The herpes simplex virus (HSV) genome contains both cis-and transacting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oris and one copy of OriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the transacting functions required for the replication of plasmids containing oris or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode transacting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either onis or ofiL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.

Herpes simplex virus 1 contains seven genes that are necessary and sufficient for origin-dependent DNA synthesis in cultured cells. We have expressed the product of one of these genes, UL9, in insect cells by using a baculovirus expression vector. The apparent size of the UL9 protein, both in insect cells and in herpes simplex virus-infected Vero cells, is 82,000 Da. By using an immunoassay for protein-DNA interaction, we have shown that UL9 protein binds specifically to the herpes simplex virus origins of DNA replication, onis and orML. DNase I "footprint" analysis has shown that the UL9 protein interacts with two related sites on onis, located on each arm of a nearly perfect palindrome. Our data strongly suggest that the origin-binding activity described (14), and the recombinant plasmid was used to generate the recombinant baculovirus AcNPV/UL9 as described (15,16).Antisera. Decapeptides corresponding to the predicted carboxyl-terminal amino acid sequence ofHSV-1 genes UL5, UL8, UL9, and UL52 were purchased from Biosearch (San Rafael, CA) and were covalently coupled to keyhole limpet hemocyanin. Approximately 0.5 mg of coupled peptide was used to immunize rabbits biweekly for a total of three injections. Sera were collected at biweekly intervals beginning one month after the first injection. A complete characterization of these sera will be published elsewhere.Preparation of Baculovirus-Infected Cell Extract. Fifteen 150-cm2 flasks of nearly confluent SF9 cells were infected with the recombinant virus AcNPV/UL9 at a multiplicity of infection of 10-20 plaque-forming units per cell. After 54 hr at 28°C, the cells were dislodged from the flasks by shaking and were washed with phosphate-buffered saline. NucleiAbbreviations: HSV, herpes simplex virus; AcNPV, Autographa californica nuclear polyhedrosis virus.

The bacterial transposable element Tn5 was observed to undergo high-frequency sequence inversion when integrated into the herpes simplex virus type 1 (HSV-1) genome. Deletion analysis of the IS50 elements through which this recombination event occurred demonstrated the absence of cis-acting signals involved in the inversion process. Several observations suggested an intimate association of the recombination mechanism with HSV-1 DNA replication, including the ability of the seven viral genes that are essential for HSV-1 DNA synthesis to mediate Tn5 inversion in the absence of any other viral functions. Comparable results were obtained by using duplicate copies of the L-S junction of the HSV-1 genome. Thus inversion of the L and S components of the HSV-1 genome during productive infection does not appear to be a site-specific process, but rather is the result of generalized recombination mediated by the complex of gene products that replicate the viral DNA.

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.

This article presents findings of an efficacy trial examining the effect of a multitiered instruction and intervention model on first grade at-risk students' reading outcomes. Schools (N = 16) were randomly assigned to the treatment or control condition. In the fall of Grade 1, students were assigned to an instructional tier on the basis of Stanford Achievement Test-10th Edition scores (31st percentile and above = Tier 1; from the 10th to the 30th percentile = Tier 2). In both conditions, students identified as at risk (i.e., Tier 2; n = 267) received 90 min of whole group instruction (Tier 1) and an additional 30 min of daily small group intervention (Tier 2). In the treatment condition, teachers were trained to enhance core reading instruction by making instruction more explicit and increasing practice opportunities for students in Tier 1. In addition, at-risk readers were provided an additional 30-min daily small group intervention with content that was highly aligned with the Tier 1 core reading program. Results indicate significant, positive effects of the intervention on students' decoding and first semester fluent reading and potentially positive effects on reading comprehension and total reading achievement.