Drosophila female precopulatory behavior is modulated by ecdysteroids.

The effect of ecdysteroid signaling on Drosophila female precopulatory behavior was investigated using two types of mutants with either globally reduced ecdysteroid availability or reduced expression of ecdysone receptors in fruitless neurons, known to control sexual behavior. While being courted by males, mutant females performed significantly less full ovipositor extrusion behavior to reject male copulation attempts. Ecdysteroid depleted females (ecdysoneless(1)) performed male-like courtship behaviors, including unilateral wing extension and song production with patterns very similar to male courtship song. These results support the hypothesis that ecdysteroids modulate female sexual behavior, perhaps acting as a regulator of sexual motivation, and as a component affecting the performance of sex specific behavior patterns.

Drosophila male courtship behavior is modulated by ecdysteroids.

Temperature-dependent induction of ecdysteroid deficiency in the ecdysoneless mutant ecd(1) adult Drosophila melanogaster results in altered courtship behavior in males. Ecdysteroid deficiency brings about significantly elevated male-male courtship behavior including song production resembling that directed toward females. Supplementation with dietary 20-hydroxyecdysone reduces male-male attraction, but does not change motor activity, courtship patterns or attraction to females. These observations support the hypothesis that reduced levels of ecdysteroids increase the probability that male fruit flies will display courtship behaviors to male stimuli.

Patterns of shaker family gene expression in single identified neurons of the American lobster, Homarus americanus.

The patterns of expression of voltage gated potassium channel genes of the Shaker family have been mapped in identified neurons of the lobster (Homarus americanus) ventral nerve cord using a single cell reverse transcriptase polymerase chain reaction procedure. Using specific oligonucleotides derived from the sequences of the shaker, shab, and shaw genes of the spiny lobster, Panulirus interruptus, we detected the corresponding potassium channel DNA fragments from Homarus americanus. The Homarus DNA fragments are 87-98% identical at the nucleotide level to the Panulirus DNA fragments. We used the Panulirus primers to measure the complement of RNAs for shaker, shab, and shaw in single identified cells that use GABA, glutamate, octopamine or serotonin as chemical messengers. Shaker and shaw RNAs were found in all four identified neuron types but shab RNA was not detected in serotonin cells under the present experimental conditions. All cells expressed alpha-tubulin RNA, which serves as an internal control suggesting that cells are intact after dissection. In glial cells that surround the neuronal cell bodies, the potassium channel genes are expressed at low to non-detectable levels.

Separation of root nodule cells and identification of tissue-specific genes.

The interior tissues of the alfalfa (Medicago sativa) root nodule differ in form and function from the peripheral layers. The interior tissues are specialized for the fixation of nitrogen in cells infected by rhizobia. In contrast, the peripheral nodule tissues perform roles that assist the interior tissues: they provide metabolic support and protect the interior tissues from damaging levels of oxygen. We used a novel microdissection technique to separate these tissue types, allowing immunological and molecular comparison between the nodule interior and periphery. Using differential mRNA display reverse transcription and polymerase chain reaction, we compared the mRNA profiles of the separated tissues and identified a transcript specific to the nodule interior, and several peripheral-specific candidate genes.

Long-Term Culture of Lobster Central Ganglia: Expression of Foreign Genes in Identified Neurons.

The ventral nerve cords of lobsters (Homarus americanus) can be cultured in vitro for at least 7 weeks. Over this period, neurons maintain their normal electrophysiological features and continue, among other measures of neuronal health, to synthesize RNA and proteins. One application of this culture system is demonstrated: the manipulation of gene expression in identified neurons. After intracellular injection of complementary RNA (cRNA) encoding green fluorescent protein (GFP), the amount of protein product measured by fluorescent confocal microscopy increases for 4 days and then decreases to background by day 10. Thus, translation of the injected message must have increased for 4 days before declining. Moreover, after injection of cRNA encoding {beta}-galactosidase, the levels of enzyme activity were measured using a fluorogenic substrate, revealing a peak of {beta}-galactosidase activity at 6 to 9 days; this activity was still detectable for at least 10 days after injection. Therefore, either GFP or {beta}-galactosidase can be used as an injectable marker, allowing in vivo quantitation of expression in individual cells over time. We measured long-lasting expression of these proteins after a single injection, suggesting that it may be possible to manipulate the levels of expression of any gene of interest.

Characterization of a nodule-enhanced glutamine synthetase from alfalfa: nucleotide sequence, in situ localization, and transcript analysis.

We have characterized two glutamine synthetase (GS) cDNA clones (pGS13 and pGS100) representing mRNA from root nodules of alfalfa. pGS13 is a full-length version of a previously isolated partial cDNA from an alfalfa nodule cDNA library, while pGS100 was previously isolated from an alfalfa suspension culture cDNA library. Using the 3' untranslated region of the two cDNAs as gene-specific probes, we have shown that the GS genes represented by pGS100 and pGS13 are expressed in all organs tested, although at varying levels. pGS13, however, represents the nodule-enhanced GS gene class. Genomic Southern blot analysis using gene-specific probes shows multiple hybridizing bands, in each case suggesting multiple genes and/or alleles for each class of cytoplasmic GS genes. In situ hybridization of alfalfa nodule sections with gene-specific antisense RNA probes has shown that the nodule-enhanced GS genes are induced in the invasion zone and that their expression is limited to the symbiotic zone, while the GS genes represented by pGS100 are induced in the early symbiotic zone and are expressed throughout the symbiotic and senescent zones. Transcripts for both sets of GS genes are localized in the infected cells and based on the spatial expression pattern it would appear that the two gene classes are induced independently of the onset of nitrogen fixation.

Sequential expression of two late nodulin genes in the infected cells of alfalfa root nodules.

The Nms-22 and leghemoglobin (Lb) genes are expressed exclusively in the infected cells of alfalfa root nodules. Expression of these two late nodulin genes originated at distinct cellular boundaries within the symbiotic region of the nodule. The Nms-22 gene was expressed in all infected cells, including those just adjacent to the meristematic region. Lb gene expression was induced in older infected cells and was most prominent in the mature region of the nodule. Despite this temporal separation of gene expression, both the Nms-22 and Lb genes were expressed in nodules elicited by bacA mutants in which bacteroid development has been blocked just after release from the infection thread.