Progress 12/01/05 to 11/30/09
Outputs
OUTPUTS: Presentations of results at the following meetings: 2008 Annual Drosphila meeting 2009 Drosophila neurobiology meeting PARTICIPANTS: David B. Morton, PhD, PI Anke Vermehren, PhD, post-doc Rachel Clemens-Grisham - technician. Shireen A. Davies, PhD, University of Glasgow, UK, Collaborator TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The goal of this project was to determine if any insect cyclic nucleotide phosphodiesterases are suitable targets for the development of pesticides. This was successful. I have data that shows that in Drosophila melanogster loss of PDE1c leads to male infertility and loss of PDE11 is lethal. Both could be used as targets for pesticides.
Publications
- No publications reported this period
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Progress 12/01/07 to 11/30/08
Outputs
OUTPUTS: The major focus of this project is to characterize the cyclic nucleotide phosphodiesterases (PDEs), using Drosophila as a model, and determine if any are likely to be a good target for the development of new pesticides. Most of the effort of the prior year has been generating mutant fly lines and characterizing their phenotypes. There are 6 PDE genes in Drosophila - PDE1c, PDE4, PDE6, PDE8, PDE9 and PDE11. We have generated fly lines that are mutant for three PDEs; PDE1c, PDE8 and PDE11 and are focusing all of our attention on the PDE1c and PDE11 mutants. PDE1c is a dual specificity (cAMP and cGMP) PDE that is regulated by calcium/calmodulin. We have a fly line that has a transposon insertion in a large intron in PDE1c. This line is homozygous male sterile and we have remobilized the transposon, which rescues the phenotype. The cause of infertility is greatly reduced sperm transfer during mating. Previous studies from other labs have identified a large number of genes required for male sterility, but only a few of these result in sterility due to reduced or no sperm transfer. We have obtained mutant alleles for these other genes and investigated possible genetic interactions between PDE1c and 3 other genes with similar phenotype. The most interesting one of these is cuckold, which fails to complement PDE1c when assessing male courtship behavior. The cuckold allele is caused by a transposon inserted in an intergenic region of DNA closest to a transcript for a micro RNA. Interestingly, PDE1c has a putative binding site for this micro RNA suggesting that infertility caused by cuckold is due to reduced expression of PDE1c. We are currently investigating this possibility in more detail. PDE11 is a dual specificity PDE with higher activity towards cGMP. We have also used FLP recombinase to generate deletion mutant fly lines that was predicted to eliminate the 5' end of the gene that includes part of the coding sequence. During the course of generating this deletion an updated version of the annotation of the Drosophila genome showed an additional transcript of PDE11 that is not affected by the deletion. Nevertheless, RT-PCR shows that the deletion lines reduce expression of PDE11 by about 50% - presumable eliminating one of the two predicted transcripts. These lines are male sterile. We are currently generating a fly line that will express PDE11 cDNA under heat shock control to rescue this phenotype. We also have obtained a fly line that expresses PDE11 double stranded RNA. When this is expressed ubiquitously the animals die soon after hatching. Expression in either the nervous system or in mesoderm also results in lethality, but in these cases the animals die during adult development. The difference in the phenotypes between the deletion and the RNAi lines is presumably because the latter eliminates both transcripts whereas the deletion lines only eliminate one transcript. We have also made additional PDE11 deletion lines that should eliminate all PDE11 transcripts and are currently screening these lines to determine if the transcripts are indeed eliminated. PARTICIPANTS: David B. Morton, PhD, PI Rachel Clemens-Grisham, Research Tech Shireen Davies, PhD, University of Glasgow, UK, Collaborator TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results generated during the course of this funding period have shown that both PDE1c and PDE11 are strong possibilities as targets for the development of novel pesticides. These fly lines will also be used to further our understanding on the roles of this signaling system in insects.
Publications
- Clemens-Grisham, R., Vermehren, A., and Morton, D.B. (2008). PDE1c and PDE11 and their role in Drosophila male fertility. 49th Annual Drosophila Research Conference, San Diego, CA.
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Progress 12/01/06 to 11/30/07
Outputs
OUTPUTS: The major focus of this project is to characterize the cyclic nucleotide phosphodiesterases (PDEs), using Drosophila as a model, and determine if any are likely to be a good target for the development of new pesticides. Most of the effort of the prior year has been generating mutant fly lines and characterizing their phenotypes. There are 6 PDE genes in Drosophila - PDE1c, PDE4, PDE6, PDE8, PDE9 and PDE11. We have fly lines that are mutant for three PDEs; PDE1c, PDE8 and PDE11. PDE1c is a dual specificity (cAMP and cGMP) PDE that is regulated by calcium/calmodulin. We have a fly line that has a transposon insertion in a large intron in PDE1c. This line is homozygous male sterile and we have remobilized the transposon, which rescues the phenotype. The cause of infertility is greatly reduced sperm transfer during mating. We have also rescued the phenotype by expressing a PDE1c cDNA under heat shock control. A heat shock soon after adult eclosion is sufficient to generate
fertile males. We have also generated a promoter driver line that shows expression primarily in the nervous system, although some expression is also seen in the male reproductive organs. We are currently generating fly lines that will express PDE1c in a tissue specific manner to determine where expression is required to rescue infertility. PDE8 is predicted to be a cAMP specific PDE although no biochemical evidence has been obtained to confirm this. There are five splice variants of the PDE8 gene and using FLP recombinase and two fly lines that have transposon insertions at either end of the gene we have generated a deletion mutant that eliminates expression of all the transcripts. These lines are viable and fertile. Dr. Shireen Davies at the University of Glasgow, UK, is collaborating with us on the characterization of the PDEs and has preliminary evidence that the PDE8 deletion lines have a weakened immune system and show higher mortality when challenged with bacterial injections.
PDE11 is a dual specificity PDE with higher activity towards cGMP. We have also used FLP recombinase to generate deletion mutant fly lines that was predicted to eliminate the 5' end of the gene that includes part of the coding sequence. During the course of generating this deletion an updated version of the annotation of the Drosophila genome showed an additional transcript of PDE11 that is not affected by the deletion. Nevertheless, RT-PCR shows that the deletion lines reduce expression of PDE11 by about 50% - presumable eliminating one of the two predicted transcripts. These lines are male sterile. We are currently generating a fly line that will express PDE11 cDNA under heat shock control to rescue this phenotype. We also have obtained a fly line that expresses PDE11 double stranded RNA. When this is expressed ubiquitously the animals die soon after hatching. Expression in either the nervous system or in mesoderm also results in lethality, but in these cases the animals die during
adult development. The difference in the phenotypes between the deletion and the RNAi lines is presumably because the latter eliminates both transcripts whereas the deletion lines only eliminate one transcript.
PARTICIPANTS: David B. Morton, PI Rachel Clemens-Grisham, Research Technician Anke Vermehren, Post-doc Shireen A. Davies, University of Glasgow, UK, Collaborator
Impacts
The results generated during the course of this funding period have shown that both PDE1c and PDE11 are strong possibilities as targets for the development of novel pesticides. These fly lines will also be used to further our understanding on the roles of this signaling system in insects.
Publications
- Clemens-Grisham, R., Vermehren, A., and Morton, D.B. (2008). PDE1c and PDE11 and their role in Drosophila male fertility. 49th Annual Drosophila Research Conference, San Diego, CA (In Press).
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Progress 12/01/05 to 12/01/06
Outputs
The overall focus of this project is to characterize the cyclic nucleotide phosphodiesterases (PDEs), using Drosophila as a model, and determine if any are likely to be a good target for the development of new pesticides. Just prior to the funding of this project, a report was published that characterized the basic biochemistry of these proteins, which was one of the original specific aims of this project. We have begun collaborative research with the lab that carried out these studies (Dr. Shireen Davies, University of Glasgow, UK) to prevent duplication of our efforts. We have been focusing on generating fly lines mutant for each PDE to determine their effect on the insect. There are 6 genes in Drosophila that code for PDEs - PDE1c, PDE4, PDE6, PDE8, PDE9 and PDE11. Only PDE4 has previously been characterized and is required for learning and memory. We have made progress in characterizing the PDE1c, PDE6, PDE8 and PDE11 genes. PDE1c is a dual specificity (cAMP and
cGMP) PDE that is regulated by calcium/calmodulin. We have obtained a fly line that has a transposon insertion in a large intron in PDE1c. The transposon has a splice acceptor site within it and hence is predicted to disrupt the splicing of the PDE1c mRNA. This line is homozygous male sterile and we have remobilized the transposon, which rescues the phenotype. This suggests that PDE1c is necessary for male fertility and disruption of the gene causes male sterility. If this is confirmed, inhibitors of PDE1c could also reduce fertility. As yet we do not know the cause of sterility - the flies mate with females and their testes appear grossly normal and contain sperm. We have also generated a promoter-reporter fly line, which we are using to determine the spatial expression pattern of the gene. We are also in the process of generating fly lines that will express a cDNA coding for PDE1c to determine whether this rescues male sterility. We have expressed PDE6 cDNA in heterologous cells and
have shown that it is a cGMP specific PDE. There are no fly lines available with transposon inserts within or near PDE6 that disrupt expression of the gene. Our collaborators have generated a fly line that expresses dsRNA complementary to PDE6 and we are using this to identify a phenotype caused by a reduced expression of the gene. PDE8 is predicted to be a cAMP specific PDE although no biochemical evidence has been obtained to confirm this. There are five splice variants of the PDE8 gene and using FLP recombinase and two fly lines that have transposon insertions at either end of the gene we have generated a deletion mutant that eliminates expression of all the transcripts. We are in the process of characterizing this line. At the present time we have shown that it is homozygous viable and fertile. PDE11 is a dual specificity PDE with higher activity towards cGMP. We have also used FLP recombinase to generate deletion mutant fly lines that are predicted to eliminate the 5' end of the
gene that includes part of the coding sequence. These will soon be tested to determine if expression of the gene is eliminated and whether it is a null mutation.
Impacts
The results generated during the first year of this project have provided us with mutant fly lines that can be used to determine whether the phosphodiesterases are viable targets for the development of novel pesticides. These fly lines will also be used to further our understanding on the roles of this signaling system in insects.
Publications
- No publications reported this period
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