Specific interference by ingested dsRNA.
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Animals↗
Biomedical subjects
L Timmons
Publications and source records attributed to L Timmons.
Green fluorescent protein/beta-galactosidase double reporters for visualizing Drosophila gene expression patterns.
We characterized 120 novel yeast Ga14-targeted enhancer trap lines in Drosophila using upstream activating sequence (UAS) reporter plasmids incorporating newly constructed fusions of Aequorea victoria green fluorescent protein (GFP) and Escherichia coli beta-galactosidase genes. Direct comparisons of GFP epifluorescence and beta-galactosidase staining revealed that both proteins function comparably to their unconjugated counterparts within a wide variety of Drosophila tissues. Generally, both reporters accumulated in similar patterns within individual lines, but in some tissues, e.g., brain, GFP staining was more reliable than that of beta-galactosidase, whereas in other tissues, most notably tests and ovaries, the converse was true. In cases of weak enhancers, we occasionally could detect beta-galactosidase staining in the absence of discernible GFP fluorescence. This shortcoming of GFP can, in most cases, be alleviated by using the more efficient S65T GFP derivative. The GFP/beta-gal reporter fusion protein facilitated monitoring several aspects of protein accumulation. In particular, the ability to visualize GFP fluorescence enhances recognition of global static and dynamic patterns in live animals, whereas beta-galactosidase histochemistry affords sensitive high resolution protein localization. We present a catalog of Ga 14-expressing strains that will be useful for investigating several aspects of Drosophila melanogaster cell and developmental biology.
Animals↗
prune/Killer of prune: a conditional dominant lethal interaction in Drosophila.
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Alleles↗
The enzymatic activity of Drosophila AWD/NDP kinase is necessary but not sufficient for its biological function.
The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female sterility requires twice as much nm23-H2 expression as awd expression. This implies that the enzymatic activity of the AWD protein is not sufficient for its biological function. The biological function may require nonconserved residues of the AWD protein that allow it to interact with other proteins.
Animals↗
The Enzymatic Activity of Drosophila AWD/NDP Kinase Is Necessary but Not Sufficient for Its Biological Function
The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female sterility requires twice as much nm23-H2 expression as awd expression. This implies that the enzymatic activity of the AWD protein is not sufficient for its biological function. The biological function may require nonconserved residues of the AWD protein that allow it to interact with other proteins.
Journal Article↗
Germline transformation using a prune cDNA rescues prune/killer of prune lethality and the prune eye color phenotype in Drosophila.
Null mutations in the prune gene of Drosophila melanogaster result in prune eye color due to reductions in red pigment accumulation. When one copy of the awd(Killer of prune) mutant gene is present in a prune background, the animals die. The cause of prune/Killer of prune lethality remains unknown. The genomic region characterized for the prune locus is transcriptionally active and complex, with multiple and overlapping transcripts. Despite the transcriptional complexity of the genomic region of prune, accumulated evidence suggests that the prune locus is small and consists of a single transcription unit, since every prune allele to date exhibits both prune eye color and prune/Killer of prune lethality. A functional prune product from a single, full-length cDNA was identified in this study that can rescue both the eye phenotype and prune/Killer of prune lethality. The DNA sequences of several mutant prune alleles along with Western blot analysis of mutant proteins provide convincing evidence that prune mutations are nulls, and that the cDNA identified in this study encodes the only product of the prune locus.
Amino Acid Sequence↗
Spread of Ixodes scapularis (Acari:Ixodidae) in Indiana: collections of adults in 1991-1994 and description of a Borrelia burgdorferi-infected population.
Collection records for the adult black legged tick, Ixodes scapularis Say, in Indiana for the period 1991-1994 are presented and a new, established population of Borrelia burgdorferi-infected I. scapularis is described. The number of I. scapularis adults collected in Indiana increased progressively from 19 in 1991 to > 200 in 1994, and the number of Indiana counties reporting at least 1 adult increased from 13 to 29. Also, during this period, 4 countries in northwestern Indiana yielded > 10 specimens each, and B. burgdorferi-infected ticks were collected in 2 of these counties. An established population of I. scapularis, as evidenced by the presence of questing larvae, nymphs, and adults, was discovered in Jasper County in 1993. Twelve of 39 adults (31%) and 4 of 44 nymphs (9%) collected with cloth drags were infected with B. burgdorferi. Three of 49 (6%) white-footed mice, Peromyscus leucopus, collected from the site were also infected with B. burgdorferi. We believe that this focus was established at least 8 yr ago, and that a tick originating from this focus was responsible for a case of Lyme disease reported from this county in 1985.
Animals↗
Point mutations in awdKpn which revert the prune/Killer of prune lethal interaction affect conserved residues that are involved in nucleoside diphosphate kinase substrate binding and catalysis.
The awd gene of Drosophila melanogaster encodes a nucleoside diphosphate kinase. Killer of prune (Kpn) is a mutation in the awd gene which substitutes Ser for Pro at position 97 and causes dominant lethality in individuals that do not have a functional prune gene. This lethality is not due to an inadequate amount of nucleoside diphosphate (NDP) kinase activity. In order to understand why the prune/Killer of prune combination is lethal, even in the presence of an adequate NDP kinase specific activity level, and to understand the biochemical basis for the conditional lethality of the awdKpn mutation, we generated second site mutations which revert this lethal interaction. All of the 12 revertants we recovered are second site mutations of the awdKpn gene. Three revertants have deletions of the awdKpn protein coding region. Two revertants have substitutions of the initiator methionine and do not accumulate KPN protein. Seven revertants have amino acid substitutions of conserved residues that are likely to affect the active site: five of these have no enzymatic activity and two have a very low level of specific activity. These data suggest that an altered NDP kinase activity is involved in the mechanism underlying the conditional lethality of the awdKpn mutation.
Amino Acid Sequence↗
The expression of the Drosophila awd gene during normal development and in neoplastic brain tumors caused by lgl mutations.
The abnormal wing discs (awd) gene of Drosophila is homologous to the nm23 gene of mammals, a gene whose expression is altered in metastatic tumors. Both awd and nm23 encode nucleoside diphosphate kinases (NDP kinases). We have examined the accumulation of AWD/NDP kinase during normal development by assaying enzyme activity in extracts. There is a nearly constant level of activity throughout larval and pupal development. We have examined the tissue-specific transcription of the awd gene by RNA in situ hybridization and by reporter gene expression. In imaginal discs and brains there is no detectable awd gene expression until the beginning of the third larval instar, despite the constant level of enzyme activity measured in extracts of larvae and pupae. The most intense awd gene expression in imaginal discs and brains occurs after the end of larval development. We have also examined awd gene expression in neoplastic brain tumors caused by mutations in the lethal giant larvae (lgl) gene. In lgl mutant brains, as in normal brains, awd gene expression begins during the third larval instar. No tumors form in brains from lgl-; awd- double mutant larva, so awd gene expression is required for tumor formation and/or proliferation. There is more accumulation of AWD/NDP kinase in lgl- mutant brains than there is in normal brains. Using an awd reporter gene, we show that this is a consequence of an increased proportion of awd gene-expressing cells in mutant brains. Using the same awd reporter gene as a marker of donor cells, we have confirmed the invasiveness of lgl-induced neuroblastomas.
Animals↗
A soluble recombinant factor VIII fragment containing the A2 domain binds to some human anti-factor VIII antibodies that are not detected by immunoblotting.
Human factor VIII (fVIII) inhibitors are pathologic antibodies that inactivate fVIII. A cDNA clone was modified to encode fVIII amino acid residues 373-740 for expression in a baculovirus vector in insect cells. The encoded protein fragment H2 was produced as a soluble, secreted protein, and it was used to test inhibitor plasmas for the presence of antibodies that were not detected by immunoblotting. Seven of 13 inhibitors that bound only to the fVIII light chain by immunoblotting also bound to fragment H2 in an immunoprecipitation assay. Thus multi-chain inhibitor reactivity of inhibitors is more frequent than previously reported. One of these inhibitors was shown to share the epitope for other inhibitors that bind to H2 within amino acid residues 373-541 in immunoblotting assays. The sensitive immunoprecipitation assay described allows determination of relative H2 binding capacity of the total IgG and epitope localization of inhibitors that cannot be similarly characterized by immunoblotting.
Animals↗
Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Escherichia coli.
Epitopes for antibodies that inhibit factor VIII procoagulant protein were analyzed by deletion mapping of factor VIII protein fragments expressed in Escherichia coli. A human factor VIII cDNA clone was used to generate E. coli expression vectors encoding fragments containing the 80-kDa factor VIII light chain (A3, C1, and C2 domains) and the 44-kDa carboxyl-terminal half of the factor VIII heavy chain (A2 domain). A series of deletions of each fragment was constructed and tested by immunoblotting for the binding of alloantibody and autoantibody inhibitors. Analysis of derivatives of the 80-kDa fragment showed that six inhibitors recognized a major epitope(s) within the carboxyl-terminal 17.3 kDa of factor VIII. These inhibitors also recognized weaker epitopes nearby and one inhibitor recognized epitopes scattered throughout the 80-kDa fragment. Deletions within the heavy chain fragment revealed one epitope-containing region confined to the amino-terminal 18.3 kDa recognized by six inhibitors. Bacterially produced factor VIII fragments containing the major epitopes were capable of neutralizing inhibitors in vitro but fragments containing weaker or no epitopes did not. These data suggest a potential therapeutic use of factor VIII fragments for neutralization of inhibitor antibodies.
Autoantibodies↗
Identification of bromotetrachlorophenol in commercial pentachlorophenol samples.
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Mass Spectrometry↗
Rapid determination of low pg/mg amounts of N-nitrosodiethylamine in rodent body fluid and tissue samples by isotope dilution high resolution mass spectrometry.
A rapid, sensitive, specific, and cost-effective method for the determination of N-nitrosodiethylamine (NDEA) in rodent tissue and body fluid samples is reported. The method involves isolation of the NDEA from the samples with subsequent detection and quantitation of observed NDEA against mass-labeled N-(15N)-nitrosodiethyl-(15N)-amine (15N2DEA) by high resolution gas chromatography/high resolution mass spectrometry selected ion monitoring. Validation of the method included linearity studies of NDEA response, calculation of the relative response factor (RRF) for NDEA versus 15N2DEA, and absolute recovery studies. Estimates of precision, detection limit, and minimum quantifiable limit are also reported.
Animals↗
Rapid determination of low pg/mg amounts of N-nitrosodimethylamine in rodent body fluid and tissue samples by low resolution mass spectrometry.
A rapid, sensitive, specific, and cost-effective method for the determination of N-nitrosodimethylamine (NDMA) in rodent tissue and body fluid samples is reported. The method involves isolation of the NDMA from the samples with subsequent detection and quantitation of observed NDMA against a mass labeled N-[15N]nitrosodimethyl-[15N]amine ([15N]NDMA) internal standard by capillary gas chromatography/mass spectrometry with selected ion monitoring. Validation of the method includes linearity studies of NDMA response, calculation of the relative response factor (RRF) for NDMA vs. [15N]NDMA, and absolute recovery studies. Estimates of precision, detection limit, and minimum quantifiable limit are also reported.
Animals↗