PubMed Health⌕ Search

Biomedical subjects

M L Pardue

Publications and source records attributed to M L Pardue.

At least 73 records · Page 4Linked to original sources
PubMed1982

Deficiency mapping of the 93D heat-shock locus in Drosophila melanogaster.

The 93D heat shock locus was mapped relative to an overlapping series of deficiencies of the 93D region by three criteria: the ability of the deleted chromosomes to puff at 93D, the ability of the deleted chromosomes to synthesize RNA from the 93D region after a temperature shift and the presence of heat shock RNA sequences at 93D as assayed by in situ hybridization. The results are essentially the same by all three criteria. Chromosomes with deficiencies that did not extend distal to 93D4 puffed and incorporated 3H-uridine after a temperature shift, and were labelled at 93D following in situ hybridization of heat shock RNA from tissue culture cells. All the other deficiency chromosomes tested failed to puff and to incorporate 3H-uridine following a temperature shift and did not show hybridization in this region after in situ hybridization with heat shock RNA. The heat shock locus was mapped to the overlapping region of Df(3R)eGp4 and Df(3R)GC14 just outside the inverted region of In(3R)GC23.

Animals↗
PubMed1981

Antibodies to left-handed Z-DNA bind to interband regions of Drosophila polytene chromosomes.

Antibodies which are specific to the Z-DNA conformation have been purified and characterized on the basis of their binding to three different DNA polymers which can form this left-handed helix. These antibodies bind specifically to polytene chromosomes of Drosophila melanogaster as visualized by fluorescent staining. The staining is found in the interband regions and its intensity varies among different interbands in a reproducible manner. This is the first identification of the Z-DNA conformation in material of biological origin.

Animals↗
PubMed1981

Chromosomal locations of two DNA segments that flank ribosomal insertion-like sequences in Drosophila: flanking sequences are mobile elements.

We report the chromosomal locations of two repetitive DNA sequences that flank ribosomal insertion-like sequences in Drosophila melanogaster. The chromocentric region of D. melanogaster contains many copies of sequences that are homologous to type 1 ribosomal insertions. These insertion-like elements are interspersed with other DNA segments that we call flanking sequences. Two distinct flanking sequences derived from the same cloned DNA molecule pDmI 101, the HindIII fragments 101E and 101F, were studied. Whole genome Southern blots with DNA from the D. melanogaster stocks Oregon R (P2), gt-1, and gt-X11 showed complex restriction patterns that differed substantially between the three stocks. This and other data show that flanking sequences are members of diverged repetitive sequence families. In situ hybridization to salivary gland chromosomes of gt-1 and gt-X11 showed that both sequences are homologous to the chromocenter and to about 5 to 8 (101E) or 25 to 30(101F) euchromatic sites in each stock. Most, if not all, of these sites differed in gt-1 and gt-X11. Both 101E and 101F are homologous to he chromocenter and very few euchromatic bands in D. simulans, but 101F is homologous to numerous bands in D. mauritiana. We conclude that the flanking sequences represented by 101E and 101F are mobile elements within the genome of Drosophila. These two sequences differ in several structural features from mobile DNA elements previously described in this organism.

Animals↗
PubMed1981

Ribosomal insertion-like elements in Drosophila melanogaster are interspersed with mobile sequences.

Sequences homologous to the type 1 ribosomal DNA insertion occur in the chromocenter and in one location on the fourth chromosome of Drosophila melanogaster. Ribosomal insertion-like elements frequently occur in the form of tandem repeats and are often interspersed with nonhomologous DNA. Sequences that interrupt of flank insertion-like elements are members of diverged repeated DNA families. Members of five of these interspersed sequence families are located at multiple euchromatic sites as well as in the chromocenter. The euchromatic sites differ greatly in the gt-1 and gt-X11 stocks of D. melanogaster, suggesting that these sequence families are mobile in the genome. No long inverted repeats were detected in any of these interspersed sequences. One sequence, called 101F, interrupts a ribosomal insertion-like element; the nucleotide sequences across the boundaries between 101F and ribosomal insertion have been determined. A stretch of 13 base pairs that is present once in the ribosomal insertion is repeated at the ends of the 101F sequence, suggesting a target site duplication. Within the 101F element, no extended direct or inverted repeat sequence exists. The interspersed repeated sequences studied are transcribed into rare, heterogeneous, poly(A)-lacking nuclear RNA molecules. In one case, we showed that both strands of a flanking sequence are transcribed, but to a different extent. With respect to structural features and the nature of their transcripts, the sequences described here are distinct from other known transposable elements in Drosophila.

Animals↗
PubMed1981

Translational control in lysates of Drosophila melanogaster cells.

Cell-free protein-synthesizing systems made from Drosophila melanogaster cells were used to study the translational control induced in these cells by heat shock. Lysates of normally growing cells, termed 25 degrees C cells, translate both normal and heat shock mRNAs. Lysates of cells heat shocked at 36 degrees C for 1 hr, termed 36 degrees C cells, translate preferentially heat-shock mRNAs and a few 25 degrees C cell mRNAs. Thus, both lysates appear to reproduce the control displayed in vivo. Both lysates are optimally active at 28 degrees C, and all translations are done at that temperature, demonstrating that, once established, the discrimination system does not require heat-shock temperature for its activity. Addition of crude ribosome fractions from 25 degrees C cell lysates to lysates from heat-shocked cells "rescues" translation of 25 degrees C cell mRNA, which suggests that the discriminating elements are associated with ribosomes. Neither the heat-shock crude ribosome supplements nor the soluble fractions have any effect on either lysate. The experiments also show that RNA selection is determined by some feature of the RNA structure that is insensitive to protease digestion and phenol/chloroform extraction. The essential structural feature may not be unique to Drosophila mRNAs because the Drosophila lysate is capable of discriminating among mRNAs from other organisms.

Actins↗
PubMed1980

Transcription and metabolism of RNA from the Drosophila melanogaster heat shock puff site 93D.

Characteristics of the major heat shock puff site 93D and the RNA transcribed from it have been investigated by hybridization to polytene chromosome preparations and to recombinant DNA. By saturation in situ hybridization, the length of the transcribed region at 93D is twice that of the mRNA coding region at the heat shock puff site 87A. From the known length of the heat shock mRNA sequence at 87A, we calculate that the minimum length of the transcribed region at 93D is 9.6 kb (kb = kilobase, i.e., 1,000 nucleotides).--The metabolism of RNA transcribed from 93D has been compared with that of RNA coding for the major heat shock protein hsp70 in cells incubated for one hour at 35 degrees C. Hsp70 mRNA sequences, assayed by hybridization to a specific recombinant DNA probe and by in situ hybridization to 87A, were found in both poly(A)+ and poly(A)- cytoplasmic RNA and were more concentrated in cytoplasmic RNA than in nuclear RNA. In contrast, sequences complementary to 93D, assayed by in situ hybridization, were more concentrated in nuclear than in cytoplasmic RNA. This implies that sequences from 93D exit from the nucleus at a lower rate and/or are turned over in the cytoplasm at a higher rate, than sequences from 87A. Site 93D is also unusual in that its transcribed region is represented in both poly(A)+ and poly(A)- nuclear RNA, even though 93D-complementary RNA in the cytoplasm is predominantly poly(A)-. Finally, only 28-58% of the length of DNA transcribed at 93D is represented in cytoplasmic RNA, indicating that only a portion of the sequences transcribed from 93D are exported from the nucleus. The transcripts from two heat shock loci, 93D and 87A, thus appear to be metabolized in significantly different ways.

Animals↗
PubMed1980

Translational control of protein synthesis in response to heat shock in D. melanogaster cells.

In response to elevated temperature, Drosophila cells synthesize a small set of proteins known as the heat-shock proteins, while synthesis of most other proteins ceases. In vitro translation has been used to demonstrate that the messenger RNAs encoding the normal (25 degrees) spectrum of proteins are not broken down or irreversibly inactivated in response to the temperature change. During the heat shock only the heat-shock mRNAs plus a small number of preexisting mRNAs are translated, while most other messages are stored and can be reactivated upon return of the cells to their normal temperature. After recovery from heat shock, cells translate both the normal mRNA and the remaining heat-shock mRNA. The translational control operating in intact cells has been reproduced in cell-free translation systems directed by purified mRNA from normal and heat-shocked cells. Lysates prepared from heat-shocked Drosophila cells preferentially translated the heat-shock messages, while the lysate made from normally growing Drosophila cells indiscriminately translated both normal and heat-shock messages. Therefore there must be stable alterations in the translational components of heat-shocked cells which are capable of causing selective translation of the heat-shock messages. In addition there must be information encoded in the heat-shock messages that allows their selection.

Animals↗
PubMed1980

A new type of virus from cultured Drosophila cells: characterization and use in studies of the heat-shock response.

An infectious virus isolated from the cultured Drosophila melanogster cell line, Schneider 2-L, appears to be a member of a new group of animal viruses. The virus, HPS-1 has a genome composed of a single segment of double-stranded RNA approximately 6 kb in length. Virions are particles approximately 36 nm in diameter. They contain only two proteins and no lipid coat. The major protein, presumably the viral coat protein, is 120,000 daltons. A 200,000 dalton protein is present in much lower quantities. Two other proteins, synthesized in virus-producing cells, are encoded in the viral genome but not included in the mature virion. Synthesis of viral proteins is not affected by the heat-shock-induced translational control that inhibits translation of most normal mRNAs but allows protein synthesis on heat-shock mRNAs. The viral mRNAs thus appear to share the structural features of heat-shock mRNAs which permit heir translation in heat-shocked cells. Viral RNA serves as a probe to study heat-shock translational control. Cells heat-shocked in the presence of actinomycin D cannot transcribe heat-shock mRNA and therefore cannot make heat-shock proteins. Although these cells are making neither heat-shock mRNA nor heat-shock protein, the translational control appears fully induced. The normal cell proteins are not made in these cells but viral proteins are synthesized. These results indicate that the heat shock-induced proteins are not components of the translational control mechanism.

Animals↗
PubMed1980

DNA-protein interactions in the Drosophila melanogaster mitochondrial genome as deduced from trimethylpsoralen crosslinking patterns.

The location of proteins on the mitochondrial DNA (mtDNA) of Drosophila melanogaster was investigated by trimethylpsoralen photoreaction of embryos disrupted by gentle homogenization. After photoreaction, the mtDNA was isolated and the pattern of DNA crosslinking was determined by electron microscopy of the DNA under totally denaturing conditions. In contrast to nuclear DNA, which showed periodic crosslinks indicative of a nucleosome structure, most of each mtDNA molecule exhibited uniformly heavy crosslinking. A 10% region of the mtDNA was, however, protected from psoralen crosslinking in a distinctive manner: five uncrosslinked segments were closely clustered in the mtDNA. Four were 394 +/- 13 (SD) base pairs in size, while the fifth measured about 200 base pairs. These protected segments mapped within the A+T-rich region of the mtDNA, extending from the end of the A+T-rich region near the Bg1 II cleavage site to the center of the A+T-rich region. Protection of this part of the mtDNA from crosslinking was interpreted to be the result of association with proteins in the mitochondrion because mtDNA that was deproteinized before the photoreaction was uniformly crosslinked over its entire length. The origin of replication of the mtDNA is also located at the center of the A+T-rich region, which suggests that the protection from the psoralen photoreaction may be due to proteins involved in membrane attachment or replication.

Animals↗
PubMed1979

Cell-free protein synthesis in lysates of Drosophila melanogaster cells.

A procedure is described for preparing cell-free protein synthesizing lysates from Drosophila melanogaster tissue culture cells and embryos. Preparation of translationally active lysates from tissue culture cells is dependent on the presence of rat liver supernatant during cell lysis to inhibit ribonuclease activity. After micrococcal nuclease treatment of the lysate, protein synthesis is dependent on the addition of exogenous messenger RNA. The fidelity of translation is very high. The conditions for optimal translation have been determined. In addition, the effects on translation of a variety of supplements, including rat liver supernatant, have been analyzed. The products of translation by the Drosophila lysate have been compared with those of wheat germ extracts and of micrococcal nuclease treated rabbit reticulocyte lysates. Translation in vitro of bovine parathyroid hormone messenger RNA yielded two products tentatively identified as preproparathyroid hormone and proparathyroid hormone, as well as an unidentified third product. This result suggests that insect enzymes can accurately process mammalian precursor proteins.

Animals↗
PubMed1979

Multiple actins in Drosophila melanogaster.

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.

Actins↗
PubMed1978

A portion of all major classes of histone messenger RNA in amphibian oocytes is polyadenylated.

Ovaries of the amphibian Triturus viridescens contain a considerable amount of 7 to 12 S RNA which fractionates with poly(A)+ RNA on oligo(dT)-cellulose column chromatography and directs the synthesis of all five classes of histones in a wheat germ cell-free system. The polyadenylate tracts associated with this 7 to 12 S poly (A)+ RNA are heterogeneous in length, ranging from approximately 60 to 120 nucleotides. Partially purified subfractions of histone mRNA templates were isolated from this RNA by preparative polyacrylamide gel electrophoresis. The correlation between the poly(A) content, the template activity in vitro, and the cell-free products encoded by these RNA subfractions suggests that Triturus ovary RNA contains poly(A)+ histone mRNA. The 7 to 12 S poly(A)+ RNA, but not 7 to 12 S poly(A)- RNA, is an effective template for the avian myeloblastosis virus reverse transcriptase-directed synthesis of complementary DNA (cDNA) in the presence of oligo(dT) primer. In the absence of primer, virtually no cDNA is synthesized. When cDNA complementary to 7 to 12 S poly(A)+ RNA is hybridized in situ to Drosophila melanogaster polytene chromosome preparations. cDNA hybrids are found primarily in the region 39D-E, the locus of the histone genes in Drosophila. This cDNA also hybridizes to the sea urchin histone gene sequences contained in the chimeric bacterial plasmids pSp2 and pSp17. These results provide strong evidence for the reality of polyadenylated histone mRNA in Triturus ovary.

Animals↗
PubMed1978

Myogenesis in primary cell cultures from Drosophila melanogaster: protein synthesis and actin heterogeneity during development.

Muscle cell cultures from Drosophila melanogaster were obtained by plating dissociated gastrula stage embryo cells on protamine-treated culture dishes. They myogenic cells in these cultures fuse to form multinucleated pulsating cells by 15 hr after plating. An analysis of protein synthesis during myogenesis in these cultures, as measured by the incorporation of 35S-methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis, showed profound changes in the pattern of protein synthesis. This analysis enabled us to identify three distinct classes of proteins. Class A proteins, the most abundant, are synthesized continuously throughout myogenesis, class B proteins are those proteins whose synthesis is initiated during myogenesis and continued throughout development; class C proteins are those synthesized at specific times during development. In addition, three forms of actin have been identified in these cultures. Actin I, which shows increased synthesis concomitant with the myogenic development in these cultures, is apparently a muscle-specific form of actin. Actin II, the predominant "cytoplasmic" form of actin in the nonmuscle Schneider cell line 2, is also the major form in the gastrula cultures before differentiation begins. Synthesis of this actin continues in the myogenic cultures. Actin III is a rapidly turning over form of actin which does not accumulate in either the Schneider cells or the myogenic cultures.

Actins↗