A chromosome bin map of 2148 expressed sequence tag loci of wheat homoeologous group 7.

The objectives of this study were to develop a high-density chromosome bin map of homoeologous group 7 in hexaploid wheat (Triticum aestivum L.), to identify gene distribution in these chromosomes, and to perform comparative studies of wheat with rice and barley. We mapped 2148 loci from 919 EST clones onto group 7 chromosomes of wheat. In the majority of cases the numbers of loci were significantly lower in the centromeric regions and tended to increase in the distal regions. The level of duplicated loci in this group was 24% with most of these loci being localized toward the distal regions. One hundred nineteen EST probes that hybridized to three fragments and mapped to the three group 7 chromosomes were designated landmark probes and were used to construct a consensus homoeologous group 7 map. An additional 49 probes that mapped to 7AS, 7DS, and the ancestral translocated segment involving 7BS also were designated landmarks. Landmark probe orders and comparative maps of wheat, rice, and barley were produced on the basis of corresponding rice BAC/PAC and genetic markers that mapped on chromosomes 6 and 8 of rice. Identification of landmark ESTs and development of consensus maps may provide a framework of conserved coding regions predating the evolution of wheat genomes.

Dissociation of DDB1-binding and transactivation properties of the hepatitis B virus X protein.

The hepatitis B virus (HBV) X protein (HBx) is a transactivator encoded by mammalian hepadnaviruses, and is thought to stimulate transcription by interacting with one or more host cell factors. Numerous cellular proteins have been reported to interact with HBx including a component of the nucleotide excision repair complex called ultraviolet damaged DNA binding (UV-DDB, or DDB1) protein. Recent studies have identified a role for DDB1 in transcription, raising the possibility that HBx may acquire its broad transcriptional properties by interacting with DDB1. A panel of HBx mutant proteins, some of which no longer bind to DDB1, was used to test this hypothesis. Plasmid DNAs encoding HBx wildtype and mutant derivatives were transfected into HepG2 cells, and their ability to transactivate a cotransfected reporter plasmid tested. Results from the transactivation assays in HepG2 cells were then compared with data obtained from HBx-DDB1 binding studies performed in yeast. Several HBx mutant proteins unable to bind DDB1 remained competent for transactivation, indicating that HBx binding to DDB1 is not required for HBx transactivation of the ETS1 promoter. It remains possible that a subset of HBx transactivation function targets an as yet undefined DDB1-specific pathway.

Identification of the minimal replicase and the minimal promoter of (-)-strand synthesis, functional in rotavirus RNA replication in vitro.

An in vitro replication system supporting the initiation and synthesis of complete rotavirus (-)-strands on (+)-strand template RNA (Chen et al., J Virol 68: 7030, 1994) was used to examine several parameters related to rotavirus RNA replication. Coexpression of VP1/2/3 in all possible combinations from baculovirus vectors revealed: [i] Virus-like particles (VLPs) were formed only if VP2 was present, and [ii] VP1/2 and VP1/2/3 VLPs had replicase activity in the in vitro system whereas VP2/3 and VP2 VLPs did not. Thus, the minimal replicase is composed of VP1 and VP2 and replicase activity is associated with VP1. In vitro replication reactions, using T7 transcripts of porcine rotavirus OSU genome segment 9 as reporter template, were performed to map cis-acting elements that regulate replication. Internal deletions and terminal truncations of the reporter RNA localized a replication signal, conferring full template activity, to the 5'-terminal 27 nucleotides (nt 1-27) and the 3'-terminal 26 nucleotides (nt 1037-1062). Further analysis showed that a minimal promoter of (-)-strand synthesis was contained in the 3'-terminal 7 nucleotides (nt 1056-1062); the sequence conserved at the 3'-terminus of all rotavirus genes. Hybrid constructs with this promoter had minimal, but detectable, template activity. This result indicated that upstream sequences between nucleotides 1037-1055 positively regulate the activity of the minimal promoter.

The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis.

We used an in vitro template-dependent replicase assay (D. Chen, C. Zeng, M. Wentz, M. Gorziglia, M. Estes, and R. Ramig. J. Virol. 68:7030-7039, 1994) to identify the cis-acting signals required for replication of a genome segment 9 template from the group A rotavirus strain OSU. The replicase phenotypes for a panel of templates with internal deletions or 3'-terminal truncations indicated that no essential replication signals were present within the open reading frame and that key elements were present in the 5' and 3' noncoding regions. Chimeric constructs containing portions of viral sequence ligated to a nonviral backbone were generated to further map the regions required for in vitro replication of segment 9. The data from these constructs showed that the 3'-terminal seven nucleotides of the segment 9 mRNA provided the minimum requirement for replication (minimal promoter). Analysis of additional chimeric templates demonstrated that sequences capable of enhancing replication from the minimal promoter were located immediately upstream of the minimal promoter and at the extreme 5' terminus of the template. Mutational analysis of the minimal promoter revealed that the 3'-terminal -CC residues are required for efficient replication. Comparison of the replication levels for templates with guanosines and uridines at nucleotides -4 to -6 from the 3' terminus compared with levels for templates containing neither of these residues at these positions indicated that either or both residues must be present in this region for efficient replication in vitro.

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.

Template-dependent, in vitro replication of rotavirus RNA.

A template-dependent, in vitro rotavirus RNA replication system was established. The system initiated and synthesized full-length double-stranded RNAs on rotavirus positive-sense template RNAs. Native rotavirus mRNAs or in vitro transcripts, with bona fide 3' and 5' termini, derived from rotavirus cDNAs functioned as templates. Replicase activity was associated with a subviral particle containing VP1, VP2, and VP3 and was derived from native virions or baculovirus coexpression of rotavirus genes. A cis-acting signal involved in replication was localized within the 26 3'-terminal nucleotides of a reporter template RNA. Various biochemical and biophysical parameters affecting the efficiency of replication were examined to optimize the replication system. A replication system capable of in vitro initiation has not been previously described for Reoviridae.