Genes for male accessory gland proteins in drosophila...

Details Genes for male accessory gland proteins in drosophila... Genes for male accessory gland proteins in drosophila...

1998-12-23

2002-04-30

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C530S350000, C536S023100, C435S006120

Reexamination Certificate

active

06380159

ABSTRACT:

BACKGROUND OF THE INVENTION
After mating, behavioral and physiological changes are seen in the female insect. Compared to virgins, mated
Drosophila melanogaster
females are largely unreceptive to further mating, lay eggs at an elevated rate, live less long, and store and efficiently utilize sperm (reviewed in Hall, 1994; Chen, 1996; Kubli, 1996; Wolfner, 1997). These changes in the female occur because she receives, via seminal fluid, secretions from her mate's accessory gland and also sperm (see Chen, 1996; Kubli, 1996; Wolfner, 1997 for reviews and for original references). Products of the predominant cell type of the accessory gland, the main cells, are necessary for changes in the female's egg-laying rate and receptivity on the first day after mating. Stored sperm cause these effects to persist for up to 11 days following mating. Accessory gland main cell secretions also shorten the life span of the mated female. In addition, they play a role in the storage of sperm and in the competition between sperm from sequential matings.
Knowledge of how accessory gland products mediate these changes is important in understanding the control of insect fertility and the mechanisms of peptide hormone action. Once genes encoding Accessory gland proteins (Acps) are identified, genetic and molecular genetic techniques uniquely possible in Drosophila can be used to dissect the role of each protein in reproduction. In a few cases, it is possible to identify the functions of Acps by injecting purified fractions into unmated female flies and observing behavioral effects. For example, in
D. melanogaster
, a “sex peptide” (“SP”) of 36 amino acids was purified and shown to stimulate egg-laying and depress receptivity to mating for one day (Chen, 1988). SP was cloned and shown to derive from a single gene at chromosomal position 70A (Chen, 1988). A sex peptide and a second peptide, ovulation-stimulating substance (OSS), with similar activities have also been purified from D. suzukii (Ohashi, 1991; Schmidt, 1993).
Only Acps which can be purified or synthesized in active forms, act on their own and act via the hemolymph, can be identified by such assays. In order to identify Acp genes without presupposition of function, differential cDNA hybridization can be used to isolate RNAs expressed only in accessory glands (Schäfer, 1986; DiBenedetto, 1987; Monsma and Wolfner, 1988). cDNA hybridization screens are more likely to isolate abundant RNAs in a tissue. Thus, they are biased towards RNAs expressed in main cells of the accessory gland (96% of the secretory cells of the accessory gland; Bertram, 1992) rather than the rarer secondary cells (4% of the secretory cells of the gland; Bertram, 1992). Previous differential cDNA hybridization screens for genomic clones encoding male-specific transcripts identified three genomic regions encoding Acps (Schäfer, 1986; DiBenedetto, 1987). Of these, the 95EF region encodes a small secreted Acp (DiBenedetto, 1990), 57D contains a gene cluster encoding three small peptides (Simmerl, 1995), and the 51F locus has not yet been characterized. In addition to these genes, a region encoding two Acps has been identified by screening a “chromosomal walk” for accessory gland-specific transcription units (Monsma and Wolffer, 1988). In this region, only 20 bases separate the gene for Acp26Aa, an ELH-similar prohormone-like molecule (Monsma and Wolfner, 1988) that stimulates egg-laying in the mated female fly (Herndon and Wolfner, 1995), from the gene for Acp26Ab, a small peptide of as yet unknown function (Monsma and Wolfner, 1988). The previously-isolated Acp genes are only a small subset of Acp genes, though the total number of Acp genes is difficult to estimate from prior protein electrophoretic data (e.g. Ingman-Baker and Candido, 1980; Stumm-Zollinger and Chen, 1985; Whalen and Wilson, 1986; Coulthart and Singh, 1988) as summarized and discussed in Chen (1991). This is because on the one hand in the electrophoretic studies small peptides were not resolved, while on the other hand some Acps run as multiple bands on SDS gels (Monsma and Wolfner, 1988). To gain a more complete picture of the spectrum of proteins produced by the accessory gland, a differential screen aimed directly at accessory gland-specific RNAs was performed.
SUMMARY OF THE INVENTION
The present invention provides an isolated nucleic acid molecule encoding an accessory gland protein from Drosophila which has the biological property of an insect toxin.
The invention further provides an isolated nucleic acid molecule encoding an amino acid sequence sufficiently duplicative of the accessory gland protein encoded by the nucleic acid molecule of SEQ. ID. No. 2 so that a polypeptide expressed from the nucleic acid molecule has the biological property of an insect toxin.
Another embodiment of the invention is an isolated
Drosophila melanogaster
insect toxin protein.
The invention also provides a method of reducing an insect's life span. The insect is contacted with an isolated Drosophila insect toxin protein under conditions effective shorten the insect's life span.
Yet another embodiment of the invention is a method of reducing an insect's life span by contacting the insect with an expression vector containing a nucleic acid molecule encoding a Drosophila insect toxin protein under conditions effective to express the protein.
The invention also provides isolated nucleic acid molecules having the nucleotide sequences of SEQ. ID. No. 2, SEQ. ID. No. 5, SEQ. ID. No. 8, SEQ. ID. No. 13, SEQ. ID. No. 18, SEQ. ID. No. 21, SEQ. ID. No. 24, SEQ. ID. No. 27, or SEQ. ID. No. 30, or a nucleic acid molecules which hybridizes under stringent conditions to a nucleic acid molecule having a nucleic acid sequence of SEQ. ID. No. 2, SEQ. ID. No. 5, SEQ. ID. No. 8, SEQ. ID. No. 13, SEQ. ID. No. 18, SEQ. ID. No. 21, SEQ. ID. No. 24, SEQ. ID. No. 27, or SEQ. ID. No. 30.
The present invention also provides isolated proteins having an amino acid sequence of SEQ. ID. No. 4, SEQ. ID. No. 7, SEQ. ID. No. 11, SEQ. ID. No. 12, SEQ. ID. No. 16, SEQ. ID. No. 17, SEQ. ID. No. 20, SEQ. ID. No. 23, SEQ. ID. No. 26, SEQ. ID. No. 29, or SEQ. ID. No. 32.
Yet another embodiment of the invention is a method for determining whether a female
Drosophila melanogaster
has recently mated. An antibody, fragment thereof, or probe which recognizes a protein having a sequence provided in SEQ. ID. No. 4, SEQ. ID. No. 7, SEQ. ID. No. 11, SEQ. ID. No. 12, SEQ. ID. No. 16, SEQ. ID. No. 17, SEQ. ID. No. 20, SEQ. ID. No. 23, SEQ. ID. No. 26, SEQ. ID. No. 29, or SEQ. ID. No. 32 is provided. The antibody, fragment thereof, or probe is bound to a label effective to permit detection of the protein upon binding of the antibody, fragment thereof, or probe to the protein. The labeled antibody is contacted with a fluid or tissue sample from
Drosophila melanogaster
under conditions effective to permit binding of the antibody, fragment thereof, or probe to the protein. The presence of any of the protein in the biological sample is detected by detecting the label.


REFERENCES:
Chen. The accessory gland proteins in male Drosophila: structural, reproductive and evolutionary aspects. Experientia 52:503-510, 1996.*
Wolfner et al. New genes for male accessory gland proteins in Drosophila melanogaster. Insect Biochem. Molec. Biol. 27(10):825-834, Oct. 1997.*
Park et al., “Two Tightly-Linked Drosophila Male Accessory Gland Transcripts with the Same Developmental Expression Derive from Independent Transcription Units,”Mechanisms of Development, 48:51-57 (1994).
Herndon et al., “Mating and Hormonal Triggers Regulate Accessory Gland Gene Expression in Male Drosophila,”J. Insect Physiol., 43(12):117-1123 (1997).
Tsaur et al., “Positive Selection and the Molecular Evolution of a Gene of Male Reproduction, Acp26Aa of Drosophila,”Mol. Biol. Evol., 14(5):544-549 (1997).
Tram et al., “Seminal Fluid Regulation of Female Sexual Attractiveness in Drosophila melanogaster,”Proc. Natl. Acad. Sci. USA, 95:4051-4054 (1998).
Coulthart et al., “Differing Amounts of

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