Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68 to 79 % overall sequence similarity to one another. Phylogenetic analysis ofnucleofide sequences derived from part of the gene encoding a non-structural protein (NS-5) has provided evidence for six major genotypes of HCV amongst a worldwide collection of 76 samples from HCV-infected blood donors and patients with chronic hepatitis. Many of these HCV types comprised a number of more closely related subtypes, leading to a current total of 11 genetically distinct viral populations. Phylogenetic analysis of other regions of the viral genome produced relationships between published sequences equivalent to those found in NS-5, apart from the more highly conserved 5' non-coding region in which only the six major HCV types, but not subtypes, could be differentiated. A new nomenclature for HCV variants is proposed in this communication that reflects the twotiered nature of sequence differences between different viral isolates. The scheme classifies all known HCV variants to date, and describes criteria that would enable new variants to be assigned within the classification as they are discovered.

The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. To further understand biochemically the functions of the Myb complex, we fractionated Drosophila embryo extracts relying upon affinity chromatography. We found that E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, we found that the transcriptional repressor protein, lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, associated with the Myb-MuvB complex. Members of the Myb-MuvB complex were localized to promoters and were shown to corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription.

Myb-MuvB (MMB)/dREAM is a nine-subunit complex first described in Drosophila as a repressor of transcription, dependent on E2F2 and the RBFs. Myb, an integral member of MMB, curiously plays no role in the silencing of the test genes previously analyzed. Moreover, Myb plays an activating role in DNA replication in Drosophila egg chamber follicle cells. The essential functions for Myb are executed as part of MMB. This duality of function lead to the hypothesis that MMB, which contains both known activator and repressor proteins, might function as part of a switching mechanism that is dependent on DNA sites and developmental context. Here, we used proliferating Drosophila Kc tissue culture cells to explore both the network of genes regulated by MMB (employing RNA interference and microarray expression analysis) and the genomic locations of MMB following chromatin immunoprecipitation (ChIP) and tiling array analysis. MMB occupied 3538 chromosomal sites and was promoter-proximal to 32% of Drosophila genes. MMB contains multiple DNA-binding factors, and the data highlighted the combinatorial way by which the complex was targeted and utilized for regulation. Interestingly, only a subset of chromatin-bound complexes repressed genes normally expressed in a wide range of developmental pathways. At many of these sites, E2F2 was critical for repression, whereas at other nonoverlapping sites, Myb was critical for repression. We also found sites where MMB was a positive regulator of transcript levels that included genes required for mitotic functions (G2/M), which may explain some of the chromosome instability phenotypes attributed to loss of Myb function in myb mutants.[Keywords: Drosophila; Myb-MuvB/dREAM; transcription] Supplemental material is available at http://www.genesdev.org. These DNA modules thus serve as sites for processing information that define the epigenetic state of the cell (Ptashne 2007).For purposes of classification, there are many examples of "modular enhancers" where the cis-acting elements are composed of clustered submotifs with flexible spacing between the DNA-binding sites that recruit different DNA-binding factors combinatorially. A model for such enhancer elements is the eve stripe 2 enhancer in Drosophila (Small et al. 1991). "Enhanceosomes" are another distinct type of enhancer element, where again, different DNA-binding factors are recruited to DNA. However, the exact spacing and geometry of the motifs are critical and when bound by proteins, allow for the creation of a higher-order nucleoprotein structure that is

Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction. DmORC displays at best six-fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA-binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30-fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator-initiator interactions and that in vivo other proteins must determine ORC's localization.

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.