Progress 08/01/08 to 07/31/13

Outputs
OUTPUTS: For additional information, please contact David Schooley at 775-784-4136 or schooley@unr.edu PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
For additional information, please contact David Schooley at 775-784-4136 or schooley@unr.edu

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Two years ago our collaborator, Paul Taghert at Washington University, deleted a gene from Drosophila which encodes the precursor for the calcitonin-like diuretic hormone of Drosophila. This 31 amino acid peptide weakly activates the 4395 receptor. However, deleting this gene also appears to knock out the production of active 4395 ligand. This suggests the 4395 ligand is made from the same precursor as Drome-DH31. We have had great difficulty synthesizing the two isoforms of the large peptide which likely is the native ligand for the CG4395 receptor. There are two possibilities, one with 81 amino acids and another with 82 amino acids. We have synthesized these in small quantity, and they prove to both activate the CG4395 receptor with EC50 values below 1 nM; a very high level of potency, suggesting one of these is likely the natural ligand. We processed a batch of about 50,000 Drosophila heads, and fractionated this on a greatly improved ion exchange HPLC system which has resolution far superior to the schemes used in prior attempts. We have now been able to get a tentative ID of one fragment of the bioactive peptide, which shows it to in fact contain the DH31 sequence. We have to do some polish up work on this project to get it ready for publication. PARTICIPANTS: Derek Jensen is a graduate student who has worked full time on this project. He has learned a very great deal during his graduate work, especially on the techniques for synthesis of very large peptides, and for HPLC separation of peptides and proteins. He has been offered a job by the Hamilton company of Reno as an HPLC specialist, and will start there May 1, 2012. Lilly Ng is a technician who assisted in many non-research aspects of this project, but she retired from UNR in September. Dr. Paul Taghert of the Department of Anatomy & Neurobiology, Washington University, is a key collaborator in this multi-institutional grant. we have frequent e mail communications, and less frequent telephone calls, to discuss problems and their solutions. He also has a senior technician, Jennifer Trigg, who conducts the assays of peptides or purification fractions for their ability to stimulate the CG4395 receptor. TARGET AUDIENCES: The results of this project should be acceptable in Proceedings of the National Academy of Sciences, and scientists of a broad range of disciplines worldwide should be interested in the results. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This research will yield information of fundamental importance in understanding mating in insects, as knocking out the gene for the receptor, CG4395, causes male flies to court males instead of females. This receptor is expressed far more abundantly in heads of males than females.

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Progress on this project continues on two fronts. Last year our collaborator, Paul Taghert at Washington University, deleted a gene from Drosophila which encodes the precursor for the calcitonin-like diuretic hormone of Drosophila. This 31 amino acid peptide weakly activates the 4395 receptor. However, deleting this gene also appears to knock out the production of active 4395 ligand. This suggests the 4395 ligand is made from the same precursor as DH31. We have had great difficulty synthesizing the very large peptides may correspond to the native ligand for the CG4395 receptor. There are two possibilities, one with 81 amino acids and another with 82 amino acids. We have synthesized these in small quantity, and they prove to both activate the CG4395 receptor with EC50 values below 1 nM; a very high level of potency, suggesting one of these is likely the natural ligand We also accumulated 190,000 heads of Oregon R Drosophila, and have utilized an improved purification scheme to attempt to obtain the native peptide. It is quite hydrophobic, which makes recovery poor in the purification schemes which require repetitive steps of HPLC prior to identification of the peptide by LC-MS-MS analysis. One sample submitted for analysis several months ago was identified as containing insulin, yet it was the same insulin used in a calibration standard for the mass spectrometer. We suspect that there was some contamination of our extract in the MS lab. We will soon attempt to determine which one of the two isoforms of peptides synthesized may have migration properties on HPLC identical with the peptide we are isolating from head extracts. We have found that ion exchange chromatography gives optimal separation of the different isoforms, due to their containing different numbers of basic amino acid residues. PARTICIPANTS: Derek Jensen is a graduate student who has worked full time on this project. He has matured and learned a great deal in the last year, especially on the techniques for synthesis of very large peptides. He has made important intellectual contributions to overcoming some of our great difficulties in chemical synthesis of these very large peptides. Lilly Ng is a technician who has assisted in many non-research aspects of this project. Dr. Paul Taghert of the Department of Anatomy & Neurobiology, Washington University, is a key collaborator in this multi-institutional grant. we have frequent e-mail communications, and less frequent telephone calls, to discuss problems and their solutions. He also has a senior technician, Jennifer Trigg, who conducts the assays of peptides or purification fractions for their ability to stimulate the CG4395 receptor. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project will provide information on fundamental importance on a peptide-receptor found largely in male fruitflies, which appears crucial in controlling mating behavior. This may have significane in a large number of species, but is best studied in the model organism Drosophila melanogaster.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Shortly after last years report, we isolated the peptide to what we believed was a homogeneous state from an extract of 60,000 heads of Oregon R strain of Drosophila melanogaster. We digested this extract with trypsin, and analyzed it by MALDI-TOF-TOF mass spectrometry, and searched the results of tandem MS analysis against the Drosophila genome. Unfortunately, the sample contained a contaminant, which was identified by MS-MS as bovine serum albumin. There was so much more than the peptide that the peptide could not be unambiguously identified. We isolated in parallel a peptide which activates the Drosophila receptor CG8422, and a significant UV active peak was obtained and clearly identified by MALDI-TOF-TOF as Drome-DH44, the known ligand for this receptor, using identical techniques. It appears that the content of 4395 ligand is at least 1-2 orders of magnitude lower than Drome-DH44, which is likely why we could not identify it given the contamination. We accumulated 190,000 heads of Oregon R Drosophila, and have utilized an improved purification scheme. At year end we are quite close to having the peptide pure enough for identification. Our collaborator, Paul Taghert at Washington University, did experiments where they deleted a gene from Drosophila which encodes the precursor for the calcitonin-like diuretic hormone of Drosophila. This 31 amino acid peptide weakly activates the 4395 receptor. However, deleting this gene also knocks out the production of active 4395 ligand. This suggests the 4395 ligand is made from the same precursor as DH31. We have therefore synthesized two possibilities for an active peptide resulting from different enzymes processing the precursor. One of these contains 77 amino acids and the other 82. The peptides are currently being purified to send to Taghert for biological testing. PARTICIPANTS: Derek Jensen,a third year graduate student, has worked on this project this year. Several undergraduate students, including Alexander Fiannaca and Kyle Bridgewater, assisted in rearing insects. TARGET AUDIENCES: All scientist with interests in insect physiology and biochemistry should be interested in the results of these studies. PROJECT MODIFICATIONS: Based on our observations that the content of the 4395 ligand is very low in Drosophila heads, we submitted a request for an administrative supplement to this grant to purchase a high end microtiter plate reader capable of doing fluorescence immunoassays which require measurement with time resolved fluorescence. This supplement for $55,527 was awarded with ARRA funds. This will allow use of assays able to detect far smaller amounts of peptide than we could have detected with our existing microtiter plate reader.

Impacts
The identification of the ligand for this receptor, and its biological response, will result in an important paper publishable in a high impact scientific journal.

Publications

• No publications reported this period

Progress 08/01/08 to 12/31/08

Outputs
OUTPUTS: The purpose of this project is to isolate and identify a peptide in brains of Drosophila melanogaster which stimulates a receptor with accession number CG4395. This is related to the calcitonin-like DH receptor, and is a collaboration with Paul Taghert at Washington U. and Scott Waddell at U. of Mass. They have shown that this receptor is expressed predominantly in male fruit fly brains, and that it is involved in courtship behavior. We have already prepared an extract of 60,000 heads of Oregon R strain of Drosophila melanogaster, and have put the peptide through six purification steps; one ion exchange and five reversed-phase liquid chromatographic purifications. We have made faster than expected progress and may be close to having the peptide in a pure state. Once the peptide is pure it will be identified by mass spectral sequencing, as we do not anticipate having enough peptide to identify it by Edman degradation. Then it will be synthesized. PARTICIPANTS: Graduate students Derek Jensen and Alaine Terrell. TARGET AUDIENCES: International scientists interested in insect neuropeptides, and those interested in courtship and reproductive behavior in the Insecta and perhaps other classes of organism. PROJECT MODIFICATIONS: No changes.

Impacts
My collaborator Taghert has created a Drosophila knockout strain which lacks the receptor gene which we are studying. This has a very interesting impact on the courtship behavior of the resultant adult Drosophila males. The knockout males court other male flies instead of female flies. Thus this gene is very important in courtship and reproduction in Drosophila. Knowledge of the sequence of the peptide ligand which activates this receptor is vital to understanding the courtship behavior of Drosophila, and perhaps other organisms.

Publications

• No publications reported this period