The courtless gene is expressed throughout Drosophila development, and its expression is developmentally regulated. Two transcripts, 1.1 and 1.3 kb, are present in equimolar amounts in embryos. The smaller (1.1 kb) transcript is probably composed of a mixture of the two classes of crl transcripts, which are similar in size (classes 1b and 2). Expression of these transcripts declines in larvae but increases at the pupal stage, and even more at the adult stage. The level of expression of the crl transcripts was compared in males and females of crl/crl, crl/CyO, and wild-type flies by Northern blot. When the entire class 1a cDNA was used as a probe (potentially capable of detecting all three transcripts), the same two bands were visible in both sexes, although higher levels were found in males than in females in all three genotypes. Densitometry indicates that in homozygous crl males the level of expression is four times lower than in wild-type males. When the membrane is stripped and reprobed with the fourth intron, it becomes evident that classes 1a and 1b transcripts that retain this intron are male specific. The only 1.3-kb cDNA isolated corresponds to the transcript that retains the fourth intron. Such a transcript is not present in females, yet females do have a 1.3-kb transcript. This indicates that at least one additional 1.3-kb crl transcript has yet to be identified (Orgad, 2000).
The spatial distribution of crl transcripts was determined by in situ hybridization of digoxigenin-labeled RNA probes corresponding to class 1a cDNA. Early in the blastoderm stage (~ 2 hr of development), crl transcripts are present both in the egg yolk, suggesting a maternal origin, and in the peripheral blastoderm cells, reflecting either maternal contribution or zygotic expression. Subsequently (3 hr of development), they are found in the mesoderm and in the cephalic furrow, and later (5 hr of development) in the extending germ band, in the neuroblasts, and in the stomodial invagination. As development proceeds, in 15-hr-old embryos, expression is confined to the central nervous system (Orgad, 2000).
Class 1a transcript was modified to include a His-tag, and was expressed in Escherichia coli. The tagged protein was used to immunize rabbits. Western blot analysis using the polyclonal antibodies and extracts from male flies of the crl/crl, crl/CyO, and wild-type genotypes reveals elevated amounts of the Crl protein in the mutant flies. Homozygous and heterozygous crl males express at least seven and three times, respectively, more of the Crl protein than the wild type. When this experiment was repeated using extracts from embryos and larvae of one of the homozygous lethal excision lines of crl, no difference was observed in the expression of the Crl protein between the homozygous mutant and heterozygotes at the embryonic stage, as expected of a transcript that is maternally deposited. However, when extracts of first and second instar larvae were used, the Crl protein was detected in the heterozygous larvae, but almost no protein was present in the homozygous mutant larvae. The lack of the Crl protein may account for the lethal phase of the excision line and provides a genetic control for the specificity of the antibodies (Orgad, 2000).
The spatial distribution of the Crl protein was determined using antibodies that were raised against the two different putative carboxy termini of the Crl proteins. In general, the two antibodies revealed a similar pattern of Crl expression, which was comparable to the distribution of the crl transcript, as determined by in situ hybridization. However, at the blastoderm stage the Crl protein is localized to the poles, while the transcripts are present in the egg yolk and in all the peripheral blastoderm cells (Orgad, 2000).
Blind or olfaction-defective mutants in Drosophila are able to court and mate with a C.I. of ~50% that of the wild type. Double mutants that are blind and olfaction defective have a C.I. of only 7% that of wild type. It was of interest to exclude the possibility that the low C.I. obtained for crl homozygous males was due to a combined effect of defective vision and olfaction. The visual response of the crl mutants was tested. Homozygous crl flies are attracted to light to the same extent as wild-type flies. The number of flies attracted to light was 17 ± 2 for wild-type flies, 16 ± 2 for crl/CyO, and 15 ± 2 for crl/crl. A similar conclusion was drawn from examination of electroretinograms of crl homozygous mutant flies and crl/CyO flies as compared to wild-type flies. Likewise, no gross defect in the olfactory response of crl homozygous males was recorded, using the trap assay. Two olfactory attractants were used, Drosophila culture medium and wild-type virgin females. In both cases no difference was observed in the olfactory response between crl homozygous, crl/CyO, and wild-type (Canton-S) flies, suggesting that olfaction defects are not a major cause of the altered courtship behavior in crl (Orgad, 2000).
To rule out the possibility that the defective courtship behavior is due to general sluggishness of the mutant flies, locomotor activity of crl homozygous flies was compared to that of crl/CyO and wild-type flies. No difference was observed between the locomotor activities of the three genotypes (Orgad, 2000).
In Drosophila spermatogenesis, four gonial mitotic divisions of the primary spermatogonial cell produce a cohort of 16 cells, which remain connected by cytoplasmic bridges throughout spermatocyte development and spermatid differentiation. Two consecutive meiotic divisions result in a cyst containing 64 haploid spermatids. Many intracellular morphogenetic events take place, leading to a dramatic change in the shape of the spermatids, whereby both the cells and the nuclei elongate. Nuclear elongation is accompanied by chromatin condensation, and in the mature sperm the nucleus is shaped as a slightly curved needle. The last stage of spermatogenesis is individualization and coiling (Orgad, 2000).
Microscopic examination indicates that in homozygous crl males the four mitotic divisions of the primary spermatogonial cells occur normally, and cysts containing 16 primary spermatocytes are evident. However, the two consecutive meiotic divisions that should follow do not take place, and no cysts with 32 or 64 haploid spermatids are found. The primary spermatocytes undergo an immediate transition to elongated spermatids that have rather long tails, and heads that are larger in size and different in shape from those of normal spermatids. The bundles of spermatids are not as well organized in the mutant cysts as in wild-type males (Orgad, 2000).
Aguilera, M., et al. (2000). Ariadne-1: a vital Drosophila gene is required in development and defines a new conserved family of ring-finger proteins. Genetics 155(3): 1231-1244. 10880484
Ardley, H. C., et al. (2001). Features of the parkin/ariadne-like ubiquitin ligase, HHARI, that regulate its interaction with the ubiquitin-conjugating enzyme, Ubch7. J. Biol. Chem. 276(22): 19640-7. 11278816
Bays, N. W., et al. (2001). Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation. Nat. Cell Biol. 3(1): 24-9. 11146622
Biederer, T., Volkwein, C. and Sommer, T. (1996). Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin-proteasome pathway. EMBO J. 15(9): 2069-76. 8641272
Cenci, G., et al. (1997). UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior. Genes Dev. 11: 863-875. 9106658
Cook, W. J., et al. (1997). Crystal structure of a class I ubiquitin conjugating enzyme (Ubc7) from Saccharomyces cerevisiae at 2.9 angstroms resolution. Biochemistry 36(7): 1621-7. 9048545
Fang, S., et al. (2001). The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum. Proc. Natl. Acad. Sci. 98(25): 14422-7. 11724934
Friedlander, R., et al. (2000). A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat. Cell Biol. 2(7): 379-84. 10878801
Gilon, T., Chomsky, O. and Kulka, R. G. (2000). Degradation signals recognized by the Ubc6p-Ubc7p ubiquitin-conjugating enzyme pair. Mol. Cell. Biol. 20(19): 7214-9. 10982838
Goebl, M. G., et al. (1988) The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science 241: 1331-1335. 2842867
Hochstrasser, et al. (1991). The short-lived MAT2alpha transcriptional regulator is ubiquitinated in vivo. Proc. Natl. Acad. Sci. 88: 4606-4610. 1647011
Huang L., et al. (1999). Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science 286(5443): 1321-6. 10558980
Jentsch, S., McGrath, J. P. and Varshavsky, A. (1987). The DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329: 131-134. 3306404
Kay, G. F., et al. (1991). A candidate spermatogenesis gene on the mouse Y chromosome is homologous to ubiquitin-activating enzyme E1. Nature 354: 486-489. 1749428
Kopski, K. M. and Huffaker, T. C. (1997). Suppressors of the ndc10-2 mutation: a role for the ubiquitin system in Saccharomyces cerevisiae kinetochore function. Genetics 147(2): 409-20. 9335582
Lenk, U. and Sommer, T. (2000). Ubiquitin-mediated proteolysis of a short-lived regulatory protein depends on its cellular localization. J. Biol. Chem. 275(50): 39403-10. 10991948
Lin, H. and Wing, S. S. (1999). Identification of rabbit reticulocyte E217K as a UBC7 homologue and functional characterization of its core domain loop. J. Biol. Chem. 274(21): 14685-91. 10329663
Moynihan, T. P., et al. (1999). The ubiquitin-conjugating enzymes UbcH7 and UbcH8 interact with RING finger/IBR motif-containing domains of HHARI and H7-AP1. J. Biol. Chem. 274(43): 30963-8. 10521492
Muralidhar, M. G. and Thomas, J. B. (1993). The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron 11: 253-266. 8394720
Naidoo, N., et al. (1999). A role for the proteasome in the light response of the timeless clock protein. Science 285: 1737-1741. 10481010
Orgad, S., Rosenfeld, G., Greenspan, R. and Segal, D. (2000). courtless, the Drosophila UBC7 Homolog, is involved in male courtship behavior and spermatogenesis. Genetics 155: 1267-1280. 10880487
Schwarz, S. E., Rosa, J. L. and Scheffner, M. (1998). Characterization of human hect domain family members and their interaction with UbcH5 and UbcH7. J. Biol. Chem. 1998 May 15;273(20):12148-54. 9575161
Shimura, H., et al. (2000). Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nat. Genet. 25(3): 302-5. 10888878
Shimura, H., et al. (2001). Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease. Science 293(5528): 263-9. 11431533
Sung, P., Parkash, S. and Parkash, L. (1988). The RAD6 protein of Saccharomyces cerevisiae poly-ubiquitinates histones, and its acidic domain mediates this activity. Genes Dev. 2: 1476-1485. 2850263
Swanson, R., Locher, M., Hochstrasser, M. (2001). A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation. Genes Dev. 15(20): 2660-74. 11641273
Wang, M., et al. (2001). Developmental changes in the expression of parkin and UbcR7, a parkin-interacting and ubiquitin-conjugating enzyme, in rat brain. J Neurochem 2001 Jun;77(6):1561-8. 11413239
Yokouchi, M., et al. (1999). Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7. J. Biol. Chem. 274(44): 31707-12.
Zheng, N., et al. (2000). Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 102(4): 533-9. 10966114
date revised: 3 March 2002
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.