Isolation and chromosomal localization of ecdysterone-responsive genes in a Drosophila cell line

Cultured Kc 0% cells of Drosophila melanogaster are responsive to ecdysterone treatment. A library of lambda phages carrying segments of Drosophila genomic DNA was differential screened using poly(A)⁺RNAs from control and ecdysterone-treated cells. Nine independent recombinant phages that hybridized more intensely with poly(A)⁺ RNA from treated cells and six that hybridized most strongly with poly(A)⁺RNA from untreated cells were selected. Genomic localization of these inducible and repressible sequences was determined by hybridization in situ. These results suggest that expression of several unique genes is increased by the hormone. The six repressible sequences each contained DNA that hybridized to multiple chromosomal sites and appeared to be mobile elements, suggesting that the steroid hormone might be acting on the transposable elements. These probes will be useful for the study of positive and negative steroid regulation within the same cell.     

Molecular cloning of DNA complementary to Drosophila melanogaster alpha-amylase mRNA

Several lambda clones containing cDNAs from Drosophila melanogaster were identified in a lambda cDNA bank using two different approaches: (i) cross-species hybridization using a mouse amylase cDNA probe, and (ii) probing with a differential probe, generated from Drosophila RNA. An amylase cDNA fragment was used, in turn, for the isolation and characterization of amylase genomic clones. The size of the Drosophila amylase mRNA was estimated at 1650 b. This is comparable with the size of the murine amylase messenger that encodes a protein of similar molecular weight. In Drosophila larvae, amylase mRNA can account for as little as 0.01% of the poly(A)+ RNA under conditions of dietary glucose repression or greater than 1% of poly(A)+ RNA under derepressing dietary conditions.     

Molecular Cloning of α-Amylase Genes from DROSOPHILA MELANOGASTER. I. Clone Isolation by Use of a Mouse Probe

A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.     

Ecdysterone induction of actin synthesis and polymerization in a Drosophila melanogaster cultured cell line

Actin pools have been evaluated in Drosophila melanogaster Kc 0% cells, through an actin assay based on differential inhibition of DNase I by globular (G) and filamentous (F) actin. Total actin represents about 4 % of total proteins and 54 % is G-actin. In ecdysterone treated cells (0.1 μM), the total actin content increases up to 9 % of total proteins after 3 days of treatment. Ecdysterone induces increase of G-actin as well as F-actin. Increase of both actins, detectable after only 24 hrs of treatment, is roughly parallel during the first two days of treatment. For longer hormonal treatment, actin polymerization is more important than accumulation of G-actin. Indirect immunofluorescence microscopy with antibodies to exogeneous DNase I suggests that actin is widely distributed in the whole cytoplasm before and after ecdysterone treatment. These results suggest that ecdysterone induces actin synthesis and polymerization in Drosophila melanogaster cells.     

mRNA usage during Drosophila melanogaster embryonic development. Analysis of nine cloned DNA segments

A set of nine phage lambda clones containing inserts from Drosophila melanogaster which are complementary to cDNA made from oocyte poly(A)+ RNA were selected from a larger group. These cloned elements code for a range of middle abundant RNA sequences which show no appreciable change in abundance during Drosophila embryogenesis. Seven of the nine clones are complementary to two oocyte RNAs, one to three RNAs and one to four RNAs. This study describes the changes that occur in these RNAs during embryonic development in the polysomal and non-polysomal fraction, and in the poly(A)+ RNA and poly(A)- RNA fraction. In all nine of these clones, greater than 70% of the complementary RNA is found in the polysomal region of a sucrose gradient. This proportion increases somewhat during development. Specific changes have been found during development in the proportion of RNA that is poly(A)+. Depending to the cloned sequence, this proportion may increase, decrease, or remain unchanged. For those clones that show a change, most of this change occurs between 8 and 19 h of development. Our data suggest, furthermore, the presence of a class of non-adenylated RNA being utilized during embryogenesis.     

Genetic properties of phage lambda recombinant molecules. The effect of an inserted fragment on the hybrid yield and recombination with respect to the h- and vir-markers

The yield of mature phages in lambda gt-lambda Dm34, lambda gt-lambda DM225 and lambda gt-lambda Dm234 is 2, 8 and 8 times lower (respectively) as compared to the wild type. The output of hvir-recombinants in lambda gt-lambda Dm225 is more than an order lower as compared to the other phages. As distinct from two other hybrids, the decrease in lambda gt-lamba DM225 yield cannot be explained by red-genotype and a shortening of the phage genome as well as the decrease in the output hvir-recombinants cannot be attributed to a decrease in the yield of mature particles and to a shortening of the distance between markers. The data obtained show a contribution of Drosophila DNA to the observed effects.     

The expression and genomic organization of randomly selected cloned Drosophila melanogaster genes

A lambda recombinant DNA library containing Drosophila melanogaster nuclear DNA inserts was screened with cDNA made from oocyte and gastrula poly(A)+ RNA. 124 clones were isolated which represented sequences complementary to a distribution of abundancies of their RNAs. The clone set was then used as probes to identify those whose RNA abundancies changed during embryonic development. The vast majority of clones showed little difference during development. Four different clones were identified whose poly(A)+ RNAs were quantitatively regulated; two were oocyte-specific, and two were embryonic-specific. 44 clones were chosen for in situ hybridization to salivary gland polytene chromosomes. The location and distribution of their sites are described. A class of clones, identified by in situ hybridization to the nucleolus, is further described. These clones contain a scrambled array of ribosomal intervening sequences.     

Two closely linked transcription units within the 63B heat shock puff locus of D. melanogaster display strikingly different regulation

We report the isolation and characterization of a cloned DNA of D. melanogaster, Dm4L, that is derived from the major heat shock puff site at 63B. This segment contains two closely linked genes that are each present once per Drosophila haploid genome. One of these, the hsp 83 gene, encodes an abundant heat shock mRNA that, unlike other major heat shock mRNAs, is also abundant in uninduced (23 degrees) kco cells. Although only a slight increase in the level of total hsp 83 RNA can be detected after heat shock in Kco cells, the level of hsp 83 poly(A)+ mRNA increases more than 6-fold and the level of pulse-labeled hsp 83 RNA in total cellular RNA increases 11-fold relative to uninduced cells. In contrast, the levels of total, poly(A)+, and pulse-labeled RNA homologous to the second gene, 63B-T2, are approximately the same in both induced and uninduced cells. Hence, even though these genes are separated by only one thousand base pairs, and, from in situ hybridization to polytene chromosomes, both lie within the heat shock puff, they display strikingly different regulatory properties, These results demonstrate that close linkage of a gene to a heat shock puff is not sufficient to render its expression heat inducible.     

Cloning and cytogenetic localization of evolutionarily conserved genomic sequences which are expressed in the head of the Drosophila melanogaster imago

Screening of Drosophila melanogaster genomic library was carried out using mouse brain polysomal poly(A)+RNA. As a result, 100 clones were selected, among which 14 clones were picked up after hybridization with fly head poly(A)+RNA. It follows therefore, that these clones contain evolutionary conserved sequences which are expressed in Drosophila fly heads. Analysis of these 14 clones revealed RNA-coding fragments. Comparison of their expression in heads and bodies of Drosophila was carried out. Using in situ hybridization we determined the localization of selected 14 sequences on polytene chromosomes. The possibility of further analysis of some clones to study developmental and functional processes in neural system of Drosophila is discussed.     

Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured Drosophila cells.

We have directly compared the ability of four promoters and three polyadenylation (poly(A)) signals to direct heterologous gene expression in stably transfected Drosophila melanogaster S2 cells. We compared two constitutive Drosophila promoters, the actin 5C distal promoter and the alpha 1-tubulin promoter, with the tightly regulated Drosophila metallothionein (Mtn) promoter and the Bombyx mori fibroin promoter. We find that the actin 5C and induced Mtn promoters generate comparable high levels of RNA and protein in this system. The alpha 1-tubulin promoter generates about four-fold lower levels, and the fibroin promoter shows no detectable activity in S2 cells. Interestingly, genes expressed from the constitutive actin 5C and alpha 1-tubulin promoters are consistently present at three- to four-fold lower copy numbers than genes expressed from the inducible Mtn promoter or the inactive fibroin promoter. Poly(A) signals of both mammalian (SV40) and Drosophila (Mtn) origin efficiently directed stable RNA synthesis in S2 cells, and, as in mammalian cells, the SV40 late poly(A) signal was more efficient than the SV40 early poly(A) signal. Thus the process of polyadenylation appears to be conserved between mammalian and Drosophila cells.     

Clusters containing different mobile dispersed genes in the genome of Drosophila melanogaster.

Ten clones containing the actively transcribed mobile dispersed gene Dm255 and its flanking sequences were selected from the HindIII bank of the Drosophila melanogaster genome. The Dm225 sequences present in these clones were identical while the flanking sequences were different in all of the clones analysed. Four of them contained, in addition to Dm225, other DNA sequences binding high amounts of cytoplasmic poly(A) + RNA. The properties of these new genes are similar to those of Dm255: they are also actively transcribed, multiple in copies, scattered throughout the genome, and located at varying genome sites which also were scattered throughout the whole genome of D. melanogaster. Thus, different mobile dispersed genes often appear as closely apposing units forming gene clusters in the genome.     

The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

Concentrations of individual RNA sequences in polyadenylated nuclear and cytoplasmic RNA populations of Drosophila cells.

Steady state concentrations of individual RNA sequences in poly(A) nuclear and cytoplasmic RNA populations of Drosophila Kc cells were determined using cloned cDNA fragments. These cDNAs represent poly(A) RNA sequences of different abundance in the cytoplasm of Kc cells, but their steady state concentrations in poly(A) hnRNA was always lower. Of ten different sequences analysed, eight showed some four-fold lower concentration in hnRNA mRNA, two were underrepresented in hnRNA relative to the others. The obvious clustering of mRNA/hnRNA ratios is discussed in relation to sequence complexity and turnover rates of these RNA populations.