The 5S genes of Drosophila melanogaster.

We have cloned embryonic Drosophila DNA using the poly (dA-DT) connector method (Lobban and Kaiser, 1973) and the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975) as a cloning vehicle. Two clones, containing hybrid plasmids with sequences complementary to a 5S RNA probe isolated from Drosophila tissue culture cells, were identified by the Grunstein and Hogness (1975) colony hybridization procedure. One hybrid plasmid has a Drosophila insert which is comprised solely of tandem repeats of the 5S gene plus spacer sequences. The other plasmid contains an insert which has about 20 tandem 5S repeat units plus an additional 4 kilobases of adjacent sequences. The size of the 5S repeat unit was determined by gel electrophoresis and was found to be approximately 375 base pairs. We present a restriction map of both plasmids, and a detailed map of of the5S repeat unit. The 5S repat unit shows slight length and sequence heterogeneity. We present evidence suggesting that the 5S genes in Drosophila melanogaster may be arranged in a single continuous cluster.     

The 5S ribosomal genes in the Drosophila melanogaster species subgroup. Nucleotide sequence of a 5S unit from Drosophila simulans and Drosophila teissieri

The 5S genes of the eight species of the D. melanogaster subgroup have been mapped. The spacers, in contrast with coding regions, differ markedly between most species. One 5S gene unit has been sequenced for both D. simulans and D. teissieri. The mature 5S RNA region in these two species is identical to the corresponding region of D. melanogaster. Only 5 nucleotide variations occur between the D. melanogaster and D. simulans 5S gene spacers. The spacer in D. teissieri is very different. Only two segments, located one at each side of the coding region, are clearly homologous to corresponding sequences of D. melanogaster and D. simulans.     

Dosage of 5 S and ribosomal genes during oogenesis of Drosophila melanogaster

During growth, the Drosophila egg chamber increases its DNA content over a thousandfold, mainly by polyploidization of the nurse cell nuclei. We wanted to determine if 5 S and ribosomal genes are replicated to the same extent as the remaining DNA. Egg chambers were mass fractionated to represent different size classes and, therefore, different stages of oogenesis. Nucleic acids were extracted from each class of egg chambers, and after removal and quantitation of the RNA, the content of 5 S and ribosomal genes in the different DNA fractions was assayed by filter hybridization. Diploid DNA and DNA from polytene salivary gland cells served as references. It was concluded that: (1) Ribosomal genes become underreplicated as oogenesis proceeds, but to a much lower extent than in polytene chromosomes of salivary glands of the same organism. (2) By contrast, 5 S genes are equally replicated in egg chambers of all stages of oogenesis. (3) Notwithstanding the large increase in DNA content of egg chambers during oogenesis, the increase in total RNA content (mostly ribosomal RNA) is over 15 times as large.     

The location of 5S (ribosomal) RNA genes in Drosophila hydei

The location of the 5S ribosomal RNA cistrons in band 2-23B1,2 of the polytene (salivary gland) chromosomes of Drosophila hydei was indicated by in situ hybridization of tritiated low molecular weight RNA fractionated from total in vivo synthesized larval RNA or from in vitro synthesized salivary gland RNA and competition of the hybridization of this RNA by 5S RNA obtained from calf lens ribosomes. -- At the submicroscopic level, band 2-23B1,2 in salivary gland chromosomes shows a compact organization. The adjacent region, 23B2, is slightly puffed and displays typical RNP particles, some of which may be observed close to band 2-23B1,2.     

Cloned complementary deoxyribonucleic acid of Drosophila cells. Relationship of genome copy number to messenger ribonucleic acid abundance

Recombinant DNA plasmids containing DNA sequence complementary to poly(adenylic acid) [(poly(A)] containing RNA from the cytoplasm of Drosophila Kc tissue culture cells were constructed. The reiteration frequency in the genome of the RNA homologous to the 20 randomly selected clones was determined by two rapid methods. Of the 20, 17 were determined to be single copy, 2 were repeated several (2-4) times, and 1 was repeated approximately 10 times. The steady-state level of mRNAs homologous to the 20 cDNAs was quantitated and varied more than 160-fold. The RNAs ranged from 0.16% to less than 0.001% of the poly(A)-containing RNA.     

The effect of polyamines on the synthesis of ribonucleic acid by Drosophila melanogaster larvae.

1. To elucidate further the possible role of polyamines in the synthesis of nuclei acids, a study of the effect of exogenously administered amines on the synthesis of RNA by Drosophila melanogaster larvae was undertaken. This system was chosen because of the previous investigations [Dion, A.S. & Herbst, E.J. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 2367-2371; Herbst, E.J. & Dion, A.S. (1970) Fed. Proc. Fed. Am. Soc. Exp. Biol. 29, 1563-1567] relating putrescine and spermidine to growth and development of Drosophila.     

Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster: Purification and properties

Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster has been purified 1400-fold over a crude 230,000g supernatant fraction. The optimum activity of the enzyme occurs at magnesium concentrations above 10 mm at 37 °C and pH 7.5. At a 50 mm Mg²⁺ concentration, NH₄⁺ stimulates the ATP-PP₁ exchange reaction as much as 2-fold. Ammonium chloride causes an increase in the V with no change in the K_m with phenylalanine as substrate. Homologous (Drosophila) tRNA, in the presence of NH₄⁺, further stimulates the ATP-PP_i, exchange reaction but inhibits the reaction in the absence of NH₄⁺.In the presence of its substrates the enzyme is inactivated by NEM to varying degrees depending upon the substrate or combinations of substrates used. In the presence of phenylalanine the enzyme is partially protected but both ATP and tRNA make the enzyme more susceptible to inactivation. NEM together with ATP and tRNA or all three substrates results in near-total inactivation.     

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.     

Proportional polyploidization of 5S RNA genes in the ovary of Drosophila melanogaster mutants containing three 5S RNA gene loci

The 5S RNA gene content of polyploid cells of the ovary of Drosophila melanogaster has been compared in animals with two or three gene clusters. The amount of 5S RNA genes is exactly proportional to the number of gene clusters as determined by DNA-RNA filter hybridization. In contrast, the number of rDNA genes in endomitotic cells remains constant regardless of different numbers of nucleolus organizer regions (Spear, 1974).     

The sequence ofTetrahymena thermophila 5S ribosomal ribonucleic acid

The primary structure ofTetrahymena thermophila 5S rRNA is reported. A secondary structure model is presented which can encompass most published eukaryotic 5S rRNA sequences. Unlike other eukaryotic 5S rRNAs,Tetrahymena is found to contain the sequence-CGAAC- beginning at position 40. The presence of this segment had previously been thought to be an exclusive characteristic of eubacterial 5S rRNAs.     

Variability in the molecular organization of the 5S RNA genes among strains of Drosophila melanogaster.

The organization of the 5S RNA cluster has been analyzed in four strains of Drosophila melanogaster by the Southern technique. In some of the strains the 5S RNA cluster appears to be interrupted by an unrelated sequence. In other strains a continuous cluster is present.     

Structure, translation, and metabolism of the cytoplasmic copia ribonucleic acid of Drosophila melanogaster

We have characterized the copia RNA in the cytoplasm of cultured Drosophila cells. Copia RNA was detected and purified by hybridization to DNA of the plasmid cDm 1142, which contains the copia sequence. A large fraction (2.2%) of the total cytoplasmic poly(A)+ RNA was found to be copia RNA. Cytoplasmic copia RNA displays all the characteristics expected for a messenger RNA. It possesses a poly(A) tract identical in length with that of total poly(A)+ cytoplasmic RNA. It is associated with polysomes and can be released from this association by treatment with EDTA. When purified copia RNA is added to an mRNA-dependent rabbit reticulocyte lysate, three polypeptides of 51000, 33000, and 21000 daltons are seen. We have not determined if these are different polypeptides or if the two smaller polypeptides are fragments of the 51000-dalton polypeptide. The half-life of copia cytoplasmic RNA was determined in pulse--chase experiments to be 9.5 h; this is 1.6 times longer than the half-life of the intermediate decay class of total poly(A)+ cytoplasmic RNA. These properties provide strong evidence that copia RNA functions in vivo as a messenger RNA.     

Maintenance of the copy number of retrotransposon MDG3 in the Drosophila melanogaster genome]

The genomes of laboratory stocks and natural population of Drosophila melanogaster contain 8-12 copies of retrotransposon MDG3 detected by in situ hybridization. Construction of genotypes with decreased MDG3 copy number using X-chromosome and chromosome 3 free of MDG3 copies results in appearance of hybrid genomes carrying up to 7-10 copies, instead of 2-4 copies expected. New MDG3 copies are detected in different genome regions, including the 42B hot spot of their location. The chromosomes, where new clusters of MDG3 were observed, carry conserved "parental pattern" of MDG1 arrangement. The data obtained suggest the existence of genomic mechanism for maintenance of retrotransposon copy number on a definite level.