scholarly journals Cloning and molecular characterization of a metabolic gene with development functions in Drosophila. I. Analysis of the head function of Punch.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 1007-1019
Author(s):  
J R McLean ◽  
R Boswell ◽  
J O'Donnell
Keyword(s):  
Drosophila Melanogaster ◽  
Young Adult ◽  
Biosynthetic Pathway ◽  
In Vitro Translation ◽  
Initial Examination ◽  
Vital Functions ◽  
Eye Pigmentation ◽  
Adult Head

Abstract In an effort to understand the functions of pterins throughout development we have been studying Punch (Pu), the structural gene for the enzyme GTP cyclohydrolase in Drosophila melanogaster. This enzyme catalyzes the first step in the pterin biosynthetic pathway. The Pu gene product is required for vital functions at two distinct stages in embryogenesis, and a pigmentation function in the eye of the young adult. We have localized the Pu region to 29 kb of DNA through the analysis of lesions present in Pu mutants. Since all of the mutations that were mapped affect the eye pigmentation function of Pu, and since this function is the best defined biochemically, we have concentrated on identifying and characterizing Pu products required for eye pigmentation in our initial examination of the cloned region. Four different transcripts from this region are expressed in the adult head. We show that one of these transcripts, the 1.7-kb species, is responsible for the pigmentation function through the analysis of mutant transcripts and the use of an in vitro translation assay. A 2-kb region lying within the locus is specifically required for this eye pigmentation function.

scholarly journals THE CHARACTERIZATION OF α-GLYCEROPHOSPHATE DEHYDROGENASE MUTANTS IN DROSOPHILA MELANOGASTER

Genetics ◽  
1983 ◽  
Vol 105 (2) ◽  
pp. 387-407
Author(s):  
Michael A Kotarski ◽  
Sally Pickert ◽  
Deborah A Leonard ◽  
Gregory J LaRosa ◽  
Ross J MacIntyre
Keyword(s):  
Drosophila Melanogaster ◽  
Protein A ◽  
Apparent Molecular Weight ◽  
In Vitro Translation ◽  
Strong Correlations ◽  
Nonsense Mutations ◽  
Flight Ability ◽  
Glycerophosphate Dehydrogenase

ABSTRACT Thirty mutants of α-glycerophosphate dehydrogenase (αGPDH, EC 1.1.1.8) from Drosophila melanogaster were produced with the chemical mutagen ethyl methanesulfonate (EMS). These mutants and nine others previously obtained have been characterized with respect to level of enzymatic activity, viability, flight ability, and presence of cross-reacting material (CRM). The presence of αGPDH mRNA in several of the mutants has been tested by in vitro translation. There are strong correlations between the level of enzyme activity, viability and flight ability. Thirteen of the mutants are CRM- by solution immunoprecipitation experiments, but of these, only three are CRM- by a more sensitive 125I-protein A-based radioimmune gel assay. The viability of the three CRM- mutants suggests that the absence of αGPDH protein is not a lethal condition. The immunoprecipitated protein of the low activity mutant, αGpdhnGL3, has a smaller apparent molecular weight on polyacrylamide-SDS gels than does the protein from wild type. Criteria for the identification of nonsense mutations in Drosophila are discussed.

scholarly journals Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2543
Author(s):  
Ruidong Ni ◽  
Suzeeta Bhandari ◽  
Perry R. Mitchell ◽  
Gabriela Suarez ◽  
Neel B. Patel ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fatty Acid ◽  
Apis Mellifera ◽  
Chemical Synthesis ◽  
Acid Amide ◽  
Fatty Acid Amides ◽  
Fatty Acid Amide

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.

scholarly journals Characterization of gossypol biosynthetic pathway

2018 ◽  
Vol 115 (23) ◽  
pp. E5410-E5418 ◽  
Author(s):  
Xiu Tian ◽  
Ju-Xin Ruan ◽  
Jin-Quan Huang ◽  
Chang-Qing Yang ◽  
Xin Fang ◽  
...  
Keyword(s):  
Plant Disease Resistance ◽  
Biosynthetic Pathway ◽  
Virus Induced Gene Silencing ◽  
Defense Compounds ◽  
Oxidative Reactions ◽  
Substrate Conversion ◽  
Transcriptome Comparison ◽  
Tight Correlation

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion.

scholarly journals Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler's murine encephalomyelitis virus

2019 ◽  
Vol 47 (15) ◽  
pp. 8207-8223 ◽  
Author(s):  
Sawsan Napthine ◽  
Susanne Bell ◽  
Chris H Hill ◽  
Ian Brierley ◽  
Andrew E Firth
Keyword(s):  
Virus Infection ◽  
Rna Structure ◽  
Encephalomyocarditis Virus ◽  
In Vitro Translation ◽  
Theiler's Murine Encephalomyelitis Virus ◽  
Ribosomal Frameshifting ◽  
Theiler’S Murine Encephalomyelitis Virus ◽  
Encephalomyelitis Virus

AbstractMany viruses utilize programmed –1 ribosomal frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘slippery’ sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.

In vitro translation of human pheochromocytoma messenger RNAs: Characterization of tyrosine-hydroxylase and dopamine-β-hydroxylase

Biochimie ◽  
1985 ◽  
Vol 67 (6) ◽  
pp. 589-595 ◽  
Author(s):  
Caroline Benlot ◽  
Joséphine Antreassian ◽  
Jean-Pierre Henry ◽  
Jean-Claude Legrand ◽  
François Gros ◽  
...  
Keyword(s):  
Tyrosine Hydroxylase ◽  
In Vitro Translation ◽  
Messenger Rnas

scholarly journals Two forms of Drosophila melanogaster Gs alpha are produced by alternate splicing involving an unusual splice site.

1990 ◽  
Vol 10 (3) ◽  
pp. 910-917 ◽  
Author(s):  
F Quan ◽  
M A Forte
Keyword(s):  
Drosophila Melanogaster ◽  
Splice Site ◽  
Sodium Dodecyl ◽  
Receptor Activation ◽  
In Vitro Translation ◽  
Alternate Splicing ◽  
Molecular Weights ◽  
Intron Structure ◽  
Gs Alpha

G proteins are responsible for modulating the activity of intracellular effector systems in response to receptor activation. The stimulatory G protein Gs is responsible for activation of adenylate cyclase in response to a variety of hormonal signals. In this report, we describe the structure of the gene for the alpha subunit of Drosophila melanogaster Gs. The gene is approximately 4.5 kilobases long and is divided into nine exons. The exon-intron structure of the Drosophila gene shows substantial similarity to that of the human gene for Gs alpha. Alternate splicing of intron 7, involving either use of an unusual TG or consensus AG 3' splice site, results in transcripts which code for either a long (DGs alpha L) or short (DGs alpha S) form of Gs alpha. These subunits differ by inclusion or deletion of three amino acids and substitution of a Ser for a Gly. The two forms of Drosophila Gs alpha differ in a region where no variation in the primary sequence of vertebrate Gs alpha subunits has been observed. In vitro translation experiments demonstrated that the Drosophila subunits migrate anomalously on sodium dodecyl sulfate-polyacrylamide gels with apparent molecular weights of 51,000 and 48,000. Additional Gs alpha transcript heterogeneity reflects the use of multiple polyadenylation sites.

scholarly journals THE GENETICS OF DOPA DECARBOXYLASE IN DROSOPHILA MELANOGASTER. II. ISOLATION AND CHARACTERIZATION OF DOPA-DECARBOXYLASE-DEFICIENT MUTANTS AND THEIR RELATIONSHIP TO THE α-METHYL-DOPA-HYPERSENSITIVE MUTANTS

Genetics ◽  
1976 ◽  
Vol 84 (2) ◽  
pp. 287-310
Author(s):  
Theodore R F Wright ◽  
Glenn C Bewley ◽  
Allen F Sherald
Keyword(s):  
Drosophila Melanogaster ◽  
Recombination Frequency ◽  
Dopa Decarboxylase ◽  
Isolation And Characterization ◽  
Methyl Dopa ◽  
Level Of Activity ◽  
The Right ◽  
Balancer Chromosome

ABSTRACT Of 84 lethals isolated over the dopa decarboxylase (DDC) deficiency Df(2L)50, 8 have been identified as DDC-deficient alleles on the basis of their effect on DDC activity when heterozygous over the CyO balancer chromosome with activities ranging from 28% to 53% of controls. Some of the Ddc-deficient alleles exhibit intracistronic complementation. Most of the complementing pairs of alleles are much reduced in viability, e.g. < 5% of expected, and express a common syndrome of mutant phenes which can reasonably be inferred to derive from inadequately sclerotinized cuticle. Individuals heterozygous for the noncomplementing allele, Ddcn7, over the 12-band DDC deficiency, Df(2L)130, die at the end of embryogenesis as unhatched larvae with unpigmented mouth parts. The Ddc alleles and the l(2)amd α-methyl dopa (αMD) hypersensitive alleles are both located within the 11 band region 37B10-C7. The l(2)amd locus is immediately to the right of hk(2-53.9).Ddc has been mapped within 0.004 Map Units to the right of l(2)amd with a maximum estimated recombination frequency of 0.01%. None of the Ddc/CyO strains are sensitive to the dietary administration of α-methyl dopa (αMD), and complementation occurs between the Ddc deficient alleles and the l(2)amd alleles both on the basis of viability and DDC activity. No effect on DDC by the amd alleles has been found to date. Even in the complementing heterozygote, amdH1/amdH89, the level of activity, thermostability, and in vitro αMD inhibition of DDC remains unaffected. Although no biochemical phene has yet been established for the αMD hypersensitive amd alleles, it seems likely that the two groups of mutants are functionally related.

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