Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids.

The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization. Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3. Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death. Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex). Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive. The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids. p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment. bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids. We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans. Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression. Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway.     

Evidence that the human X chromosome is enriched for male-specific but not female-specific genes

There is increasing evidence that X chromosomes have an unusual complement of genes, especially genes that have sex-specific expression. However, whereas in worm and fly the X chromosome has a dearth of male-specific genes, in mice genes that are uniquely expressed in spermatogonia are especially abundant on the X chromosome. Is this latter enrichment true for nongermline, male-specific genes in mammals, and is it found also for female-specific genes? Here, using SAGE data, we show (1) that tissue-specific genes tend to be more abundant on the human X chromosome, (2) that, controlling for this effect, genes expressed exclusively in prostate are enriched on the human X chromosome, and (3) that genes expressed exclusively in mammary gland and ovary are not so enriched. This we propose is consistent with Rice's model of the evolution of sexually antagonistic alleles.     

Evidence for genomic regulation of the telomeric activity in Drosophila melanogaster

The structural integrity of TART elements has been used as reporter of instability at chromosomal ends in numerous Drosophila stocks and over time in an unstable stock. The results show that telomeric activity is a regulated process that may differ between the stocks as well as over time within a stock. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hox genes and the regulation of movement in Drosophila

Many animals show regionally specialized patterns of movement along the body axis. In vertebrates, spinal networks regulate locomotion, while the brainstem controls movements of respiration and feeding. Similarly, amongst invertebrates diversification of appendages along the body axis is tied to the performance of characteristically different movements such as those required for feeding, locomotion, and respiration. Such movements require locally specialized networks of nerves and muscles. Here we use the regionally differentiated movements of larval crawling in Drosophila to investigate how the formation of a locally specialized locomotor network is genetically determined. By loss and gain of function experiments we show that particular Hox gene functions are necessary and sufficient to dictate the formation of a neuromuscular network that orchestrates the movements of peristaltic locomotion.     

Evidence for rolling circle replication of tandem genes in Drosophila

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It is found in various organisms and contains sequences derived primarily from repetitive chromosomal DNA. Using 2D gel electrophoresis, we have previously detected eccDNA composed of chromosomal tandem repeats throughout the life cycle of Drosophila. Here, we report for the first time evidence suggesting the occurrence of rolling circle replication of eccDNA in Drosophila. We show, on 2D gels, specific structures that can be enriched by benzoylated naphthoylated DEAE-cellulose chromatography and were identified in other systems as rolling circle intermediates (RCIs). These RCIs are homologous to histone genes, Stellate and Suppressor of Stellate, which are all organized in the chromosomes as tandem repeats. RCIs are detected throughout the life cycle of Drosophila and in cultured fly cells. These structures are found regardless of the expression of the replicated gene or of its chromosomal copy number.     

Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis

The pleiotropic functions of small GTPase Rho present a challenge to its genetic analysis in multicellular organisms. We report here the use of the MARCM (mosaic analysis with a repressible cell marker) system to analyze the function of RhoA in the developing Drosophila brain. Clones of cells homozygous for null RhoA mutations were specifically labeled in the mushroom body (MB) neurons of mosaic brains. We found that RhoA is required for neuroblast (Nb) proliferation but not for neuronal survival. Surprisingly, RhoA is not required for MB neurons to establish normal axon projections. However, neurons lacking RhoA overextend their dendrites, and expression of activated RhoA causes a reduction of dendritic complexity. Thus, RhoA is an important regulator of dendritic morphogenesis, while distinct mechanisms are used for axonal morphogenesis.     

Evidence for de novo evolution of testis-expressed genes in the Drosophila yakuba/Drosophila erecta clade

The mutational origin and subsequent evolution of de novo genes, which are hypothesized to be genes of recent origin that are not obviously related to ancestral coding sequence, are poorly understood. However, accumulating evidence suggests that such genes may often function in male reproduction. Here we use testis-derived expressed sequence tags (ESTs) from Drosophila yakuba to identify genes that have likely arisen either in D. yakuba or in the D. yakuba/D. erecta ancestor. We found several such genes, which show testis-biased expression and are often X-linked. Comparative data indicate that three of these genes have very short open reading frames, which suggests the possibility that a significant number of testis-biased de novo genes in the D. yakuba/D. erecta clade may be noncoding RNA genes. These data, along with previously published data from D. melanogaster, support the idea that many de novo Drosophila genes function in male reproduction and that a small region of the X chromosome in the melanogaster subgroup may be a hotspot for the evolution of novel testis-biased genes.     

Definition of the carbohydrate response element of the rat S14 gene. Evidence for a common factor required for carbohydrate regulation of hepatic genes.

The 5'-flanking region of the S14 gene from -4316 to +18 contains regulatory sequences responsible for activation of promoter activity in response to elevated carbohydrate metabolism in primary hepatocytes. To map these sequences, a series of constructs containing various internal deletions of the S14 5'-flanking sequence were assayed in primary hepatocytes. The region from -1601 to -1395 was found to be essential for this response. Comparison of the sequence of this S14 region to a region of the L-type pyruvate kinase gene that has been shown to mediate carbohydrate regulation (Thompson, K. S., and Towle, H. C. (1991) J. Biol. Chem. 266, 8679-8682) revealed a segment with 9 out of 10 identity. In both cases, this conserved sequence aligned with a DNase I footprint formed with hepatic nuclear extract. Oligonucleotides (approximately 30 base pairs) from either S14 or pyruvate kinase genes containing the conserved element bound to a hepatic nuclear factor(s) that gave identical complexes by mobility shift assay. Furthermore, these two oligonucleotides cross-competed for binding to the nuclear factor(s), suggesting that a common factor(s) binds to this conserved element. Reinsertion of the S14 oligonucleotide into an unresponsive S14 promoter construct restored the carbohydrate control. Moreover, this oligonucleotide could confer a glucose response when fused to a heterologous promoter. Thus, the S14 segment from -1457 to -1428 is a carbohydrate response element essential for the binding of nuclear factor(s) regulated by increased carbohydrate metabolism. This factor(s) may be common to the carbohydrate regulation of the S14 and pyruvate kinase genes.     

Apical spectrin is essential for epithelial morphogenesis but not apicobasal polarity in Drosophila.

Changes in cell shape and position drive morphogenesis in epithelia and depend on the polarized nature of its constituent cells. The spectrin-based membrane skeleton is thought to be a key player in the establishment and/or maintenance of cell shape and polarity. We report that apical beta(Heavy)-spectrin (beta(H)), a terminal web protein that is also associated with the zonula adherens, is essential for normal epithelial morphogenesis of the Drosophila follicle cell epithelium during oogenesis. Elimination of beta(H) by the karst mutation prevents apical constriction of the follicle cells during mid-oogenesis, and is accompanied by a gross breakup of the zonula adherens. We also report that the integrity of the migratory border cell cluster, a group of anterior follicle cells that delaminates from the follicle epithelium, is disrupted. Elimination of beta(H) prevents the stable recruitment of alpha-spectrin to the apical domain, but does not result in a loss of apicobasal polarity, as would be predicted from current models describing the role of spectrin in the establishment of cell polarity. These results demonstrate a direct role for apical (alphabeta(H))(2)-spectrin in epithelial morphogenesis driven by apical contraction, and suggest that apical and basolateral spectrin do not play identical roles in the generation of apicobasal polarity.     

Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy.

The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy.     

The Mu transpositional enhancer can function in trans: requirement of the enhancer for synapsis but not strand cleavage.

The phage Mu transpositional enhancer has been previously shown to stimulate the initial rate of the Mu DNA strand transfer reaction by a factor of 100. We now show that the Mu enhancer can function in trans on an unlinked DNA molecule. This activity is greatly facilitated by the presence of a free DNA end proximal to the enhancer element. Function of the enhancer in trans does not alter either the requirement for donor DNA supercoiling or for the two Mu ends to be in their proper orientation on the donor plasmid. An important consequence of these findings is that we have been able to evaluate directly the step in the transposition reaction for which the enhancer is required. We show that the role of the enhancer is limited to promoting productive synapsis; efficient strand cleavage can occur in the absence of the enhancer.     

Evidence for differential regulation of genes in the chondroitin sulfate utilization pathway of Bacteroides thetaiotaomicron.

Expression of the chondroitin sulfate utilization (csu) genes of Bacterioides thetaiotaomicron is regulated by chondroitin sulfate. We have now found, however, that the csu genes are not all regulated in the same way. In particular, the gene encoding beta-glucuronidase (csuE) is expressed under two different conditions that do not lead to expression of other csu genes.     

Stage-specific regulation of actin genes in Drosophila wing cells

Extreme and rapid changes in the synthesis of messenger RNAs and proteins accompany differentiation in wing tissues of Drosophila. Of the six actin genes, at least three are expressed in wing cells, some during the most extreme changes in cell shape. However, different messages of the set appear, decay, and reappear on a regulated temporal program. These results show that actin expression is stage-specific in a single cell type.     

Evidence for Drosophila P element transposase activity in mammalian cells and yeast

Drosophila P element transposase expression is limited to the germline by tissue-specific splicing of one of its three introns. Removal of this intron by mutagenesis in vitro has allowed both P element excision and transposition to be detected in Drosophila somatic tissues. In order to determine if P element transposase can function in other organisms, we have expressed modified P elements either lacking one intron or lacking all three introns in mammalian cells and yeast, respectively. Using an assay for P element excision, we have detected apparent excision events in cultured monkey cells. Furthermore, expression of the complete P element cDNA is lethal to Saccharomyces cerevisiae cells carrying a mutation in the RAD52 gene, indicating that double-stranded DNA breaks are generated, presumably by transposase action.     

Drosophila abl and genetic redundancy in signal transduction.

Genetic studies on Drosophila Abl and, more recently, on mouse c-Abl and c-Src indicate that the functions of these non-receptor tyrosine kinases may duplicate activities of other molecules within signal transduction pathways. In Drosophila, second-site mutations have been recovered that disrupt the redundant functions so that the Abl tyrosine kinase is essential to the formation of axonal connections in the embryonic central nervous system and for attachment of embryonic muscles to the body wall. Molecular isolation and analysis of the genes identified by these second-site mutations should define the molecular basis for the genetic redundancy.     

Drosophila goosecoid participates in neural development but not in body axis formation.

In vertebrate embryos, the homeobox gene goosecoid (gsc) is expressed in the gastrula organizer region and in later arising embryonic tissues including the foregut anlage. Ectopic expression and loss-of-function studies have demonstrated that Xenopus gsc elicits a dorsalizing activity that contributes to body axis formation. Here we report that the gsc gene is conserved in invertebrates. In Drosophila, D-gsc is expressed most strongly in the foregut anlage, which gives rise to the foregut proper and the stomatogastric nervous system (SNS). D-gsc expression overlaps with one of the three SNS precursor groups invaginating from the foregut anlage. Embryos mutant for D-gsc gastrulate normally but show disrupted invagination in the SNS primordium and lack one specific SNS ganglion. In addition, D-gsc mutant embryos show a less well defined defect in foregut arrangement. Our results indicate that this invertebrate homolog of gsc is not required for gastrulation but plays a role in neurogenesis in post-gastrula Drosophila embryos.