Mary Lou Pardue

Chapter 1 Nucleic Acid Hybridization to the DNA of Cytological Preparations

Publisher Summary This chapter highlights in situ nucleic acid hybridization. Many conventional cytological procedures can be used to make slides that give good results in in situ hybridization experiments. The best preparations are those, which are well spread and very flat. For cell suspensions, tissue culture cells are gently spun out of the medium and resuspended in a hypotonic solution composed of 1 part medium and 3 parts distilled water. The amount of hypotonic solution added should be about 40 times the volume of the cell pellet. After being resuspended very gently in the hypotonic solution, the cells are left for 5 minutes at room temperature. Endogenous RNA, which might act as a competitor for the hybridizing RNA, is removed by treating the squashes with pancreatic RNase. Moreover, all the agents, such as alkali, heat, and organic solvents that denature purified DNA also denature the DNA of cytological preparations, as measured by in situ hybridization. The conditions of ionic strength, nucleic acid concentration, temperature, and length of time for hybridization are chosen for each experiment. The conditions chosen determine the stringency of the hybridization conditions and set limits on the amount of sequence mismatching that can occur.

Messenger RNA in heat-shocked Drosophila cells

The rate of production of cytoplasmic, heterogenously sedimenting, poly(A)-containing messenger RNA is greatly reduced when cultured Drosophila cells are subjected to heat shock. A large number of discrete RNA species which do continue to appear in the cytoplasm when cells are treated at 37°C have been characterized. Twelve of these poly(A)-containing RNAs are products of the mitochondrial genome, while another five RNAs lacking poly(A) probably represent Drosophila histone mRNAs since those which have been tested hybridize specifically to the region of the polytene chromosomes which contain the histone coding sequences. Six electrophoretically separable fractions of RNA whose appearance in the cytoplasm is greatly stimulated by heat-treatment comprise the majority of the polyadenylated RNA labeled during a heat shock. In all likelihood these RNAs code for the new proteins synthesized by heat-shocked cells since they are associated almost entirely with polysomes and hybridize in situ to regions of the Drosophila chromosomes which puff in response to heat-treatment. The kinetics of synthesis of these mRNAs and the effect of different temperatures on their production have been investigated. A complex relationship has been found to exist between the chromosomal sites which form heat shock puffs and the temperature-induced mRNAs. Three sites, 63C, 67B and 95D, each show hybridization in situ mainly with unique fractions of heat-induced mRNA as expected if a one-to-one relationship between puffs and mRNAs exists. The most abundant mRNA species labels two sites, 87A and 87C, and these loci react to a lesser extent with many other RNA fractions. All the mRNAs show some complementarity with DNA in or very near to the chromocenter. These observations, together with the finding that at least 25 euchromatic chromosomal sites which do not form visible heat shock puffs also hybridize in situ to some degree with RNA labeled in heat-shocked cells, are discussed.

Distribution of 18+28S ribosomal genes in mammalian genomes

In situ hybridization with 3H 18S and 28S ribosomal RNA from Xenopus laevis has been used to study the distribution of DNA sequences coding for these RNAs (the nucleolus organizing regions) in the genomes of six mammals. Several patterns of distribution have been found: 1) A single major site (rat kangaroo, Sebas fruit bat), 2) Two major sites (Indian muntjac), 3) Multiple sites in centromeric heterochromatin (field vole), 4) Multiple sites in heterochromatic short arms (Peromyscus eremicus), 5) Multiple sites in telomeric regions (Chinese hamster). — The chromosomal sites which bind 3H 18S and 28S ribosomal RNA correspond closely to the sites of secondary constrictions where these are known. However, the correlation is not absolute. Some secondary constrictions do not appear to bind 3H ribosomal RNA. Some regions which bind ribosomal RNA do not appear as secondary constrictions in metaphase chromosomes. — Although the nucleolus organizing regions of most mammalian karyotypes are found on the autosomes, the X chromosomes in Carollia perspicillata and C. castanea carry large clusters of sequences complementary to ribosomal RNA. In situ hybridization shows that the Y chromosome in C. castanea also has a large nucleolus organizing region.

Analysis of drosophila mRNA by in situ hybridization: sequences transcribed in normal and heat shocked cultured cells.

Messenger RNA transcribed in cultured Drosophila cells adapted for growth under conditions permitting labeling to high specific acitivty has been analyzed by the technique of in situ hybridization. Poly(A)-containing cytoplasmic RNA binds specifically and reproducibly to about 50 bands in the salivary gland polytene chromosomes. In addition heavy labeling of the beta-heterochromatin associated with each of the chromosome arms is observed. The species which are detected probably belong to the more abundant classes of RNA. When the cultured Drosophila cells are subjected to heat shock immediately before labeling with 3H-uridine, there is a drastic alteration in the pattern of gene transcription detected by in situ hybridization. Most of the mRNA synthesis which could be detected in the normal cell is shut off. Newly synthesized RNA hybridizes strongly to seven new sites which do not bind mRNA from control cells. The new loci correspond almost exactly to the regions of Drosophila polytene chromosomes which puff when intact larvae are subjected to an identical heat treatment.

Drosophila telomeres: new views on chromosome evolution

In Drosophila, chromosome ends (telomeres) are composed of telomere-specific transposable elements (the retroposons HeT-A and TART). These elements are a bona fide part of the cellular machinery yet have many of the hallmarks of retrotransposable elements and retroviruses, raising the possibility that parasitic transposable elements and viruses might have evolved from mechanisms that the cell uses to maintain its chromosomes. It is striking that Drosophila, the model organism for many discoveries in genetics, development and molecular biology (including the classical concept of telomeres), should prove to have chromosome ends different from the generally accepted model. Studies of these telomere-specific retrotransposable elements raise questions about conventional wisdom concerning not only telomeres, but also transposable elements and heterochromatin.

Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster

Cytoplasmic RNA from imaginal discs of Drosophila melanogaster, labeled by uridine incorporation in organ culture, has been assayed by hybridization to cytological preparations of polytene chromosomes. RNA labeled during the early stages (first four hours) of ecdysone stimulation was compared to RNA labeled in the absence of the hormone. For the poly(A)-containing fraction (oligo-dT bound), several loci hybridize only RNA labeled in the presence of ecdysone; one locus hybridizes only control RNA. The majority of hybridizing loci are unaffected by the hormone. Of the loci hybridizing RNA not bound to oligo-dT, several appear specific for the ecdysone-treated sample, though most are labeled more heavily with this RNA than with the control. None of the ecdysone-sensitive loci visualized by in situ hybridization are the sites of salivary gland puffs induced by ecdysone on the same time scale.

The effect of heat shock on RNA synthesis in Drosophila tissues

Total RNA from Drosophila imaginal discs, labeled under conditions of heat shock, is analyzed by hybridization in situ to salivary gland polytene chromosomes. Grain densities over the hybridizing bands are compared, showing that the response to heat shock is similar for several disc types and fly stocks. Alteration of the culture medium used for labeling during heat shock results in the specific induction (in discs, salvary glands, or fat body) of one of the heat-shock loci to a level far beyond that normally seen. We discuss the implications of this specific induction regarding the mechanism of response. We also discuss the observed difference between transcription labeling of salivary gland chromosomes and labeling by in situ hybridization.

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates

When Drosophila tissue culture cells are shifted from 25 to 36 degrees C (heat shocked) the pre-existing mRNAs (25 degrees C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25 degrees C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding alpha-tubulin, beta-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25 degrees C. Increased temperature should lead to increased output of protein per ribosome. However, the 25 degrees C proteins are actually synthesized at less than 10% of 25 degrees C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25 degrees C mRNAs than they are on heat shock mRNAs in heat-shocked cells.

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Telomeres and telomerase: more than the end of the line.

Abstract. Early studies of telomerase suggested that telomeres are maintained by an elegant but relatively simple and highly conserved mechanism of telomerase-mediated replication. As we learn more, it has become clear that the mechanism is elegant but not as simple as first thought. It is also evident that, although many species use similar, sometimes identical, DNA sequences for telomeres, these species express their own individuality in the way they regulate these sequences and, perhaps, in the additional tasks that they have imposed on their telomeric DNA. The striking similarities between telomeres in different species have revealed much about chromosome ends; the differences are proving to be equally informative. In addition to the differences between species that use telomerase, there are also a few exceptional organisms with atypical telomeres for which no telomerase activity has been detected. This review addresses recent studies, the insights they offer, and, perhaps more importantly, the questions they raise.