Drosophila Tissue Culture Cells Research Articles

Similarities in structure and function of calf thymus and Drosophila casein kinase II.

Both calf and Drosophila contain a type II casein kinase with similar molecular structure and catalytic activity. Purified calf thymus casein kinase II is composed of three subunits of Mr = 44,000 (alpha), 40,000 (alpha'), and 26,000 (beta) (Dahmus, M.E. (1981) J. Biol. Chem. 256, 3319-3325), whereas the Drosophila enzyme is composed of two subunits of Mr = 36,700 (alpha) and 28,200 (beta) (Glover, C. V. C., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 3258-3265). The native form of the enzyme is an alpha 2 beta 2 tetramer. Polyclonal antibodies prepared against each enzyme react with both the alpha and beta subunits of the homologous enzyme and cross-react with both subunits of the heterologous enzyme. Reaction of polyclonal antibodies with proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis establishes that no significant difference in subunit molecular weight exists between the purified enzymes and the enzyme present in initial cell extracts. Each antibody effectively inhibits the in vitro activity of the homologous enzyme and causes a slight inhibition in the activity of the heterologous enzyme. Peptide maps derived from purified subunits indicate that the alpha and beta subunits are unique and that there is extensive primary sequence homology between the corresponding subunits of the calf and Drosophila enzyme. Casein kinase II from both sources phosphorylates the same subunits of calf thymus RNA polymerase II and an identical set of proteins in a complex mixture of acid-soluble proteins from Drosophila tissue culture cells. The striking similarity in molecular structure and catalytic activity between the calf and Drosophila enzyme suggests that casein kinase II has been highly conserved in evolution.

Circular extrachromosomal copia-like transposable elements in Drosophila tissue culture cells

We find that in the circular extrachromosomal DNA from Drosophila tissue culture cells the transposable elements copia, 412, 297, and mdg 1 are present in variable amounts. There is no detectable circular DNA homologous to B104. From the relationship between the intra- and extrachromosomal forms it appears that the amount of different circular elements is not related to the amount of the respective chromosomal elements.

Replicative and conservative transpositional recombination of insertion sequences.

We have presented the results of experiments with IS903- and IS10- derived transposons that have led us to the following conclusions: The predominant mechanism of transpositional recombination of these IS elements is a donor-suicide process that results intermolecularly in a simple IS insertion. This process presumably involves little or no replication of the IS. Intramolecular transposition by this process normally results in nonviable products. However, in the particular situation where the transpositional target lies within the transposon, viable products are obtained; these are deletions and deletion-inversions. Deletions between an IS and a target lying outside the element (the conventional "adjacent deletion") occur by a fully replicative process analogous to the formation of cointegrate molecules in intermolecular transposition. The ability of an IS to promote adjacent deletions correlates closely with its ability to fuse replicons into a cointegrate. Before transposition can occur, a complex of the transposase and both IS ends is probably formed. Requirement for such a pretranspositional complex is suggested by the effect on transpositional frequency of changing the distance between the ends. Our results do not support any of the asymmetrical models for transposition. They are, however, compatible with a modified version of the symmetric model proposed by Shapiro (1979). It is interesting to note the similarity between the structures generated by intramolecular simple transposition of an inverse transposon and the circular structures apparently formed by retroviral and copia autointegrative transposition. Shoemaker et al. (1981a,b) and Flavell and Ish-Horowicz (1983) have characterized circular molecules from retrovirally infected cells and Drosophila tissue-culture cells, respectively. The structures of some of the circular molecules resemble deletions and deletion-inversions (Fig. 3B). To our knowledge, a circular species containing two long terminal repeats (LTRs) and an adjacent deletion, which we predict could only occur by a fully replicative process given the similarity in geometry of an LTR to an IS, have not been found. It would appear, then, that the molecule containing two LTRs acts as an inverse transposon, integrating into itself. Shoemaker et al. (1981b) and Flavell and Ish-Horowicz (1983) have also suggested that these products arise from molecules containing two LTRs. We suggest that the two inside LTR ends interact in a conservative, intramolecular, simple transpositionlike event.

Extrachromosomal replication of copia-based vectors in cultured Drosophila cells

The functional analysis of cloned Drosophila genes in vivo has so far not been possible because of the lack of an appropriate cultured cell transformation system. We have developed a transformation method which uses the Escherichia coli gene coding for xanthine guanine phosphoribosyl transferase (gpt)1, inserted into a cloned copia2 transposable element, and a medium selective for transformed cells expressing the gpt gene. We show that the resulting plasmids (pCVgpt) propagate extrachromosomally in transformed cells, apparently without integrating into the genome. This suggests that the extrachromosomal copia circles found in Drosophila tissue-culture cells may also replicate semi-conservatively.

Nonpackaging and packaging proteins of hnRNA in Drosophila melanogaster

Monoclonal antibodies have previously been raised against chromosomal proteins of Drosophila. Using a biochemical fractionation method for the isolation of large hnRNA-containing structures (hnRNP) of Drosophila tissue culture cells, we show that seven of these antibodies recognize different antigens, and that these antigens are associated with RNA. Analysis of the sedimentation behavior of antigen-containing structures in sucrose gradients reveals that the antigens are differentially distributed with respect both to one another and to pulse-labeled RNA. We demonstrate that the antigens are minor components of hnRNP and are different from the major Drosophila hnRNP packaging proteins, which we have also identified. The antigens are probably involved in the processing of hnRNA in the nucleus.

Acetylation and methylation patterns of core histones are modified after heat or arsenite treatment of Drosophila tissue culture cells.

Exposure of Drosophilamelanogaster tissue culture cells to 37 degrees C (heat shock) or to arsenite induces a severe deacetylation of core histones and blocks the methylation of histone H(3) and H(4). Heat shock induces the methylation of histone H(2b). These results are discussed in view of chromatin structure and function.Images

Heat shock induces rapid dephosphorylation of a ribosomal protein in Drosophila.

Ribosomes isolated from Drosophila melanogaster tissue culture cells labeled in vivo with 32Pi contain a single, heavily phosphorylated, ribosomal protein. As much as 40% of this protein is phosphorylated in cells cultured at 25 degrees C. The molecular weight and other characteristics of this protein suggest possible homology with ribosomal protein S6. Following a shift-up to 37 degrees C, the protein is specifically and quantitatively dephosphorylated. The kinetics of this dephosphorylation are rapid with a half-time on the order of a few minutes. These kinetics closely parallel the heat shock-induced breakdown of the preexisting polysome population.

The proteins expressed by different isolates of Drosophila C virus.

Isolates of Drosophila C virus (DCV) from Drosophila flies obtained in geographically different regions were adapted to growth in Drosophila tissue culture cells. The viruses, purified from tissue culture cells, were shown to be serologically related to one of the isolates ("O" from Ouarzazate, Morocco). Analysis of the structural proteins by polyacrylamide gel electrophoresis demonstrated differences between the isolates. Labelling intracellular proteins of infected Drosophila melanogaster cells with 35S-methionine at 28 degrees C demonstrated the presence of the virus structural proteins and their immediate precursors. Raising the temperature to 37 degrees C both before and during the pulse period inhibited the processing of the high molecular weight proteins and resulted in a greater "shut-off" of host cell proteins than viral induced proteins. This allowed the precursor proteins to be compared as well as the structural proteins of the different strains. It was possible to clearly distinguish differences between the isolates on the basis of the induced proteins, although limited proteolysis of corresponding proteins showed marked similarities. Hence it is possible to distinguish between different isolates of the "same" small RNA-virus of insects from geographically different regions.

Immunofluorescence localization of a small heat shock protein (hsp 23) in salivary gland cells of Drosophila melanogaster.

An aggregate present in cell-free extracts of Drosophila melanogaster tissue culture cells, sedimenting at 20 to 30S, contains hsps 23, 26 and 27. Hsp 23 was purified from this aggregate and a monospecific antibody was raised against it. Immunofluorescence microscopy showed the presence of hsp 23 preferentially in nuclei after heat shock, while on return to 25 degrees C, hsp 23 was reduced in nuclei and increased in the cytoplasm. Thus the immunofluorescence observations reported here unambiguously confirm for hsp 23 earlier reports that heat shock proteins are mainly found in nuclei after heat shock and that upon return to 25 degrees C, they move to the cytoplasm.

Transcription of the major Drosophila heat-shock gene in vitro

Active eukaryotic genes are more accessible to some proteins than bind DNA than are inactive genes. In order to probe the accessibility of the Drosophila heat-shock genes we have isolated nuclei from Drosophila tissue culture cells and have used these nuclei as templates for Escherichia coli RNA polymerase. With nuclei isolated from cells that had not been heat shocked, the synthesis of heat-shock RNA was not detected by hybridization to a DNA clone containing sequences from the major heat-shock region. In contrast, approximately 0.22% of the RNA synthesized in nuclei isolated from cells that had been previously heat shocked hybridized to the heat-shock clone. The synthesis of heat-shock RNA was DNA dependent, was sensitive to rifampicin and to actinomycin D, and represented a 70-fold enrichment over random transcription of the Drosophila genome. Transcription showed an extraordinary preference for a region 5' distal to the structural gene. These results demonstrate that preferential transcription by the bacterial RNA polymerase is indicative of the active state of Drosophila genes.

The polypeptides induced in Drosophila cells by Drosophila C virus (strain Ouarzazate)

The Ouarzazate strain of Drosophila virus (DCV 0) was grown in Drosophila melanogaster tissue culture cells, and [ 35S]methionine-labeled virions were found to contain a group of major structural proteins with a molecular weight of ≌30,000 as well as several minor proteins of higher molecular weight and a protein of ≌10,000 daltons. Using a range of pulses, chases and gel systems, examination of the intracellular proteins induced by DCV 0 showed the presence of 17 polypeptides not found in uninfected cells. The synthesis of virus-induced polypeptides was extremely asymmetric with a rapid appearance of the major virus structural proteins and a much slower appearance of the lowest molecular weight structural protein (VP4). Processing of virus-induced proteins including the appearance of VP4 was demonstrated using pulse-chase after pulsing with [ 35S]methionine. While the highest molecular weight induced protein found in infected cells was 146,000, pretreatment of cells with iodoacetamide resulted in the appearance of a protein with a molecular weight of ≌200,000. The evidence presented in this paper supports the inclusion of DCV 0 in the Picornaviridae group.

Inhibition of the induction of heat shock proteins in Drosophila melanogaster cells infected with insect picornaviruses

Infection of cells by mammalian picornaviruses results in an inhibition of ‘shut-off’ of host cell protein synthesis to varying extents depending upon the virus used [ 11. The recently characterised picornaviruses of insects have been shown to act similarly, with Cricket paralysis virus (CrPV) being extremely efficient in inhibitingDrosophila cell protein synthesis [2,3], whereas the related Drosophila C virus (DCV) is relatively inefficient [4]. Effort has been concentrated on the elucidation of the shut-off mechanisms although this has not led to a satisfactory model being established. Work has been hindered by having to look at the inhibition of a broad spectrum of cellular proteins and also by the absence of a method to differentiate between the effects on mRNA existing before infection and de novo expression of genes during the course of infection. This means that while theories of shut-off at the level of translation may serve to explain events occuring early in the infection process, they may not necessarily be valid models for later shut-off. To overcome both these problems, we have utilised the phenomenon of heat-shock in Drosophila in which elevated temperature induces the expression of a novel set of genes. This was first recognised in vivo as a new puffing pattern in Drosophila melanogaster salivary gland chromosomes and has since been demonstrated in many of the tissues of this fly, as well as in Drosophila tissue culture cells [S]. Elevation of the temperature of Drosophila tissue culture cells shifts the pattern of protein synthesis from a normal broad spectrum of proteins to a small number of previously unexpressed proteins. This makes it possible to examine the effect of infection on a small number of proteins which are synthesised at a known chromosomal site

Actin acetylation in Drosophila tissue culture cells.

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.

The 5' termini of RNAs encoded by the transposable element copia.

The 5' termini of copia-specific RNAs in Drosophila melanogaster tissue culture cells were determined by S1 nuclease mapping and cap analysis. Both major copia RNAs share an identical set of heterogeneous 5' ends. Three major cap 1 structures M7GpppCmpUp, M7GpppCmpCp and M7GpppGmpUp together with several other minor caps were found. Almost all the 5' termini, as judged by S-1 nuclease mapping, were located either in a pyrimidine-rich part of the terminal direct repeat or apparently outside of the copia element, suggesting that a proportion of copia transcripts derive from promoters external to the genetic element.

Translation and developmental regulation of RNA encoded by the eukaryotic transposable element copia.

copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.

Differential localization of ribosomal proteins S14 and 7/8 in egg chambers of D. melanogaster

The localization of Drosophila melanogaster ribosomal proteins S14, and 7/8 during oogenesis was studied by indirect immune fluorescence microscopy. The acidic proteins S141 and 7/81 were isolated from D. melanogaster embryonic ribosomal proteins by carboxymethylcellulose chromatography (Chooi 1980). Antibodies raised against each of these proteins were applied to ovariole squashes, and the position of each antibody was localized by fluorescein labeled sheep antirabbit IgGs. Anti-S14 was found predominantly in nurse cell nuclei, follicle cell nuclei, oocytes and, to a much lesser degree, in nurse cell and follicle cell cytoplasm. In contrast, anti-7/8 was found in major quantities in nurse cell and follicle cell cytoplasm, and oocytes. Anti-7/8 in the nurse cell and follicle cell nuclei was either not detectable or at a strikingly lower level than that found in the corresponding cytoplasm. The ‘egg chamber’ patterns of localization of these two proteins were also found in salivary gland cells. However, in Drosophila tissue culture cells, these patterns were altered; both anti-S14 and anti-7/8 were detected only in the cytoplasm.

Localization of the heat shock-induced proteins in Drosophila melanogaster tissue culture cells

The exposure of Drosophila melanogaster tissue culture cells to 37°C induces the synthesis of a new set of proteins, the heat shock proteins, while the synthesis of most of the proteins actively made before the heat shock is strongly repressed. Cellular localization of the heat shock proteins has been studied in different cell fractions and by electron microscope autoradiography of cell sections. The results indicate that the heat shock proteins of 22, 23, 26, and 27 kdaltons and at least a fraction of the 68- and 70-kdalton proteins are found in the nucleus after the heat shock, where they are present in chromatin and nucleoli preparations. When the temperature is shifted back to 25°C, the synthesis of the heat shock proteins regresses and stops after several hours, while the proteins normally synthesized at 25°C reappear. At 25°C, a large proportion of the heat shock proteins leaves the nucleus and is predominantly localized in the cytoplasm.

Investigation of the function of the heat shock proteins in Drosophila melanogaster tissue culture cells.

The effect of inhibitors of protein synthesis on RNA synthesis was investigated before and during heat shock. The results indicate that proteins specifically made following heat shock might be required for the resumption, after heat shock, of the synthesis of the RNA normally made at 25 degrees C. It has previously been shown that the heat shock proteins, with the exception of hsp 84 are found in the nucleus bound to chromatin at 37 degrees C, and that they move to the cytoplasm on further incubation of the cells at 25 degrees C (Arrigo et al., 1980). Taken together, these results suggest that some protein(s) synthesized during heat shock may be involved in the regulation of RNA synthesis. However evidence is presented showing that the newly synthesized proteins at 37 degrees are not involved in repressing the transcription of most of the genes active before the heat shock.

Transposition of elements of the 412, copia and 297 dispersed repeated gene families in drosophila

The stability of elements of three different dispersed repeated gene families in the genome of Drosophila tissue culture cells has been examined. Different amounts of sequences homologous to elements of 412, copia and 297 dispersed repeated gene families are found in the genomes of D. melanogaster embryonic and tissue culture cells. In general the amount of these sequences is increased in the cell lines. The additional sequences homologous to 412 copia and 297 occur as intact elements and are dispersed to new sites in the cell culture genome. It appears that these elements can insert at many alternative sites. We also describe a DNA sequence arrangement found in the D. melanogaster embryo genome which appears to result from a transposition of an element of the copia dispersed repeated gene family into a new chromosomal site. The mechanism of insertion of this copia element is precise to within 90 bp and may involve a region of weak sequence homology between the site of insertion and the direct terminal repeats of the copia element.

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.