cDNA libraries from identified neurons.

One approach to studying the changes in gene expression which underlie differentiation is to construct cDNA libraries from different tissues or at different stages of development. However, generating representative cDNA libraries from heterogeneous tissues such as the nervous system is often a real problem. Here, we describe a reproducible method for the construction of large and complex cDNA libraries from a few leech Retzius or P neurons (equivalent to about 50 pg of mRNA) using polymerase chain reaction-based technology.

The relationship between mRNA half-life and gene function in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae (Sc) mRNAs have been described as falling into two major classes with respect to mRNA half-life [Santiago et al., Nucleic Acids Res. 14 (1986) 8347-8360].

Insulin normalization as an approach to the pharmacological treatment of obesity.

Hyperinsulinemia and exaggerated insulin response to glucose are among the hallmarks of obesity. However, the role of hyperinsulinemia in the etiology and maintenance of obesity has been controversial. If hyperinsulinemia plays a critical role as proposed, then its reversal may have therapeutic potential.

The Flybrain Project.

The large amounts of neuroanatomical data and images that can be difficult to publish by conventional means because of the lack of space and financial constraints can now be made available electronically. The Flybrain Project represents a concerted effort to generate an on-line atlas and database for the structure and function of the Drosophila nervous system. In this article Heisenberg and Kaiser describe the background to the project and its aims for the future.

The coactivator p15 (PC4) initiates transcriptional activation during TFIIA-TFIID-promoter complex formation.

We have analyzed the mechanisms underlying stimulation of transcription by the activator GAL4-AH and the recombinant coactivator p15 (PC4). We show that p15 binds to both double-stranded and single-stranded DNA. Analyses of deletion mutants correlates binding to double-stranded DNA with the ability to mediate activator-dependent transcription.

Functional dissection of the Drosophila mushroom bodies by selective feminization of genetically defined subcompartments.

Relatively little is known about the neural circuitry underlying sex-specific behaviors. We have expressed the feminizing gene transformer in genetically defined subregions of the brain of male Drosophila, and in particular within different domains of the mushroom bodies. Mushroom bodies are phylogenetically conserved insect brain centers implicated in associative learning and various other aspects of behavior.

Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns.

Phylogenetically conserved brain centers known as mushroom bodies are implicated in insect associative learning and in several other aspects of insect behavior. Kenyon cells, the intrinsic neurons of mushroom bodies, have been generally considered to be disposed as homogenous arrays. Such a simple picture imposes constraints on interpreting the diverse behavioral and computational properties that mushroom bodies are supposed to perform.

Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library.

Conventional hybridomas and combinatorial Ab libraries were used to develop neutralizing murine mAbs to human IL-5. Mice were immunized with rIL-5. Spleens from two mice were used to generate hybridomas.

Progress in Drosophila genome manipulation.

The introduction of cloned and manipulated genetic material into the germline of an experimental organism is one of the most powerful tools of modern biology. In the case of the fruit fly, Drosophila melanogaster, there is also an unparalleled range of sophisticated genetic tools to facilitate subsequent analysis. In consequence, Drosophila remains a most favourable model organism for the dissection of gene structure and function in vivo.

Characterisation of Saccharomyces cerevisiae genes encoding ribosomal protein YL6.

We have characterised a Saccharomyces cerevisiae cDNA (cDNA13), originally isolated on the basis of the short half-life of the corresponding mRNA. We show here that its sequence is closely related to that of the genes encoding ribosomal proteins K37, KD4 and K5 of Schizosaccharomyces pombe. 'mRNA13' also behaves like other mRNAs encoding ribosomal proteins, in that its abundance increases sharply when glucose is added to cells grown on ethanol (nutrient-upshift), and declines when cells are subjected to a mild heat-shock.

Abnormal regional benzodiazepine receptor uptake in the prefrontal cortex in patients with panic disorder.

The neuroanatomical networks involved in the initiation of panic attack and the maintenance of panic disorder are poorly understood. This study aimed to elucidate the possible abnormalities in benzodiazepine receptor uptake in the brain of patients with panic disorder. Seventeen unmedicated patients with panic disorder were investigated using 123I-iomazenil single photon emission tomography (SPET).

Enhanced access to rare brain cDNAs by prescreening libraries: 207 new mouse brain ESTs.

To use single-pass cDNA sequencing to characterize low-frequency cDNA clones from a region of the brain that includes the primary site of neurodegeneration in human Parkinson disease, we have developed a prescreening procedure using single brain region first-strand cDNA probes. Selection of cDNA clones giving low hybridization signals allowed the elimination of clones resulting from abundant messages and enrichment for clones corresponding to low-copy messages. Comparative sequencing of standard and prescreened cDNA libraries (191 and 124 clones, respectively) showed that this procedure raised the frequency of novel sequences encountered from 54 to 81%.

A novel mediator of class II gene transcription with homology to viral immediate-early transcriptional regulators.

Our investigations of mammalian class II gene transcription resulted in identification, purification, and cloning of the corresponding cDNA of a cellular factor (p15) that mediates the effects of several distinct activators on transcription in vitro. Functional deletion analyses revealed a bipartite structure of p15 comprising an amino-terminal regulatory domain and a carboxy-terminal cryptic DNA-binding domain. We provide evidence that activity of p15 is controlled by protein kinases that target the regulatory domain.

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).

A novel role for the nitric oxide-cGMP signaling pathway: the control of epithelial function in Drosophila.

The nitric oxide (NO) signaling pathway plays major roles in the vertebrate vascular, nervous, and immune systems. Here we present evidence that all the elements in the NO pathway are present in, and act to control epithelial fluid secretion by, the Malpighian tubules of an insect, Drosophila melanogaster. This finding will allow both a physiological and a molecular genetic dissection of the NO pathway in the same tissue.

A Drosophila melanogaster chromosome 2L repeat is expressed in the male germ line.

We describe the initial characterisation of a Drosophila melanogaster locus, Mst40 (Male-specific transcript), that was cloned on the basis of its male-specific transcription during the third larval instar. Corresponding low molecular weight poly(A)+ mRNAs are abundant in primary spermatocytes, but in no other larval or adult tissue. During early embryogenesis Mst40 expression is complex; initially transcription is detected during early cleavage stages.

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.

Identification of genes up-regulated in dedifferentiating Nicotania glauca pith tissue, using an improved method for constructing a subtractive cDNA library.

Pith explants of Nicotiana glauca grown in vitro in synthetic medium supplemented with 2,4 dichlorophenoxyacetic acid (2, 4 D), are induced to dedifferentiate. Treatment with actinomycin D within the first 4-8 h of culture (but not later) is lethal and the explants die, implying a requirement for de novo transcription. The genes expressed during the initial period of culture are presumably critical for subsequent cell survival and proliferation, but so far their identity is unknown.

The determination of biogenic amines in four strains of the fruit fly Drosophila melanogaster.

A range of biogenic amines were measured in the heads from four strains of Drosophila melanogaster. Quantitation was carried out using gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS) with stable isotope dilution. The principal amines detected in the heads were dopamine, noradrenaline and 5 HT with small amounts of p- and m-tyramine; p-octopamine could not be detected in samples of 25 heads with a limit of detection of 10 pg per sample.