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BIOLOGICAL OVERVIEW

The courtless (crl) mutation disrupts early steps of courtship behavior in Drosophila males, as well as the development of their sperm. Most of the homozygous crl mutant males (78%) do not court at all. Only 5% perform the entire ritual and copulate, yet these matings produce no progeny. The crl gene encodes two proteins that differ in their carboxy termini and are the Drosophila homologs of the yeast ubiquitin-conjugating enzyme UBC7. The crl mutation is caused by an insertion of a P element into the 3' UTR of the gene, which probably disrupts translational regulatory elements. As a consequence, the homozygous mutants exhibit a six- to seven-fold increase in the level of the Crl protein. The crl product is essential, and deletions that remove the crl gene are lethal. During embryonic development crl is expressed primarily in the CNS. These results implicate the ubiquitin-mediated system in the development and function of the nervous system and in meiosis during spermatogenesis (Orgad, 2000).

When wild-type mature males are presented with virgin females, they initiate the courtship ritual almost instantaneously, spending most of their time performing the different courtship steps, culminating in a copulation rate of >90%. However, in homozygous crl males, 78% of the males tested did not court at all, and only 17% performed some of the courtship steps. Of these courting mutant males (13%) most performed only the early steps of courtship (orienting, following, and wing extension). Only 5% of the crl/crl males eventually copulated, but none of these matings produced progeny. The courtship index (C.I.) representing the fraction of the observation time that each male actually spent courting was 72% for wild-type (Canton-S) males and only 12% for crl homozygous males (breaking down this value for the noncourting and courting mutant males gives a C.I. value of 0 for noncourting and 23% for courting mutant males). In contrast, crl/crl females are fertile when mated to wild-type males, behave normally, and are as receptive to males as are wild-type females (Orgad, 2000).

Genes at the top of the sex determination hierarchy are involved in the control of courtship. Besides these, known mutants that act earliest in the ritual are those that affect the courtship song -- as in cacophony, dissonance, and croaker. The crl mutation blocks courtship at the very beginning of the sequence. Most homozygous crl males do not court at all and those few that do generally fail to progress beyond the early steps of following and wing extension. These early steps in courtship have been associated with male-specific development in the dorsal posterior brain (Orgad, 2000).

Mutants that are generally defective in locomotor behavior are also often defective in courtship. This sometimes manifests itself as a sluggish phenotype with a low probability of initiating courtship. However, the behavioral defect in crl homozygous males is not due to general sluggishness since their locomotor activity is as high as that of their crl/CyO sibling or wild-type males (Orgad, 2000).

Visual, rhythm, as well as learning and memory mutants have been found to court less rigorously than wild-type males and to have prolonged latency of the initiation of this behavior. However, the visual and olfactory systems in crl homozygous flies appear to be normal and cannot account for the mutant phenotype of crl. Therefore, the abnormal courtship behavior of the crl mutant may be attributed to defects in those parts of the central nervous system (CNS) that are responsible for this behavior. A general feature of courtship mutants is their pleiotropic nature. However, the abnormalities they engender are usually not global but rather are restricted to a small number of particular behaviors. For example, two mutants, cacophony and dissonance, isolated in a screen for mutants affecting the male courtship song were later, and rather surprisingly, found to be allelic to previously identified visual mutations. The former, although not visually defective, is an allele of the nbA gene, which, when mutated, causes poor performance in optomotor and phototactic tests, and the latter is allelic to the nonA mutation, which leads to absence of the light-on and light-off transient spikes, but does not affect the courtship song. Two additional examples for pleiotropy among courtship-defective mutants are fruitless and period, both of which display at least two distinct behavioral abnormalities: one is a defect in courtship song, which is common to both mutants, and the other is abnormal circadian rhythm (in the case of per) or display of homosexual behavior (in fru). Given that courtship behavior is complex, utilizing most of the sensory modalities, it is not surprising that mutations affecting vision, olfaction, and audition lead to aberrant courtship behavior (Orgad, 2000).

The courtless mutation is pleiotropic too, affecting distinct systems such as the nervous system and spermatogenesis. While homozygous crl females behave normally, are as receptive to males as wild-type females, and are fertile, homozygous crl males hardly court virgin females and produce abnormal sperm. The defect in spermatogenesis in crl homozygous males appears to be very similar to the phenotype reported for the ms(1)413, ms(1)RD11, and ms(1)682 mutants. In all three, mitochondrial aggregation occurs prematurely, meiotic spindles are not formed, and the primary spermatocytes are transformed directly into tetraploid spermatids. These mutants and crl fall into a distinct phenotypic class of spermatogenic mutants, which includes mutants in the genes for the cell cycle regulatory phosphatase twine, and the cell cycle kinase cdc2. In both mutants certain meiotic events are skipped, yet spermatid differentiation proceeds. As a result, testes of twine or cdc2 males contain bundles of 16 4N-cells that grow flagellar axonemes and undergo DNA condensation and nuclear shaping, as has been observed in crl males. A similar pleiotropy affecting both the nervous system and the reproductive tract has been reported for the dunce mutation. Several male sterile mutations affecting mitochondrial aggregation during spermatogenesis display, in addition, a common behavioral defect: they shake their appendages abnormally. Another example of a gene that encodes a component of the ubiquitin pathway is UbcD1 (effete: Cenci, 1997). effete mutants affect meiosis in males (Orgad, 2000 and references therein).

For normal courtship to occur, certain changes must take place in the CNS during its pupal-adult metamorphosis, and the subsequent reproductive maturation of the newly eclosed fly may entail specifically the dorsal posterior brain and the thoracic ganglion, known to be involved in normal sexual behavior. The molecular details of these processes are largely unknown (Orgad, 2000).

The courtless gene encodes the Drosophila homolog of the ubiquitin-conjugating enzyme UBC7. A distinctive property of the ubiquitin-mediated system is its remarkable functional diversity. It is implicated in various cellular functions including DNA repair (Jentsch, 1987), cell cycle control (Goebl, 1988), and transcription (Hochstrasser, 1991). The individual components of this system display remarkable functional specialization, suggesting narrow substrate specificities (Orgad, 2000).

The courtless gene potentially encodes two proteins that share a conserved UBC domain, but have different carboxy extensions. The C-terminal extensions of UBCs are believed to contribute to the substrate specificity of these enzymes and to their intracellular localization (Sung, 1988). The crl gene products are involved, at least, in two different processes, CNS development/function and spermatogenesis. The results suggest that the level of the Crl protein is critical for courtship. crl/+ males have approximately three times as much Crl protein as have wild-type males, and their courtship is normal. However, crl/crl males have at least seven times the Crl protein that the wild-type males have and are highly defective in courtship. One speculation may be that excess Crl protein may be compatible with normal courtship, as long as its level does not reach a certain threshold (Orgad, 2000).

The ben product was the first to exemplify the involvement of the ubiquitin-mediated system in the development and function of the CNS of Drosophila (Muralidhar, 1993). Recently this system was implicated in the regulation of the circadian feedback loop of the fly (Naidoo, 1999). The results with respect to courtless suggest that the ubiquitin-mediated system is involved in additional aspects of CNS function, in those parts of the brain important for courtship behavior (mushroom bodies, antennal lobes, etc.). This system, via the role of crl, is implicated in spermatogenesis as well. These data strengthen the finding that a mouse gene A1s9, which has been implicated in spermatogenesis, is homologous to the ubiquitin-activating enzyme E1 from yeast (Kay, 1991) and that the phenotype of mice knocked out for the mHR6B gene, the homolog of the yeast RAD6 ubiquitin-conjugating enzyme, is male infertility (Orgad, 2000).

In Drosophila a single regulatory hierarchy controls all aspects of somatic sexual differentiation, including those parts of the CNS involved in sexual behavior. Differentiation of those aspects of the CNS responsible for male-specific courtship occurs during the middle of the pupal period, when the CNS undergoes extensive reorganization, concomitant with the accumulation of crl transcripts. This set of genes, which determines the innate property of sexual behavior, has to be active continuously in the adult to maintain normal courtship behavior (Orgad, 2000).

In adults, sexual differences were found in crl expression, as determined by Northern blots, and the transcripts that retain the fourth intron are male specific. In embryos the crl transcripts are found throughout the CNS with no obvious sex-specific expression. In situ hybridization to sectioned CNS from pupae and adults should allow further studies to determine whether crl is expressed in those parts of the brain known to be involved in sexual behavior (Orgad, 2000).

The P element in the original crl mutant has inserted into the 3' UTR of the crl gene 200 bp upstream of the end of the transcription unit. The 3' UTR of various genes is involved in regulation of the stability of the transcript as well as its translation. In mammals, as well as in Drosophila, AU-rich sequences residing in the 3' UTR act as negative regulatory elements to facilitate degradation of the transcript. In Drosophila, several additional motifs that negatively regulate transcript stability and its translation efficiency were identified in the 3' UTR of various proneural genes of the acaete-scute complex, including Brd-box (CAGTTTAA), GY-box (GTCTTCC), and K-box (TGTGAT). The crl transcript contains in its 3' UTR two Brd-like boxes, one K-box, and several AU-rich motifs, suggesting regulation of its stability and translation efficiency. Indeed, the insertion of the P element in the crl mutant into the 3' UTR causes both reduction in the level of the crl transcript and elevation in the level of its translation. Although there is as yet no explanation for this phenomenon, such a situation has been reported for the 3' UTR of the growth factor TGF-ß1 gene. This UTR contains a CG-rich region that has been identified as being responsible for decrease in transcription, rather than instability of the mRNA, as well as stimulation of translation (Orgad, 2000 and references therein).

The primary role of ubiquitination is to target proteins for degradation. However, existence of stable ubiquitinated proteins has been documented, as well as of proteins that are reversibly ubiquitinated. This suggests that the ubiquitin-mediated system plays a role also in modification of protein function. Thus, the function of the crl-encoded UBC, in ensuring proper development and function of specific parts of the fly's CNS that are important for male sexual behavior, could conceivably be accomplished as follows. It may stably ubiquitinate a substrate protein(s) that is directly involved in patterning of the relevant connections in the CNS as was shown for arthrin, which is a stable actin-ubiquitin conjugate involved in the assembly or function of thin filaments in the Drosophila flight muscles. Alternatively, crl may mark specific substrate proteins for degradation, affording the means for close regulation of the function of proteins by modulating their half-life. This may enable these proteins to act as on/off switches of CNS functions. Overexpression of the Crl protein in the crl mutant may shift the delicate balance between Crl and its specific substrate required for normal development and proper activity of the CNS, culminating in the manifested phenotype of the crl mutant. Yet loss of function of the crl gene is fatal and causes lethality at the larval stage. Identification and characterization of genes that genetically interact with crl, and therefore could serve as a target substrate for Crl, may help in understanding the molecular mechanisms that underlie this complex behavior. It should also lead to better understanding of the increasingly sophisticated role the ubiquitin-mediated system is turning out to play in normal development and function of the CNS and of the neural diseases brought about by its malfunction (Orgad, 2000 and references therein).

GENE STRUCTURE

The crl gene spans only 1.65 kb and is composed of five exons separated by small (50–150 bp) introns. The P element responsible for the initial courtless mutation is inserted 200 bp upstream from the end of the transcription unit, in the 3' untranslated region (UTR) of the gene (Orgad, 2000).

Sequencing of 5 different cDNA clones reveals the existence of three classes of cDNAs. Classes 1a and 1b (1.3 and 1.1 kb long, respectively) both retain the fourth intron, and have the same open reading frame, which is capable of encoding a protein of 200 amino acids with a calculated molecular weight of 22,344 D. The two cDNA classes differ in their 3' untranslated region, with class 1b having a shorter 3' UTR. Class 2 (1.1 kb long) is an alternatively spliced species, which is the result of splicing out of the fourth intron that is retained in the other two classes. Thus, the deduced class 2-derived protein is shorter: 185 amino acids long with a molecular weight of 20,390. The 3' UTR of class 2 is identical to that of class 1a. The two predicted protein products of the crl gene differ in their carboxy termini (Orgad, 2000).

Primer extension experiment assigned the transcription start site to the adenine at position 0. The predicted TATA box is located 58 nucleotides upstream of the transcription start site. Several AU-rich sequences, one K-like box, and two Brd-like boxes are present in the 3' UTR. These elements are known to confer a short half-life to transcripts. The Brd-box has been shown to reduce translation as well (Orgad, 2000).

cDNA clone length - 1330bp

Bases in 5' UTR - 193

Exons - 5

Bases in 3' UTR - 434

PROTEIN STRUCTURE

Amino Acids - 200

Structural Domains

Analysis of the predicted protein product of crl reveals that it is highly homologous to the yeast ubiquitin-conjugating enzyme UBC7. This enzyme is a member of a large family of proteins that are found in all eukaryotes and function in the covalent transfer of ubiquitin to specific protein substrates. A comparison of the sequence of the Crl protein and UBC7 proteins from different organisms reveals that the Crl sequence is 60% identical to the yeast UBC7, and taking into account conservative substitutions raises the similarity to 72%. It is 52% and 46% identical to the UBC7 of Arabidopsis and wheat, respectively, indicating high evolutionary conservation. The conserved cysteine residue at position 89 is the putative active site for formation of a thiolester bond with ubiquitin, which is essential for the transfer of ubiquitin to the substrate (Orgad, 2000).

date revised: 3 March 2002

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