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.     

Cellular titers and subcellular distributions of abundant polyadenylate-containing ribonucleic acid species during early development in the frog Xenopus laevis.

The distribution of cytoplasmic messenger ribonucleic acids (RNAs) in translationally active polysomes and inactive ribonucleoprotein particles changes during early development. Cellular levels and subcellular distributions have been determined for most messenger RNAs, but little is known about how individual sequences change. In this study, we used hybridization techniques with cloned sequences to measure the titers of 23 mitochondrial and non-mitochondrial polyadenylate-containing [poly(A)+]RNA species during early development in the frog Xenopus laevis. These RNA species were some of the most abundant cellular poly(A)+ RNA species in early embryos. The concentrations of most of the non-mitochondrial (cytoplasmic) RNAs remained constant in embryos during the first 10 h of development, although the concentrations of a few species increased. During neurulation, we detected several new poly(A)+ RNA sequences in polysomes, and with one possible exception the accumulation of these sequences was largely the result of new synthesis or de novo polyadenylation and not due to the recruitment of nonpolysomal (free ribonucleoprotein) poly(A)+ RNA. We measured the subcellular distributions of these RNA species in polysomes and free ribonucleoproteins during early development. In gastrulae, non-mitochondrial RNAs were distributed differentially between the two cell fractions; some RNA species were represented more in free ribonucleoproteins, and others were represented less. By the neurula stage this differential distribution in polysomes and free ribonucleoproteins was less pronounced, and we found species almost entirely in polysomes. Some poly(A)+ RNA species transcribed from the mitochondrial genome were localized within the mitochondria and were mapped to discrete fragments of the mitochondrial genome. Much of this poly(A)+ RNA was transcribed from the ribosomal locus. Nonribosomal mitochondrial poly(A)+ RNA species became enriched in polysome-like structures after fertilization, with time courses similar to the time course of mobilization of cytoplasmic poly(A)+ RNA.     

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.     

The occurrence of families of repetitive sequences in a library of cloned cDNA from human lymphocytes

A library of cloned cDNAs representative of lymphocyte total poly(A)+ RNA was screened with total DNA probes at high clone density. 10% of the recombinants showed the presence of sequences which are repeated in the genome. Further analysis of six such isolated cDNA clones indicated that they contain different families of repetitive sequences with reiteration frequencies of between 150 and 45,000 copies per haploid genomes. Five of the six clones were found to contain single copy sequences as well as a repetitive sequence. cDNA clones containing repetitive sequences have been found to be derived from high, intermediate and low abundance classes of lymphocyte poly(A)+ RNA.     

Cloning of cDNA sequences derived from poly(A)+ nuclear RNA ofXenopus laevis at different developmental stages: Evidence for stage specific regulation

Summary Nuclear poly(A)⁺ RNA was isolated from gastrula and early tadpole stages ofXenopus laevis, transcribed into cDNA and integrated as double stranded cDNA by the G-C joining method into the Pst cleavage site of plasmid pBR 322. After cloning inE. coli strain HB 101 the clone libraries were hybridized to³²P labelled cDNA derived from nuclear poly(A)⁺ RNA of the two different developmental stages. About 20% of the clones gave a positive hybridization signal thus representing RNA molecules of high and medium abundance. From these clones, some individual clones were identified containing sequences which are not present at the oocyte and gastrula stages but which are transcribed at the early tadpole stage of embryonic development.     

Isolation of genes preferentially expressed during Bacillis subtilis spore outgrowth.

From the Charon 4A library of Ferrari et al (J. Bacteriol. 146:430-432, 1981) we isolated three genes involved in Bacillus subtilis spore outgrowth by screening the library by hybridization with labeled RNA from outgrowing spores in the presence of an excess of unlabeled vegetative RNA. Hybridization competition experiments with purified clones showed that the clones contained sequences that were transcribed only during spore outgrowth or preferentially during spore outgrowth. Fragments of the cloned DNAs were subcloned in plasmid pJH101, and by using plasmid integration and PBS1 transduction the chromosomal loci were mapped. The three loci which we mapped are outG and outH, which are located between polC and dnaA, and outI, which is located near pycA. Using the cloned DNAs and derived plasmids in dot hybridization experiments with RNA extracted from cells at different developmental stages, we defined for two clones a region that is transcribed only during the outgrowth phase.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24 degrees C are transferred rapidly to 32 degrees C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)+RNA is 24 degrees C and 35% at 32 degrees C. Kinetic analysis of cDNA-poly(A)+RNA hybridizations reveals three abundance classes of poly(A)+RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24 degrees C and 13000 at 32 degrees C. The intermediately abundant and highly abundant classes are represented by 100-200 and 1-3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)+RNA with increase in temperature.     

Actin gene expression is modulated by ecdysterone in a Drosophila cell line

The steroid hormone ecdysterone induced characteristic and specific changes of morphology, enzymatic activities and protein synthesis in a Kc 0% Drosophila melanogaster cell line. To study the ecdysterone action at a molecular level, a Drosophila genomic library was screened by differential hybridization to poly(A)+ RNA from control and ecdysterone-treated cells. Two recombinant phages were selected for hybridizing very intensively with poly(A)+ RNA of ecdysterone-treated cells and very weakly with poly(A)+ RNA of untreated ones. These two clones (lambda Dm 1632 and lambda Dm A5A1) mapped at the 5 C locus on polytene chromosomes; they overlap for a 9000 base-pair sequence that contains an abundantly transcribed region in ecdysterone-treated cells of about 2000 base-pairs. This region permits the selection of mRNA that gives, after translation in vitro, two polypeptides identified as cytoplasmic actin II and III. We demonstrated that these two recombinant phages, hybridizing preferentially with poly(A)+ RNA of ecdysterone-treated cells, contain the 5 C actin gene. Poly(A)+ RNA prepared from various times of treatment of cells were electrophoresed on agarose gels, transferred to nitrocellulose paper and then hybridized with the cloned actin probe. Results of these experiments indicate that there is a sharp increase in the level of RNA coding for actin after ecdysterone treatment of the cell, and that there are two forms of actin-specific RNA in the D. melanogaster cells. Using genomic blots with specific probes derived from lambda Dm 1632, we show that there are six actin genes per haploid Drosophila cell genome contained on six EcoRI fragments, as in Drosophila embryos, indicating that there is no rearrangement of these sequences in cultured cells. Our results suggest that the expression of actin genes in D. melanogaster Kc 0% cells is modulated by ecdysterone.     

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.     

The cloning and expression of the gene encoding organ-specific esterase S from the genome of Drosophila virilis

We have cloned the gene for the esterase S isozymes complex from the genome of Drosophila virilis in pBR322. Esterase S is an enzyme which is specifically synthesized in the ejaculatory bulbs of D. virilis adult males. The gene for the esterase S isozyme complex (estS) has been localized in band 2G5e of chromosome II. Poly(A)+ RNA prepared from ejaculatory bulbs actively hybridizes with this band. A cloned 15-kb fragment of D. virilis DNA (pVE9) also hybridizes with band 2G5e. The area encoding the poly(A)+ RNA is located in the middle part of the cloned fragment whose ends are not transcribed in vivo. Only one poly(A)+ RNA which is 1.9 kb long and complementary to pVE9 DNA can be revealed in the cytoplasm. The mRNA preselected by hybridization to pVE9 DNA was microinjected into the cytoplasm of Xenopus laevis oocytes. In other experiments, the pVE9 DNA itself was microinjected into oocyte nuclei. In both cases, esterase S is synthesized in the oocytes, and the major part of the protein is transported from the oocytes and accumulated in the incubation medium.     

The isolation of cloned cDNA sequences which are differentially expressed in human lymphocytes and fibroblasts.

Poly(A)+ RNA populations derived from normal lymphocytes and fibroblasts have been compared by hybridising each RNA to cDNA derived from the other RNA population. This indicated that approximately 75% of the sequences were common to both, and that these were present at different concentrations in the two cell types. The two RNA populations were further compared by hybridising them to a cDNA recombinant library derived from lymphocyte poly(A)+ RNA. This allowed the identification of clones containing sequences which are abundant in lymphocyte poly(A)+ RNA but absent or rare in fibroblast poly(A)+ RNA. A direct estimation of the abundance of five of these sequences in lymphocyte cDNA demonstrated that clones can be detected by such a procedure if they represent 0.2% or greater of the original cDNA population.     

Location of messenger specifying sequences in mammalian chromosomes

In situ hybridization of complementary DNA (cDNA) synthesized from total cytoplasmic polyadenylated RNA isolated from Chinese hamster cells was employed to investigate the distribution of messenger specifying sequences on mammalian chromosomes. The kinetics of cDNA-nuclear DNA annealing indicate that about 85% of the cDNA represents sequences which are transcribed from non-repetitive DNA sequences. When cDNA is hybridized back to its template RNA, the reaction kinetics show that more than 60% of the poly(A) RNA is at least 10⁴ times more complex than rabbit globin mRNA. In situ hybridization of cDNA to Chinese hamster cells fixed on slides shows no significant clustering of silver grains on interphase nuclei. On metaphase chromosomes the majority of silver grains are localized in euchromatic areas. It appears that all euchromatic segments have similar grain densities. Chromosomes 1 and 2, which have relatively little heterochromatin, do not have a higher grain density than the other chromosomes. However, the Y chromosome, which is entirely heterochromatic, contains only about 1/3 the grain density of the chromosomes 1 or 2. — When the cDNA, which anneals only to the high abundancy class of poly(A) RNA was fractionated and hybridized in situ to Chinese hamster chromosomes, the distribution of silver grains is localized in the euchromatic areas. The Y chromosome and the heterochromatic arm of the X chromosome contain less grains; telomeres of some autosomes have higher grain densities. The oligo-(dT) primer in cDNA did not affect the results of this study since no grains are found when ₃H-poly(dT) was used as probe for in situ hybridization. The majority (>90%) of the grains could be blocked by competition with excess repetitive DNA in the hybridization reaction, indicating that the in situ hybridization involved predominantly repetitive sequences.