Biomedical subjects

S F Goodwin

Publications and source records attributed to S F Goodwin.

13 recordsLinked to original sources

PubMed2005

Female receptivity phenotype of icebox mutants caused by a mutation in the L1-type cell adhesion molecule neuroglian.

Relatively little is known about the genes and brain structures that enable virgin female Drosophila to make the decision to mate or not. Classical genetic approaches have identified several mutant females that have a reluctance-to-mate phenotype, but most of these have additional behavioral defects. However, the icebox (ibx) mutation was previously reported to lower the sexual receptivity of females, without apparently affecting any other aspect of female behavior. We have shown that the ibx mutation maps to the 7F region of the Drosophila X chromosome to form a complex complementation group with both lethal and viable alleles of neuroglian (nrg). The L1-type cell adhesion molecule encoded by nrg consists of six immunoglobulin-like domains, five fibronectin-like domains, one transmembrane domain and one alternatively spliced intracellular domain. The ibx strain has a missense mutation causing a glycine-to-arginine change at amino acid 92 in the first immunoglobulin domain of nrg. Defects in the central brain of ibx mutants are similar to those observed in another nrg mutant, central brain deranged(1) (ceb(1)). However, both ceb(1) homozygous and ceb(1)/ibx heterozygous females are receptive. The expression of a transgene containing the non-neural isoform of nrg rescues both the receptivity and the brain structure phenotypes of ibx females.

Animals↗

PubMed2001

Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless.

A multibranched hierarchy of regulatory genes controls all aspects of somatic sexual development in Drosophila melanogaster. One branch of this hierarchy is headed by the fruitless (fru) gene and functions in the central nervous system, where it is necessary for male courtship behavior as well as the differentiation of a male-specific abdominal structure, the muscle of Lawrence (MOL). A preliminary investigation of several of the mutations described here showed that the fru gene also has a sex-nonspecific vital function. The fru gene produces a complex set of transcripts through the use of four promoters and alternative splicing. Only the primary transcripts produced from the most distal (P1) promoter are sex-specifically spliced under direction of the sex-determination hierarchy. We have analyzed eight new fru mutations, created by X-ray mutagenesis and P-element excision, to try to gain insight into the relationship of specific transcript classes to specific fru functions. Males that lack the P1-derived fru transcripts show a complete absence of sexual behavior, but no other defects besides the loss of the MOL. Both males and females that have reduced levels of transcripts from the P3 promoter develop into adults but frequently die after failing to eclose. Analysis of the morphology and behavior of adult escapers showed that P3-encoded functions are required for the proper differentiation and eversion of imaginal discs. Furthermore, the reduction in the size of the neuromuscular junctions on abdominal muscles in these animals suggests that one of fru's sex-nonspecific functions involves general aspects of neuronal differentiation. In mutants that lack all fru transcripts as well as a small number of adjacent genes, animals die at an early pupal stage, indicating that fru's function is required only during late development. Thus, fru functions both in the sex-determination regulatory hierarchy to control male sexual behavior through sex-specific transcripts and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development through sex-nonspecific transcript classes.

Alleles↗

PubMed2000

Spatial, temporal, and sexually dimorphic expression patterns of the fruitless gene in the Drosophila central nervous system.

The fruitless (fru) gene of Drosophila produces both sex-specifically and non-sex-specifically spliced transcripts. Male-specific fru products are believed to regulate male courtship. To further an understanding of this gene's behavioral role, we examined the central nervous system (CNS) for temporal, spatial, and sexually dimorphic expression patterns of sex-specific fru products by in situ hybridization and immunohistochemistry. For the latter, antibodies were designed to detect only male-specific forms of the protein (FRU(M)) or amino acid sequences that are in common among all translated products (FRU(COM)). Sex-specific mRNAs and male-specific proteins were first observed in mature larvae and peaked in their apparent abundances during the first half of the pupal period. At later stages and in adults, faint mRNA signals were seen in only a few neural clusters; in contrast, relatively strong FRU(M) signals persisted into adulthood. Twenty neuronal groups composed of 1700 fru-expressing neurons were identified in the midpupal CNS. These groups overlap most of the neural sites known to be involved in male courtship. Anti-FRU(COM) led to widespread labeling of neural and nonneural tissues in both sexes, but in the female CNS, only in developing ganglia in a pattern different from that of the male's FRU(M) cells. Expression of sex-specific fru mRNAs in the CNS of males analyzed from the earliest pupal stages indicated that sex-specific alternative splicing is not the exclusive mechanism regulating expression of fruitless transcripts.

Animals↗

PubMed2000

Aberrant splicing and altered spatial expression patterns in fruitless mutants of Drosophila melanogaster.

The fruitless (fru) gene functions in Drosophila males to establish the potential for male sexual behaviors. fru encodes a complex set of sex-specific and sex-nonspecific mRNAs through the use of multiple promoters and alternative pre-mRNA processing. The male-specific transcripts produced from the distal (P1) fru promoter are believed to be responsible for its role in specifying sexual behavior and are only expressed in a small fraction of central nervous system (CNS) cells. To understand the molecular etiology of fruitless mutant phenotypes, we compared wild-type and mutant transcription patterns. These experiments revealed that the fru(2), fru(3), fru(4), and fru(sat) mutations, which are due to P-element inserts, alter the pattern of sex-specific and sex-nonspecific fru RNAs. These changes arise in part from the P-element insertions containing splice acceptor sites that create alternative processing pathways. In situ hybridization revealed no alterations in the locations of cells expressing the P1-fru-promoter-derived transcripts in fru(2), fru(3), fru(4), and fru(sat) pharate adults. For the fru(1) mutant (which is due to an inversion breakpoint near the P1 promoter), Northern analyses revealed no significant changes in fru transcript patterns. However, in situ hybridization revealed anomalies in the level and distribution of P1-derived transcripts: in fru(1) males, fewer P1-expressing neurons are found in regions of the dorsal lateral protocerebrum and abdominal ganglion compared to wild-type males. In other regions of the CNS, expression of these transcripts appears normal in fru(1) males. The loss of fruitless expression in these regions likely accounts for the striking courtship abnormalities exhibited by fru(1) males. Thus, we suggest that the mutant phenotypes in fru(2), fru(3), fru(4), and fru(sat) animals are due to a failure to appropriately splice P1 transcripts, whereas the mutant phenotype of fru(1) animals is due to the reduction or absence of P1 transcripts within specific regions of the CNS.

Amino Acid Sequence↗

PubMed1999

Molecular neurogenetics of sexual differentiation and behaviour.

Sex and death. Two things that come once in a lifetime. Only after death you're not nauseous. - Woody Allen 'Sleeper'. The brain and nervous system functions that underlie sex-specific behaviour are of obvious importance to all animals, including humans. To understand behaviour related to sex, it is important to distinguish those aspects that are controlled genetically. Much of the recent progress in studies of the molecular neurogenetics of sexual differentiation and behaviour has come from the use of genetically tractable organisms (i.e. fruitflies and nematode worms) that exhibit a full range of sexually dimorphic phenotypes.

Animals↗

PubMed1999

Abnormal courtship conditioning in males mutant for the RI regulatory subunit of Drosophila protein kinase A.

The previously described site-selected P-element mutagenesis of a Drosophila gene encoding the regulatory subunit of cAMP-dependent protein kinase generates mutants that have defective behavior in the olfactory learning test. Here we describe the effect of the same mutations in a courtship conditioning assay. Wild-type males can distinguish between virgin females (which they court vigorously), and fertilized females (which they court less vigorously). After exposure to fertilized females, wild-type males modify their behavior by decreasing courtship to subsequent target virgins, an effect that may last for many hours. Like wild-type males, PKA-RI mutant males are also able to distinguish between virgin and fertilized females. PKA-RI males also modify their behavior towards virgin females after prior exposure to a fertilized female, but such an effect is short-lived, suggesting a defect in memory rather than learning. We also show that under these conditions the behavior of PKA-RI males is similar to that of amnesiac, dunce and rutabaga males.

Animals↗

PubMed1997

Defective learning in mutants of the Drosophila gene for a regulatory subunit of cAMP-dependent protein kinase.

Disruptions of a Drosophila gene encoding a regulatory subunit of cAMP-dependent protein kinase homologous to mammalian RIbeta (dPKA-RI) were targeted to the first (noncoding) exon of dPKA-RI via site-selected P element mutagenesis. Flies homozygous for either of two mutant alleles showed specific defects in olfactory learning but not in subsequent memory decay. In contrast, olfactory acuity and shock reactivity, component behaviors required for normal odor avoidance learning, were normal in these mutants. Northern and Western blot analyses of mRNA and protein extracted from adult heads have revealed a complex lesion of the PKA-RI locus, including expression of a novel product and over- or underexpression of wild-type products in mutants. Western blot analysis revealed reductions in RI protein in mutants. PKA activity in the absence of exogenous cAMP also was significantly higher than normal in homogenates from mutant adult heads. These two mutant alleles failed to complement each other for each of these phenotypic defects, eliminating second-site mutations as a possible explanation. These results establish a role for an RI regulatory subunit of PKA in Pavlovian olfactory conditioning.

Animal Structures↗

PubMed1996

Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene.

Sexual orientation and courtship behavior in Drosophila are regulated by fruitless (fru), the first gene in a branch of the sex-determination hierarchy functioning specifically in the central nervous system (CNS). The phenotypes of new fru mutants encompass nearly all aspects of male sexual behavior. Alternative splicing of fru transcripts produces sex-specific proteins belonging to the BTB-ZF family of transcriptional regulators. The sex-specific fru products are produced in only about 500 of the 10(5) neurons that comprise the CNS. The properties of neurons expressing these fru products suggest that fru specifies the fates or activities of neurons that carry out higher order control functions to elicit and coordinate the activities comprising male courtship behavior.

Age Factors↗

PubMed1996

Analysis and inactivation of vha55, the gene encoding the vacuolar ATPase B-subunit in Drosophila melanogaster reveals a larval lethal phenotype.

Vacuolar ATPases play major roles in endomembrane and plasma membrane proton transport in eukaryotes. A Drosophila melanogaster cDNA encoding vha55, the 55-kDa vacuolar ATPase (V-ATPase) regulatory B-subunit, was characterized and mapped to 87C2-4 on chromosome 3R. A fly line was identified that carried a single lethal P-element insertion within the coding portion of gene, and its LacZ reporter gene revealed elevated expression in Malpighian tubules, rectum, antennal palps, and oviduct, regions where V-ATPases are believed to play a plasma membrane, rather than an endomembrane, role. The P-element vha55 insertion was shown to be allelic to a known lethal complementation group l(3)SzA (= l(3)87Ca) at 87C, for which many alleles have been described previously. Deletions of the locus have been shown to be larval lethal, whereas point mutations show a range of phenotypes from subvital to embryonic lethal, implying that severe alleles confer a partial dominant negative phenotype. The P-element null allele of vha55 was shown also to suppress ectopic sex combs in Polycomb males, suggesting that transcriptional silencing may be modulated by genes other than those with known homeotic or DNA binding functions.

Alleles↗

PubMed1994

Reverse genetics of Drosophila brain structure and function.

A set of molecular genetic technologies are described, which will have far reaching consequences for the study of brain structure, function and development in Drosophila melanogaster. Site selected mutagenesis (a PCR-based screen for P-element insertion events) allows insertion mutants to be isolated for any cloned gene, and is being used in this laboratory to ask questions about the rolls of particular cellular components in learning and memory. Transposants have been isolated in genes encoding a regulatory (RI) and a catalytic (DCO) subunit of cAMP-dependent protein kinase, and in a gene encoding a Gi-like alpha subunit. The alternative use of I factors is described. The PKA RI homozygous mutants display a significant decrement in initial learning ability. Enhancer-trap strategies, for which the GAL-4 P-element system is particularly convenient, allow the identification of genes expressed in the developing fly brain. Strategies for the efficient detection of such events are described.

Animals↗

PubMed1994

Evidence that the 16 kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and ductin from gap junctions are the same polypeptide in Drosophila and Manduca: molecular cloning of the Vha16k gene from Drosophila.

The 16 kDa proteolipid (subunit c) of the eukaryotic vacuolar H(+)-ATPase (V-ATPase) is closely related to the ductin polypeptide that forms the connexon channel of gap junctions in the crustacean Nephrops norvegicus. Here we show that the major protein component of Manduca sexta gap junction preparations is a 16 kDa polypeptide whose N-terminal sequence is homologous to ductin and is identical to the deduced sequence of a previously cloned cDNA from Manduca (Dow et al., Gene, 122, 355-360, 1992). We also show that a Drosophila melanogaster cDNA, highly homologous to the Manduca cDNA, can rescue Saccharomyces cerevisiae, defective in V-ATPase function, in which the corresponding yeast gene, VMA3, has been inactivated. Evidence is presented for a single genetic locus (Vha16) in Drosophila, which in adults at least contains a single transcriptional unit. Taken together, the data suggest that in Drosophila and Manduca, the same polypeptide is both the proteolipid subunit c component of the V-ATPase and the ductin component of gap junctions. The intron/exon structure of the Drosophila Vha16 is identical to that of a human Vha16 gene, and is consistent with an ancient duplication of an 8 kDa domain. A pilot study for gene inactivation shows that transposable P-elements can be easily inserted into the Drosophila ductin Vha16 gene. Although without phenotypic consequences, these can serve as a starting point for generation of null alleles.

Amino Acid Sequence↗

PubMed1992

Analysis of the gene encoding a 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase from Manduca sexta midgut and tubules.

Vacuolar ATPases (V-ATPases), originally characterised as components of endomembranes, have also been implicated in epithelial ion transport, both in vertebrates and in insects. The ATPase is particularly noteworthy in lepidopteran larvae, where it generates large transepithelial potential differences and short-circuit currents across the midgut epithelium. A cDNA library from Manduca sexta larval midguts and Malpighian tubules was screened with a Drosophila melanogaster cDNA encoding the 16-kDa proteolipid subunit of the V-ATPase, and a 1.4-kb cDNA sequenced in its entirety. The sequence contains a long open reading frame, encoding a putative peptide of 156 amino acids (aa) and with an M(r) of 15,967, in close agreement with values previously suggested by sodium dodecyl sulfate-polyacrylamide gels of M. sexta midgut proteins. Correspondence of the deduced aa sequence with those of other species, particularly D. melanogaster, was extremely close. Northern blots of M. sexta midgut mRNA at high stringency revealed two transcripts of 1.4 and 1.9 kb, whereas genomic Southern blots suggest that there is only a single copy of the gene in M. sexta. The possibility that members of the 16-kDa gene family might serve multiple roles in transport and membrane communication is discussed.

Amino Acid Sequence↗

PubMed1990

"Site-selected" transposon mutagenesis of Drosophila.

Despite the wide range of techniques that can be brought to bear on the study of basic processes in Drosophila, there are still deficiencies in our armory. One of these is an ability to select mutants in cases where the gene is known and has been cloned, but where we are ignorant of the associated phenotype. We describe here a solution to this problem as applied to a model system, the singed (sn) locus. Our method is a combination of classical genetics and molecular biology: sib selection plus the polymerase chain reaction. We have used the method to isolate rare individuals with P-element-induced alleles of sn merely by recognition of the DNA structures induced at the locus by transposon insertion. Phenotypic criteria were used only retrospectively to verify our diagnoses. There are obvious implications of this technique for the mutagenesis of other organisms.

Animals↗