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Regulation of gene expression is a highly complex process that requires the concerted action of many proteins, including sequence-specific transcription factors, cofactors, and chromatin proteins. In higher eukaryotes, the interplay between these proteins and their interactions with the genome still is poorly understood. We systematically mapped the in vivo binding sites of seven transcription factors with diverse physiological functions, five cofactors, and two heterochromatin proteins at ≈1-kb resolution in a 2.9 Mb region of the Drosophila melanogaster genome. Surprisingly, all tested transcription factors and cofactors show strongly overlapping localization patterns, and the genome contains many ‘hotspots’ that are targeted by all of these proteins. Several control experiments show that the strong overlap is not an artifact of the techniques used. Colocalization hotspots are 1-5 kb in size, spaced on average by ≈50 kb, and preferentially located in regions of active transcription. We provide evidence that protein-protein interactions play a role in the hotspot association of some transcription factors. Colocalization hotspots constitute a previously uncharacterized type of feature in the genome of Drosophila, and our results provide insights into the general targeting mechanisms of transcription regulators in a higher eukaryote. [ABSTRACT FROM AUTHOR]

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms. The screen suggested functions for 393 genes for which no RNAi-mediated phenotype was known. The comparison with similar screens in worms has general implications for RNAi experiments. [ABSTRACT FROM AUTHOR]

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms. [ABSTRACT FROM AUTHOR]

The G12 type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells . However, little is known about the molecular nature of the components that act in the G12-signaling pathway in an organism. We characterized a C. elegans Gα subunit gene, gpa-12, which is a homolog of mammalian G12/G13α, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G12QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G12 participates, we screened for suppressors of the G12QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCθ/δ. TPA-1 mediates the action of the tumor promoter PMA . Expression of G12QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G12 signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G12 proteins. [Copyright &y& Elsevier]

The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible. [ABSTRACT FROM AUTHOR]

Examines the role of mutational spectrum in Caenorhabditis elegans in characterizing the adenylyl cyclase gene. Suppressors of a neuronal degeneration phenotype; Identification of the canal-associated neurons of C. elegans; Crystal structure of the catalytic domains of lyases.

Examines the role of the Escherichia coli nucleotide excision repair (NER) proteins in DNA replication. Factors influencing the viability of E. coli cells; Proteins required in the replication backup system replacing the DNA polymerase I; Impact of mutation at the N-terminal zinc finger domain of UvrA on NER.

Drosophila melanogaster Suppressor of Under-Replication (SuUR) gene encodes a protein that modulates replicative properties of heterochromatin in endocycles of polytene cells. The SuUR mutation abolishes underreplication of intercalary heterochromatin and results in partial underreplication of pericentric heterochromatin. We performed a genome- wide mapping of SUUR target genes in non-polytenic Drosophila Kc cells by using the DamID approach. We show that SUUR preferentially binds genes that are transcriptionally silent and late-replicated. Distinct subsets of SUUR targets are associated with PcG proteins (Pc and Esc; Polycomb and Extra sexcombs), heterochromatic proteins [HP1 and SU(VAR)3-9] and B-type lamin. The SUUR binding profile negatively correlates with the DNA polytenization levels of salivary gland polytene chromosomes. Finally, SUUR target genes are repressed in Drosophila embryos and gradually activated later in development. Together these results suggest that SUUR is a ubiquitous marker of heterochromatin in different cell types. [ABSTRACT FROM AUTHOR]