Metaphase chromosomal and interphase chromatin proteins from cells of two species have been compared by polyacrylamide gel electrophoresis. Consistent, common changes in the quantitative distribution of the nonhistone chromosomal proteins are observed in both species. Proteins of ca. 65,000 and 68,000 MW are enriched in interphase chromatin while proteins of ca. 50,000 and 200,000 are more prominent components of metaphase chromosomes. A group of proteins of 90,000-100,000 are also increased in metaphase chromosomes compared to interphase chromatin. By two dimensional gel analysis, the most abundant proteins from chromosomes of both cell types are similar, suggesting a structural role for these nonhistone proteins (1).

Animals↗
PubMed1979

Sequence of histone 2B of Drosophila melanogaster.

The complete sequence of histone 2B of Drosophila has been determined by using an improved Beckman sequenator. Comparing these data with those previously published by other investigators on the histone 2B of calf [Iwai, K., Hayashi, H., & Ishikawa, K. (1972) J. Biochem. (Tokyo) 72, 357--367], trout [Koostra, A., & Bailey, G. S. (1978) Biochemistry 17, 2504--2510], and Patella (a limpet) [van Helden, P. D., Strickland, W. N., Brandt, W. F., & von Holt, C. (1979) Eur. J. Biochem. 93, 71--78], it is possible to assess the evolutionary stability of this protein. There is little conservation of sequence in the N-terminal portion of the molecule (residues 1--26 numbering according to calf H2B), while the remainder of the protein, which we designate the C-terminal portion, is highly conserved. In the region of 27--125 residues, there are 9 substitutions in the composite data among the 98 positions, 8 of them conservative. These data indicate that very different selective pressures operate on the two different portions of the H2B molecule, implying the existence of two well-defined regions. Studies on the structure of the nucleosome by others have suggested that the C-terminal portion of H2B is involved in histone-histone interactions while the N-terminal portion is a relatively free "tail" binding to DNA. The sequence data indicate that the function of the C-terminal region of H2B requires considerable sequence specificity while that of the N-terminal region does not.

Amino Acid Sequence↗
PubMed1979

The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity.

We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.

Animals↗
PubMed1978

Production and characterization of antisera against three individual NHC proteins; a case of a generally distributed NHC protein.

In order to assess the selectivity of the distribution patterns of individual nonhistone chromosomal proteins (NHC proteins), immunofluorescent staining experiments were performed on Drosophila polytene chromosomes. Antisera have been prepared against three individual NHC proteins which were isolated by sequential preparative slab gel isoelectric focusing and SDS polyacrylamide gel electrophoresis. In two cases, immunofluorescent staining of the chromosomes indicated a specific limited distribution pattern; apparently the antigen in each case is present at a reproducible and distinct subset of chromomeres. This type of pattern has also been obtained with antisera prepared against molecular weight subfractions of NHC proteins (Silver and Elgin, 1977). Each selective fluorescence distribution pattern obtained so far is reproducible and unique to the antiserum under study. In a third case, an antiserum caused prominant staining at dense chromomeres and the chromocenter in a pattern mimicking DNA (and presumably histone) distribution. Indirect radioimmunostaining of SDS and isoelectric focusing gels on which total NHC proteins had been separated confirmed that this antiserum reacted specifically with a protein(s) of molecular weight 21,000 D and pI 5.2. The data in conjunction with absorption experiments indicates that the chromosomal staining is due to an interaction of antibodies with NHC protein(s) and not with histones. This finding suggests that at least one major acidic NHC protein plays a very general role (comparable to that of the histones) in maintaining chromatin structure.

Animals↗
PubMed1978

A protein released by DNAase I digestion of drosophila nuclei is preferentially associated with puffs.

Antisera have been produced against five molecular weight subfractions of the Drosophila proteins readily extracted from nuclei following limited DNAase I digestion. Immunofluorescence staining techniques were used to assess the distributions of these proteins in the polytene chromosomes of Drosophila. In three cases, the antigens were widely distributed; in one case, the antigens appeared to be slightly more concentrated at active loci; and in one case, the antigens were strongly concentrated at a defined set of loci, including puffs and most of the loci which are active (puffed) at some time during third instar larval and prepupal development. The latter distribution pattern differs from that of RNA polymerase. Nonhistone chromosomal proteins of this type may have a key role in establishing and/or maintaining the altered chromatin structure characteristic of the active state.

Animals↗