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The yield of progeny occlusion bodies from a baculovirus infection is a critical parameter governing viral population dynamics in the field. Previous evidence has suggested that the ability of the virus to block host development may be an important factor in determining yield of progeny virus. Here, we explore the relationship between yield, dose, and host developmental status at the time of infection during Autographa californica nucleopolyhedrovirus infection of final instar Heliothis virescens. The data indicate that occlusion body yield is strongly inversely dependent on the time elapsed after ecdysis before the insect is infected. The later in the instar the infective dose is received, the lower the efficiency with which the virus can block host development. The occlusion body yield from insects whose development is completely arrested is more than fourfold greater than the yield from insects that have initiated prepupal development. Dose is also an important factor, with high-dose infections more likely to lead to developmental arrest. Thus, the infection parameters that give rise to optimal progeny virus yield are infection at high dose early in the instar. Analysis of ecdysteroid titers demonstrated that only low levels of ecdysteroids are detectable in insects whose development is completely arrested. In contrast, in insects whose development was only partially arrested, extremely high ecdysteroid titers were frequently observed. These data support the hypothesis that the function of the baculovirus ecdysteroid UDP-glucosyltransferase gene is to delay or block host development, with the benefit of increasing the yield of progeny virus. Copyright 1998 Academic Press.

Autonomously replicating sequence (ARS) elements were first identified in the budding yeast Saccharomyces cerevisiae as chromosomal DNA fragments that promoted high frequency of transformation and extrachromosomal maintenance of plasmid DNA. These specific sequence elements were subsequently shown to function as origins of DNA replication. Detailed analysis of the structure and function of ARS elements has been limited largely to S. cerevisiae and more recently the fission yeast Schizosaccharomyces pombe. Characterization of ARS activity in other eukaryotes is far less complete. Here we describe the ARS assay developed in yeast and its application to the study of origin function in other eukaryotes. Other available methods for detecting autonomous replication in these systems are also presented.

Human replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, homologous recombination, and excision repair. We have previously reported the cloning and bacterial overexpression of the three RPA genes and have mapped them to chromosome 1 (RPA2), chromosome 7 (RPA3), and chromosome 17 (RPA1). We have now obtained yeast strains with artificial chromosomes carrying the three human RPA genes and report the more detailed genomic mapping of RPA. RPA1 was mapped to chromosome 17p13.3 using a combination of PCR amplification of somatic cell hybrids and radiation hybrids containing chromosome 17 fragments. RPA2 was mapped to chromosome 1p35 by PCR amplification of somatic cell hybrids of chromosome 1 and by fluorescence in situ hybridization. RPA3 was mapped to chromosome 7p22 by Southern analysis and PCR amplification of somatic cell hybrids of chromosome 7 as well as fluorescence in situ hybridization. Since RPA is an essential component of major metabolic events affecting DNA, the physical mapping of the genes for it may help elucidate the biochemical basis of genetic disorders involving DNA metabolism.

The effects of juvenile hormone (JH) and egg development neurosecretory hormone (EDNH) on ovarian ecdysteroid production during vitellogenesis in the autogenous mosquito, Aedes atropalpus, were investigated using in vitro techniques coupled with radioimmunoassay (RIA) and high-pressure liquid chromatography (HPLC). Normal females were characterized by quantitative, qualitative, and temporal patterns of in vitro ovarian ecdysteroid production. Females decapitated at emergence showed little ovarian ecdysteroid production and did not undergo vitellogenesis. A 500-ng dose of JH-I applied topically to decapitated females restored normal patterns of ecdysteroid production. In both normal and experimental females, ecdysone constituted the major portion of the ecdysteroids secreted by the ovaries in vitro. However, significant amounts of other RIA-active materials were detected, one of which was probably 20-OH-ecdysone. Fat body incubations indicated that these tissues produce little RIA-active material during the peak of vitellogenesis. During that period, the ovaries were the major source of ecdysteroid. Various doses of JH-I, applied to abdomens isolated at emergence, enhanced ovarian responsiveness to subsequent applications of head extracts containing EDNH both in vivo and in vitro. A 500-ng dose of JH-I, applied topically to blood-fed, decapitated Aedes aegypti, stimulated a significant increase in in vitro ovarian ecdysteroid production. Similarities between these data and those demonstrating prothoracicotropic effects of JH in the Lepidoptera are discussed.