Animals ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; Memory ; physiology ; Mushroom Bodies ; cytology ; metabolism ; Proteins ; genetics ; metabolism

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.
Animals ; Dopa Decarboxylase/genetics* ; Dopamine/biosynthesis* ; Drosophila melanogaster/genetics* ; Escherichia coli/metabolism* ; Humans ; Metabolic Engineering

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.
Animals ; DNA Repair ; genetics ; DNA, Single-Stranded ; genetics ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; genetics ; metabolism ; Loss of Heterozygosity ; genetics ; RecQ Helicases ; genetics ; metabolism

In most insects, polyunsaturated fatty acids (PUFAs) are mainly polyunsaturated fatty acids with a carbon-chain length less than 18 carbon atoms, hardly any long-chain polyunsaturated fatty acids such as C20 and C22 that are more valuable and bioactive. This study, by using Drosophila melanogaster (Fruit fly) as a model organism, optimized the Δ6-fatty acid elongase enzyme Elovl5 gene from mice and transferred it to fruit flies for expression. Vectors containing Elovl5 gene were successfully injected into drosophila embryo through the microscopic injection. There were enhanced green fluorescent proteins expressed in the whole developmental stage of Drosophila be means of fluorescence microscope. At the same time, expression of Elovl5 gene significantly contributed to the transformation of fruit flies C18-polyunsaturated fatty acids in the body towards the biosynthesis of longer-chain polyunsaturated fatty acids. The transgenic fruit fly model rich in long-chain polyunsaturated fatty acids such as C20 and C22 were obtained, providing a basis for further research on biosynthesis of polyunsaturated fatty acids in fruit flies.
Acetyltransferases/genetics* ; Animals ; Drosophila melanogaster/genetics* ; Fatty Acid Elongases/metabolism* ; Fatty Acids/genetics* ; Gene Transfer Techniques ; Mice

OBJECTIVETo search new clues to reveal the action mechanism of inhaled anesthetics.

METHODSThree kinds of Drosophila melanogaster were used as studied models: the wild type strain (H), the sevoflurane-sensitive strain (S), and the sevoflurane-resistant strain (R). Differential display reverse transcriptional-polymerase chain reaction method was performed to examine the differentially expressed fragments between Drosophila induced with and without sevoflurane. Rapid amplification of cDNA ends (RACE) method was used to clone the full length cDNA from positive differentially expressed fragments.

RESULTSThirty-one differentially expressed fragments were found between the two groups. Three fragments were identified as the positive differentially expressed fragments by Northern blot analysis. Two full-length cDNAs were cloned by RACE method, among which one was a 1.0 kb Drosophila calmodulin (CaM), located on Chr.2; the other was a 4.1 kb gene with unknown function (No.45), located on Chr.3.

CONCLUSIONThe two full-length cDNAs belong to the genes that related to anesthetic action pathway, which might participate in the regulation of cellular functions and signal transduction pathways. The two genes that we found should provide a novel way to study the mechanism of inhaled anesthetic action.

Anesthetics, Inhalation ; pharmacology ; Animals ; Cloning, Molecular ; DNA, Complementary ; Drosophila melanogaster ; drug effects ; genetics ; Gene Expression ; Methyl Ethers ; pharmacology ; Signal Transduction

Microsatellites or simple sequence repeats (SSRs) have been found in most organisms during the last decade. Since large-scale sequences are being generated, especially those that can be used to search for microsatellites, the development of these markers is getting more convenient. Keeping SSRs in viewing the importance of the application, available CDS (coding sequences) or ESTs (expressed sequence tags) of some eukaryotic species were used to study the frequency and density of various types of microsatellites. On the basis of surveying CDS or EST sequences amounting to 66.6 Mb in silkworm, 37.2 Mb in fly, 20.8 Mb in mosquito, 60.0 Mb in mouse, 34.9 Mb in zebrafish and 33.5 Mb in Caenorhabditis elegans, the frequency of SSRs was 1/1.00 Kb in silkworm, 1/0.77 Kb in fly, 1/1.03 Kb in mosquito, 1/1.21 Kb in mouse, 1/1.25 Kb in zebrafish and 1/1.38 Kb in C. elegans. The overall average SSR frequency of these species is 1/1.07 Kb. Hexanucleotide repeats (64.5%-76.6%) are the most abundant class of SSR in the investigated species, followed by trimeric, dimeric, tetrameric, monomeric and pentameric repeats. Furthermore, the A-rich repeats are predominant in each type of SSRs, whereas G-rich repeats are rare in the coding regions.
Animals ; Anopheles ; genetics ; Bombyx ; genetics ; Caenorhabditis elegans ; genetics ; Drosophila melanogaster ; genetics ; Expressed Sequence Tags ; Genome ; Invertebrates ; genetics ; Mice ; genetics ; Microsatellite Repeats ; genetics ; Zebrafish ; genetics

We analyzed the whole genome coding sequence of Volvariella volvacea to study the pattern utilization of codons by Codon W 1.4.2. As results, 24 optimal codons were identified. Moreover, the frequency of codons usage was calculated by CUSP program. We compared the frequency of codons usage of V. volvacea with other organisms including 6 modal value species (Homo sapiens, Saccharomys cerevisiae, Arabidopsis thalian, Mus musculus, Danio rerio and Drosophila melanogaster) and 4 edible fungi (Coprinopsis cinerea, Agaricus bisporus, Lentinula edodes and Pleurotus ostreatus). We found that there were less differences in 3 edible fungi (excluding Pleurotus ostreatus) than 6 modal value species, comparing with the frequency of codons usage of V. volvacea. With software SPSS16.0, cluster analysis which showed differences in the size of codon bias, reflects the evolutionary relationships between species, which can be used as a reference of evolutionary relationships of species. This was the first time for analysis the codon preference among the whole coding sequences of edible fungi, serving as theoretical basis to apply genetic engineering of V. volvacea.
Agaricales ; genetics ; Animals ; Arabidopsis ; genetics ; Cluster Analysis ; Codon ; DNA, Fungal ; genetics ; Drosophila melanogaster ; genetics ; Humans ; Mice ; Saccharomyces cerevisiae ; genetics ; Software ; Volvariella ; genetics ; Zebrafish ; genetics

Little is known about pre-mRNA splicing in Dictyostelium discoideum although its genome has been completely sequenced. Our analysis suggests that pre-mRNA splicing plays an important role in D. discoideum gene expression as two thirds of its genes contain at least one intron. Ongoing curation of the genome to date has revealed 40 genes in D. discoideum with clear evidence of alternative splicing, supporting the existence of alternative splicing in this unicellular organism. We identified 160 candidate U2-type spliceosomal proteins and related factors in D. discoideum based on 264 known human genes involved in splicing. Spliceosomal small ribonucleoproteins (snRNPs), PRP19 complex proteins and late-acting proteins are highly conserved in D. discoideum and throughout the metazoa. In non-snRNP and hnRNP families, D. discoideum orthologs are closer to those in A. thaliana, D. melanogaster and H. sapiens than to their counterparts in S. cerevisiae. Several splicing regulators, including SR proteins and CUG-binding proteins, were found in D. discoideum, but not in yeast. Our comprehensive catalog of spliceosomal proteins provides useful information for future studies of splicing in D. discoideum where the efficient genetic and biochemical manipulation will also further our general understanding of pre-mRNA splicing.
Alternative Splicing ; Animals ; Arabidopsis ; genetics ; Dictyostelium ; genetics ; Drosophila melanogaster ; genetics ; Genome, Protozoan ; Humans ; Phylogeny ; Ribonucleoproteins, Small Nuclear ; classification ; genetics ; Saccharomyces cerevisiae ; genetics ; Spliceosomes ; genetics ; metabolism

The heterochronic genes regulate cell proliferation and switch development stage transitions. Heterochronic genes might also play important roles in regulating the development of silkworm, but very few of their expression profiles, functions and their relationship with microRNAs are available so far. Firstly, in this work, the primers for cloning Bmlin-41 were designed based on the homologous sequence of known Drosophila melanogaster lin-41, which was used as the query to blast against SilkDB. The obtained full CDS (2 166 bp) of Bmlin-41 encodes 721 amino acids and contains B-box and NHL domains. Then, the spatiotemporal expression patterns of Bmlin-41 were characterized by RT-PCR, quantitative real time PCR as well as our lab's previous silkworm genome microarray data. Bmlin-41 was increasingly expressed from embryonic to adult stage. In diverse tissues of day-3 fifth instar, Bmlin-41 showed the highest accumulation in ovary, secondly in testis and midgut, but very low expression was observed in other tissues. Finally, 3'UTR of Bmlin-41 1 434 bp was cloned by rapid-amplification of cDNA ends (3'RACE) and was predicted to bare two binding sites of bmo-let-7 by using online RNAhybrid. To verify the binding effect, 3'UTR was cloned into psi-CHECK-2 vector and submitted to dual luciferase assay in the S2 cells in vitro. The dual luciferase assay demonstrated that Bmlin-41 was down-regulated by bmo-let-7 mimics and upregulated by bmo-let-7 antagomir, thus confirming the Bmlin-41 is negatively regulated by bmo-let-7. Our work might help further study on the roles of Bmlin-41 and bmo-let-7 and their regulation relationship involved in controlling metamorphosis of silkworm.
3' Untranslated Regions ; Animals ; Bombyx ; Cloning, Molecular ; DNA, Complementary ; Down-Regulation ; Drosophila melanogaster ; Gene Expression Regulation ; Insect Proteins ; genetics ; metabolism ; Metamorphosis, Biological ; MicroRNAs ; metabolism ; Transcription Factors ; genetics ; metabolism

Animals ; Cell Nucleus ; enzymology ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Long-Term Potentiation ; physiology ; MAP Kinase Signaling System ; physiology ; Memory Consolidation ; physiology ; Neurons ; cytology ; enzymology