The generation of cloned Drosophila melanogaster

We report here the first successful use of embryonic nuclear transfer to create viable adult Drosophila melanogaster clones. Given the generation time, cost effectiveness, and relative ease of embryonic nuclear transplant in Drosophila, this method can provide an opportunity to further study the constraints on development imposed by transplanting determined or differentiated nuclei.     

Molecular cloning and genomic organization of an allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins are neurohormones, acting on the corpora allata (where they block the release of juvenile hormone) and on the insect gut (where they block smooth muscle contraction). We screened the "Drosophila Genome Project" database with electronic sequences corresponding to various insect allatostatins. This resulted in alignment with a DNA sequence coding for some Drosophila allatostatins (drostatins). Using PCR with oligonucleotide primers directed against the presumed exons of this Drosophila allatostatin gene and subsequent 3'- and 5'-RACE, we were able to clone its cDNA. The Drosophila allatostatin preprohormone contains four amino acid sequences that after processing would give rise to four Drosophila allatostatins: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH(2) (drostatin-1), Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH(2) (drostatin-2), Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH(2) (drostatin-3), and Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2) (drostatin-4). Drostatin-2 is identical to helicostatin-2 (11-18) and drostatin-3 to helicostatin-3, two neurohormones previously isolated from the moth Helicoverpa armigera. Furthermore, drostatin-3 has previously been isolated from Drosophila itself. Drostatins-1 and -4 are novel members of the insect allatostatin neuropeptide family. The Drosophila allatostatin preprohormone gene contains two introns and three exons. The gene is located on the right arm of the third chromosome, position 96A-B. The existence of at least four different Drosophila allatostatins opens the possibility of a differential action of some of these hormones on the two recently cloned Drosophila allatostatin receptors, DAR-1 and -2. This is the first report on an allatostatin preprohormone from Drosophila.     

Transcriptional organization of the Drosophila melanogaster ribosomal RNA genes.

Five distinct DNA replicating intermediates have been separated from lysates of bacteriophage G4-infected cells pulse-labelled during the period of replicative form synthesis using propidium diiodide/caesium chloride gradients. These are a partially single-stranded theta structure that is labelled in both the viral and complementary DNA strands; partially single-stranded circles, some with an unfinished viral DNA strand (25%) and some with an unfinished complementary DNA strand (75%); replicative form II(RFII) and replicative form I(RFI) DNA labelled only in the complementary DNA strand. To explain the pulse-label data a model is proposed in which G4 replicative form replication takes place by a displacement mechanism in which synthesis of the new viral DNA strand displaces the old viral DNA strand as a single-stranded DNA loop (D-loop) and when the displacement reaches half way round the molecule (the origin of synthesis of the G4 viral and complementary DNA strands are on opposite sides of the genome, Martin &; Godson 1977) synthesis of the complementary DNA strand starts, but in the opposite direction. Strand separation of the parent helix runs ahead of DNA synthesis, releasing two partially single-stranded circles from the replicating structure which then complete their replication as free single-stranded DNA circles. No evidence was found to support a rolling circle displacement mechanism of G4 replicative form synthesis.     

Characterization and expression of collagen-like genes in Drosophila melanogaster

We have used a cloned chicken collagen cDNA sequence to help identify hypothetic members of the collagen gene family from Drosophila melanogaster. Several experimental evidences have been obtained which indicate that the Drosophila genome contains numerous collagen-like sequences. We have characterized in more detail ten distinct DNA sequences that hybridized strongly to the heterologous collagen probe. By in situ hybridization we have shown that these sequences are dispersed throughout the Drosophila genome. Two of them are shown to originate from the previously described DCg 1 and DCg 2 collagen genes. In other respects, we show that in addition to DCg 1 and DCg 2, at least five putative collagen genes are expressed during the Drosophila lifetime. These genes are unique, and some of them are seen to be transcribed into different size classes of mRNAs. Additionally, the data presented so far demonstrate that the expression of these genes is regulated temporally and/or quantitatively during the Drosophila life cycle.     

The genomic organization of HeT-A retroposons inDrosophila melanogaster

Members of theDrosophila HeT-A family of transposable elements are LINE-like retroposons that are found at telomeres and in centric heterochromatin. We recently characterized an active HeT-A element that had transposed to a broken chromosome end fewer than mine generations before it was isolated. The sequence arerangement of this element, called 9D4, most likely represents the organization of an actively transposing member of the HeT-A family. Here we assess the degree of divergence among members of the HeT-A family and test a model of telomere length maintenance based on HeT-A transposition. The region containing the single open reading frame of this element appears to be more highly conserved than the non-coding regions. The HeT-A element has been implicated in theDrosophila telomere elongation process, because frequent transpositions to chromosome ends are sufficient to counter-balance nucleotide loss due to incomplete DNA replication. The proposed elongation model and the hypothetical mechanism of HeT-A transposition predict a predominant orientation of HeT-A elements with their oligo (A) tails facing proximally at chromosome ends, as well as the existence of irregular tandem arrays of HeT-A elements at chromosome ends resulting from transposition of new HeT-A elements onto chromosome ends with existing elements. Twenty-nine different HeT-A fragments were isolated from directional libraries that were enriched in terminal DNA fragments. Sequence analyses of these fragments and comparisons with the organization of the HeT-A element, 9D4, fit these two predictions and support the model ofDrosophila telomere elongation by transposition of HeT-A elements.     

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.     

Structure and expression of ubiquitin genes of Drosophila melanogaster.

We isolated and characterized two related ubiquitin genes from Drosophila melanogaster, polyubiquitin and UB3-D. The polyubiquitin gene contained 18 repeats of the 228-base-pair monomeric ubiquitin-encoding unit arranged in tandem. This gene was localized to a minor heat shock puff site, 63F, and it encoded a constitutively expressed 4.4-kilobase polyubiquitin-encoding mRNA, whose level was induced threefold by heat shock. To investigate the pattern of expression of the polyubiquitin gene in developing animals, a polyubiquitin-lacZ fusion gene was introduced into the Drosophila genome by germ line transformation. The fusion gene was expressed at high levels in a tissue-general manner at all life stages assayed. The ubiquitin-encoding gene, UB3-D, consisted of one ubiquitin-encoding unit directly fused, in frame, to a nonhomologous tail sequence. The amino acid sequence of the tail portion of the protein had 65% positional identity with that of yeast UBI3 protein, including a region that contained a potential nucleic acid-binding motif. The Drosophila UB3-D gene hybridized to a 0.9-kilobase mRNA that was constitutively expressed, and in contrast to the polyubiquitin gene, it was not inducible by heat shock.     

Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)(6)Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2), 相似文献   

Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.     

Phototransduction genes are up-regulated in a global gene expression study of Drosophila melanogaster selected for heat resistance

The genetic architecture underlying heat resistance remains partly unclear despite the well-documented involvement of heat shock proteins (Hsps). It was previously shown that factors besides Hsps are likely to play an important role for heat resistance. In this study, gene expression arrays were used to make replicate measurements of gene expression before and up to 64 hours after a mild heat stress treatment, in flies selected for heat resistance and unselected control flies, to identify genes differentially expressed in heat resistance-selected flies. We found 108 genes up-regulated and 10 down-regulated using the Affymetrix gene expression platform. Among the up-regulated genes, a substantial number are involved in the phototransduction process. Another group of genes up-regulated in selected flies is characterized by also responding to heat shock treatment several hours after peak induction of known Hsps revert to nonstress levels. These findings suggest phototransduction genes to be critically involved in heat resistance, and support a role for components of the phototransduction process in stress-sensing mechanisms. In addition, the results suggest yet-uncharacterized genes responding to heat stress several hours after treatment to be involved in heat stress resistance. These findings mark an important increase in the understanding of heat resistance.     

Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins are a diverse group of neuropeptides that obtained their names by their inhibitory actions on the corpora allata (two endocrine glands near the insect brain), where they block the biosynthesis of juvenile hormone (a terpenoid important for development and reproduction). Chemically, the allatostatins can be subdivided into three different peptide groups: the large group of A-type (cockroach-type) allatostatins, which have the common C-terminal sequence Y/FXFGLamide; the B-type (cricket-type) allatostatins, which have the C-terminal sequence W(X(6))Wamide in common; and a single allatostatin that we now call C-type allatostatin that was first discovered in the moth Manduca sexta, and which has a nonamidated C terminus, and a structure unrelated to the A- and B-type allatostatins. We have previously cloned the preprohormones for the A- and B-type allatostatins from Drosophila melanogaster. Here we report on the cloning of a Drosophila C-type allatostatin preprohormone (DAP-C). DAP-C is 121 amino acid residues long and contains one copy of a peptide sequence that in its processed form has the sequence Y in position 4) from the Manduca sexta C-type allatostatin. The DAP-C gene has three introns and four exons and is located at position 32D2-3 on the left arm of the second chromosome. Northern blots show that the gene is strongly expressed in larvae and adult flies, but less in pupae and embryos. In situ hybridizations of larvae show that the gene is expressed in various neurons of the brain and abdominal ganglia and in endocrine cells of the midgut. This is the first publication on the structure of a C-type allatostatin from insects other than moths and the first report on the presence of all three types of allatostatins in a representative of the insect order Diptera (flies).     

Structure and genomic organization of I elements involved in I-R hybrid dysgenesis in Drosophila melanogaster.

I-R hybrid dysgenesis in D. melanogaster is controlled by transposable elements known as I factors which terminate at their 3' ends by an A-rich sequence. Inducer strains contain active I factors. Both reactive and inducer stocks possess defective I elements. We have cloned various I elements from both categories of strains. The I elements having recently transposed in inducer strains have a structure closely related to that of active I factors. However we have isolated one such I element that is truncated at its 5' end. The I elements common to reactive and inducer strains are affected by various rearrangements and many point mutations. They do not appear to be simple derivatives of complete I factors.     

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.