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Abstract: This paper presents an effective method for prioritizing areas within a country for acquisition of germplasm of a crop gene pool for ex situ conservation. The method was applied to the rare wild pepper species, Capsicum flexuosum Sendtn., in south-east Paraguay. A model to prioritize areas for collecting germplasm was constructed by combining (1) a prediction of the species' geographic distribution based on the climate at previous collection points, (2) the distribution of forest margins (the species' natural habitat) and (3) areas accessible by road. The model was then tested in the field by visiting 20 sites having both high and low predicted probability of occurrence of C. flexuosum. Six new populations were found, representing a significant improvement over two previous collecting missions for the species in the same region, undertaken without the use of GIS targeting. Using the most optimistic analysis of model performance, C. flexuosum was found at five out of seven points predicted to harbour the species and not found at four of five points predicted not to harbour the species. The model was then improved by the use of higher resolution climate surfaces. It is recommended that future explorers use more recent and higher resolution satellite images to locate suitable habitats. The method is replicable for different species in different geographic regions and is offered as a means of optimizing efficiency in financially constrained, national plant genetic resources programs.

Geographic location data for 383 accessions of five Passifloraspecies from five Andean countries (P. cumbalensis, P. mixta, P. tripartitavar. mollissima, P. natistipula, and P. manicata) were linked to interpolated continental surfaces of monthly mean rainfall, monthly mean temperature, and monthly mean diurnal temperature range. This permitted us to identify the potential distribution of each species and document its climatic adaptation. Maps are presented showing regions where the climate is similar to that in areas where each species has been found, but from which no collections have yet been made, thus representing possible geographical gaps in collections. Some species showed evidence of intra-specific variation in their climatic adaptation. The study of Passifloradiversity in Andean countries continues and these maps based on passport data are expected to be useful tools for the planning of both ex situand in situconservation activities.

The signing of the Convention on Biological Diversity at the ``Earth Summit'' in 1992, its ratification and its subsequent entering into force have highlighted the need for an approach to biodiversity conservation that employs both ex situand in situtechniques in a complementary manner. Though much research has focused on ex situtechniques, less progress has been made in developing methodologies for the conservation of genetic diversity in situ. The definition of in situconservation used in the Convention on Biological Diversity encompasses two distinct processes: conservation of wild species in genetic reserves and of crops on-farm. Of these two, the latter, where the genetic diversity of crop land races is conserved within traditional farming systems, has been the less studied and remains less well understood. While there are still relatively few practical examples of on-farm genetic resources conservation, most genetic diversity of immediate and potential use to plant breeders is found among land races, and there is evidence that it is being rapidly eroded. This paper attempts to set on-farm conservation within the context of plant genetic resource conservation as a whole, to introduces a possible generalised model for the conservation of genetic diversity on-farm and to promote debate around the science of on-farm conservation.

The P143 protein of Autographa californica nuclear polyhedrosis virus is essential for replication of viral DNA. To determine the function of P143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress P143. ATPase activity copurified with P143 protein during purification and also during gel filtration at a high salt concentration. The ATPase activity did not require the presence of single-stranded DNA, but was stimulated fourfold by the addition of single-stranded DNA. The ATPase activity of P143 had a K(m) of 60 microM and a turnover of 4.5 molecules of ATP hydrolyzed/s/molecule of enzyme, indicating moderate affinity for ATP and high catalytic efficiency. P143 unwound a 40-nucleotide primer in an ATP-dependent manner, indicating that the enzyme possesses in vitro DNA helicase activity. Based on this result, it seems likely that P143 functions as a helicase in viral DNA replication.

The use of plant genetic diversity is essential for ensuring an adequate and stable supply of diverse food crops as well as for enhancing their nutritional quality. The role of effective conservation, management, and use of plant genetic resources in ensuring the availability of a diverse range of nutritious food crops is recognized throughout the work of the International Plant Genetic Resources Institute (IPGRI). Several research activities in the Institute's programme highlight how the diversity between and within food crop species is inextricably linked to the diversity of human cultural needs, preferences, and knowledge systems with respect to the management and use of plant genetic resources. The nutritional quality of food crops is among the major considerations that are important in both the improvement and conservation of genetic resources. Specific examples from IPGRI's work are used to illustrate these linkages.

The DNA polymerase (DNApol) of Autographa californica nuclear polyhedrosis virus was purified to homogeneity from recombinant baculovirus-infected cells. DNApol was active in polymerase assays on singly primed M13 template, and full-length replicative form II product was synthesized at equimolar ratios of enzyme to template. The purified recombinant DNApol was shown to be processive by template challenge assay. Furthermore, DNApol was able to incorporate hundreds of nucleotides on an oligo(dT)-primed poly(dA) template with limiting amounts of polymerase. DNApol has moderate strand displacement activity, as it was active on nicked and gapped templates, and displaced a primer in a replication-dependent manner. Addition of saturating amounts of LEF-3, the viral single-stranded DNA-binding protein (SSB), increased the innate strand displacement ability of DNApol. However, when LEF-3 was added prior to the polymerase, it failed to stimulate DNApol replication on a singly primed M13 template because the helix-destabilizing activity of LEF-3 caused the primer to dissociate from the template. Escherichia coli SSB efficiently substituted for LEF-3 in the replication of a nicked template, suggesting that specific protein-protein interactions were not required for strand displacement in this assay.

An ecogeographic survey is required for the development of a strategy for the conservation and use of plant genetic resources. An herbarium-based ecogeographic survey is presented, supplemented by detailed field surveys of wildCoffeaspecies in the Mascarene Islands. The geographical and ecological distribution of the different Coffeaspecies in the Mascarene Islands, principally in Mauritius, is determined, and an assessment is made of the IUCN conservation status of native Coffeaspecies. Ecogeographic data were collated from herbarium specimens from four major herbaria holding collections of Mascarene Coffea. The geography, ecology, phenology and conservation status of the Coffeaspecies from Mauritius are discussed.

Alfalfa (Medicago sativaL.) is a common element of oasis agriculture in the Middle East; however, germplasm from this region has not been widely used in alfalfa breeding elsewhere. Previous research in Arizona showed that ecotypes from the southern portion of the Arabian Peninsula possess traits that may be useful in breeding very nondormant cultivars. The objectives of this research were to describe morphological and agronomic variation among 41 accessions from Oman, Yemen and southwestern Saudi Arabia, and eight accessions representing basic germplasm groups from elsewhere in the Arabian Peninsula and North Africa. Results of this research will be useful in preservation and utilization of these germplasm resources. Data collected from a 2‐yr field trial at Tucson, AZ, were evaluated by average linkage cluster and principal components analyses. Southern Arabian accessious collected below elevations of 1000 m were distinct from those collected at higher elevations. The most distinctive accessions were from low‐elevation oases in Yemen and were extremely susceptible to frost damage. Accessions from the Salalah Coastal Plain in Oman were especially variable, perhaps reflecting recent importation. Based largely on poor response to low winter temperatures, accessions from low elevations in Yemen were shown to be separable from those from Oman. Germplasm from the Batinah Coastal Plain and the Hajar Foothills in Oman could not generally be separated. The Saudi Arabian ecotype Hasawi was similar to germplasm from northern Oman. Utilization of affalfa germplasm from subtropical environments in southern Arabia may be complicated outside this region by its adaptation to intensive management under frost‐free conditions.

The 39K Autographa californica nuclear polyhedrosis virus (AcMNPV) gene is highly expressed throughout the virus life cycle and is controlled by tandem promoters that exhibit features of early and late baculovirus promoters. Late transcripts initiate at a conserved TAAG motif, while early transcripts are heterogeneous and initiate near a conserved CAGT motif. To define the nucleotide sequences that regulate early transcription of the 39K gene, a series of mutations was generated by substitution of 10-bp stretches in the 39K promoter with a BglII linker. The effects of these mutations on transcription from the early promoter were determined by transient expression and primer extension assays in the presence of the viral trans-activator IE1 gene. Mutations in the region from -15 to -44 revealed that early 39K transcription was controlled by dual TATA boxes. These TATA boxes are separated by 10 bp, which partially accounts for the heterogeneity in early 39K transcripts. Transcripts initiating at the CAGT motif (proximal transcripts) were abolished by deletion of the proximal TATA box located at -29 relative to CAGT. Proximal transcripts were not affected by alterations in the distal TATA motif located at -39 relative to the CAGT. Similarly, transcripts initiating upstream of CAGT (distal transcripts) were eliminated by mutations in the distal TATA but were unaffected by substitutions in the proximal TATA box. Proximal transcripts were not detected with a plasmid containing mutations in the CAGT motif, although the distal transcripts were unaffected by CAGT mutations. When the sequences surrounding the initiation site for the distal transcripts were altered, the start site was shifted one nucleotide, but transcription was not quantitatively affected. These results suggest that early 39K transcription is controlled by two distinct TATA elements, one that is dependent on an initiator and one in which the site of initiation is determined by the TATA element alone. Mutations in an upstream region from -45 to -68 relative to the CAGT motif had a quantitative effect but did not alter the heterogeneous pattern of early transcripts, suggesting these sequences function as an upstream regulatory region. Analysis of late transcription indicated that the TAAG element was essential, while transcription was unaffected by other mutations.

A cell-free protein synthesizing system was prepared from cells of Drosophila melanogaster line 1 and made mRNA dependent by treatment with micrococcal nuclease. The system was tested with homologous RNA from black beetle virus propagated in Drosophila cells, with Drosophila heat shock mRNA, and with various heterologous viral mRNA's. Under optimal conditions amino acid incorporation programmed with black beetle virus RNAs was 30-fold higher than endogenous incorporation. RNAs 1 and 2 primarily directed the synthesis of proteins with approximately molecular weights of 120,000 and 46,000, respectively. mRNA's, prepared by transcription from vesicular stomatitis virus or vaccinia virus, were translated efficiently and yielded products that comigrated with authentic viral proteins. Brome mosaic virus RNA and encephalomyocarditis virus RNA were translated poorly. The system retained full activity after freezing.

Gower's (1977) method for the canonical analysis of asymmetric matrices (CAA) where the same entities index both the rows and the columns is described. This technique displays the indexing entities in a series of orthogonal planes of decreasing importance in such a way as to preserve the pattern of the asymmetry between them. The analysis is applied to a half-diallel experiment and to some leaf-overtopping data, both from a study of the control of relative abundance of perennials in chalk grassland. The results suggest a common source for the asymmetry in the very different interference and overtopping data, namely that of leaf height. It is concluded that relative abundance in the field may be determined, in part, by leaf height through aboveground interference between plants.

Autographa californica nuclear polyhedrosis virus (AcMNPV) pp31 is a nuclear phosphoprotein that accumulates in the virogenic stroma, which is the viral replication center in the infected-cell nucleus, binds to DNA, and serves as a late expression factor. Considering that reversible phosphorylation could influence its functional properties, we examined phosphorylation and dephosphorylation of pp31 in detail. Our results showed that pp31 is posttranslationally phosphorylated by both cellular and virus-encoded or -induced kinases. Threonine phosphorylation of pp31 by the virus-specific kinase activity was sensitive to aphidicolin, indicating that it requires late viral gene expression. We also found that pp31 is dephosphorylated by a virus-encoded or -induced phosphatase(s), indicating that phosphorylation of pp31 is a dynamic process. Analysis of pp31 fusion proteins showed that pp31 contains at least three phosphorylation sites. The amino-terminal 100 amino acids of pp31 include at least one serine residue that is phosphorylated by a cellular kinase(s). The C-terminal 67 amino acids of pp31 include at least one threonine residue that is phosphorylated by the virus-specific kinase(s). Finally, this C-terminal domain of pp31 includes at least one serine that is phosphorylated by either a host or viral kinase(s). Interestingly, site-directed mutagenesis of the consensus threonine phosphorylation sites in the C-terminal domain of pp31 failed to prevent threonine phosphorylation, suggesting that the virus-specific kinase is unique and has an undetermined recognition site.

The rate of uptake of putrescine by Anacystis nidulans has been shown to depend on the external pH and the extracellular concentration of putrescine. Accumulation of exogenous putrescine was also proportional to the concentration of putrescine in the medium, suggesting that putrescine uptake was not subject to cellular regulation. An equation was derived to test the hypothesis that putrescine accumulation was due to ion trapping. Comparison of the predicted and observed intracellular concentrations of putrescine under various conditions showed a close correlation in support of the hypothesis of ion trapping. Under conditions leading to cell death (e.g., 150 microM putrescine, pH 9.8), the correlation did not hold as a result of leakage of accumulated putrescine.

The baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV, which is representative of the MNPV subtype in which the virions may contain many nucleocapsids within a single viral envelope) encodes a protein, v-ubi, that has 76% identity with the eukaryotic protein ubiquitin. Transcriptional mapping indicated that the gene for v-ubi was transcribed during the late phase of viral infection. Two transcriptional start sites potentially encoding v-ubi were identified. Both sites were contained within a sequence motif common to baculovirus late genes. A recombinant virus, AcUbi-beta Gal, encoding a ubiquitin-beta-galactosidase fusion protein was constructed to monitor the temporal regulation of v-ubi gene during viral infection. The fusion protein was expressed maximally at 14-18 hr postinfection, consistent with its classification as a late protein. The amount of ubiquitin-beta-galactosidase fusion protein that accumulated in AcUbi-beta Gal-infected cells by 48 hr postinfection was approximately 14% of the level of beta-galactosidase that was synthesized under control of the polyhedrin promoter. Transcriptional analysis confirmed that synthesis of the fusion protein was directed by the v-ubi gene promoter. AcUbi-beta Gal also produced normal levels of authentic viral ubiquitin message. Southern blot analysis of AcUbi-beta Gal and 15 additional isolates revealed that the fusion sequences had not recombined at the ubiquitin locus. A polyubiquitin gene was isolated and sequenced from Spodoptera frugiperda, a lepidopteran host cell line for AcMNPV. The predicted amino acid sequence of the product of the host gene is identical to animal ubiquitin.

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase alpha-primase that mediates this species specificity. To this end, we cloned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha-primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha-primase, suggesting that the primase heterodimer or one of its subunits controls host specificity. To determine whether both mouse primase subunits were required, recombinant hybrid polymerase alpha-primases containing only one mouse primase subunit, p48 or p58, together with three human subunits, were assayed for Py replication activity. Only the hybrid containing mouse p48 efficiently replicated Py DNA in depleted human cell extracts. Moreover, in a purified initiation assay containing Py T antigen, replication protein A (RP-A) and topoisomerase I, only the hybrid polymerase alpha-primase containing the mouse p48 subunit initiated primer synthesis on Py origin DNA. Together, these results indicate that the p48 subunit is primarily responsible for the species specificity of Py DNA replication in vitro. Specific physical association of Py T antigen with purified recombinant DNA polymerase alpha-primase, mouse DNA primase heterodimer, and mouse p48 suggested that direct interactions between Py T antigen and primase could play a role in species-specific initiation of Py replication.

The baculovirus Autographa californica nuclear polyhedrosis virus contains an element known as homologous region 5 (hr5) which is an enhancer of delayed-early viral gene expression. To begin to identify proteins that interact with hr5, DNA-protein interactions were analyzed by using extracts from Spodoptera frugiperda cells and a fragment of DNA containing the left half of the hr5 enhancer. This 252-bp DNA fragment contains two copies of a 30-bp direct repeat (DR30) and two copies of a 24-bp imperfect palindrome contained within a 60-bp direct repeat (DR60). Extracts prepared from normal S. frugiperda cells and cells transfected with pUC8 lacked enhancer-binding proteins. However, when gel shift assays were performed with extracts from cells transfected with a plasmid containing the viral trans-activator IE1 gene, two DNA-protein complexes were formed. Both DNA-protein complexes were specifically inhibited by competition with a 60-bp oligonucleotide corresponding to DR60 but not by competition with a different oligonucleotide corresponding to DR30. Formation of the two complexes did not appear to involve cooperative interactions between binding proteins. When DR60 was used as a probe, a single complex was formed. To measure the enhancer activity of DR60, a reporter plasmid was constructed that contained DR60 cloned upstream of the reporter chloramphenicol acetyltransferase gene under the control of the delayed-early 39K promoter. Transient expression analysis indicated that the oligonucleotide increased expression of this gene 300-fold over the level obtained in the absence of any enhancer sequences.

A review of the existing literature available on the bibliographic CD-ROM PlantGeneCD on the use of protein and molecular markers on plant material from sub-Saharan Africa was undertaken. The review showed that these molecular techniques have been used in a wide range of studies, from measurement of genetic diversity within single population to the elucidation of phylogenetic relationship among species in a genus or group of genera. The review revealed that 67% of the 146 studies examined used protein electrophoresis (58% isozymes, the rest mostly seed proteins), 20% used RFLPs, 8% RAPDs and 3% sequencing. Isozymes have been used for a whole range of applications, whereas electrophoresis of other proteins has mainly been used to study ecogeographic distribution of diversity in crops. RAPDs have been used particularly in the investigation of genetic relationship among accessions of a single species and phylogentic relationship among species. RFLPs of cpDNA have mostly been used at the interspecific level and nuclear markers at the intraspecific level. The studies covered most of the major crop species of Africa and their wild relatives, though important gaps remain, for example sweet potato. So-called “minor” crops were not well represented. Wild taxa not related to crops were also covered, in particular agroforestry and forest species.

The signing and ratification of the Convention on Biological Diversity has resulted in increased scientific and public interest in the conservation and utilisation of biological diversity. This has in turn triggered the need to develop more effective methodologies to conserve biological diversity for the benefit of all humanity. One of the first factors to be considered when conserving botanical diversity is the efficient and effective selection of the target taxa. The aim of this paper is not to set detailed priorities for the genetic conservation of the plant genetic resources of any particular crop or species complex, but to draw attention to the factors that should be considered when formulating priorities. The factors that are considered important when selecting plant genetic resource targets are: current conservation status; potential economic use; threat of genetic erosion; genetic distinctiveness; ecogeographic distribution; biological importance; cultural importance; cost, feasibility and sustainability; legislation; ethical and aesthetic considerations; and priorities of the conservation agency undertaking the conservation. Each of these factors is discussed in turn. Although it is not possible to provide a single methodology for the selection of plant genetic resource targets at this time, it is hoped that the consideration the factors discussed will make the selection of target taxa more object, make better use of the limited conservation resources and thus enhance the process of genetic conservation as a whole.

Putrescine is lethal to the cyanobacterium Anacystis nidulans at extracellular pH values at which significant concentrations of the nonprotonated diamine rapidly diffuse into the cell and accumulate as the charged form. Although over 98% of the accumulated putrescine is not metabolized, a small fraction is rendered trichloroacetic acid-insoluble, and about 90% of this is bound as putrescinie to proteins and cell structures. Various synthetic functions were studied in the presence of a bacteriostatic (40 microM) and a bacteriocidal (150 microM) concentration of putrescine at pH 9.5. Under lethal conditions, protein synthesis was completely inhibited after 45 min and CO2 fixation after 100 min, whereas nucleic acid synthesis was less affected. Spermidine was lost from the cell and its synthesis was arrested. These functions were much less inhibited at 40 microM putrescine. Ribosomes from putrescine-killed cells were found to be irreversibly dissociated into 30S and 50S subunits. Some putrescine (1-4 molecules) cosedimented with each subunit.

The transregulatory IE1 protein of Autographa californica nuclear polyhedrosis virus binds to the viral enhancer element hr5. To test whether IE1 binds independently of host cell factors, IE1 was translated in rabbit reticulocyte extracts and tested for DNA binding activity by an electrophoretic mobility shift assay. Complexes with the hr5 probe were detected with translation reaction mixtures primed with ie1 RNA but not with control translation reaction mixtures. However, the DNA-protein complexes formed with IE1 translated in vitro migrated more slowly than complexes formed with IE1 that was transiently expressed in insect cells. Phosphatase treatment of the translation reactions resulted in an increase in the mobility of the DNA-protein complexes, suggesting that hyperphosphorylation was responsible for the altered migration. To further verify that IE1 was capable of binding DNA in the absence of host cell factors, an N-terminal truncation of IE1 was synthesized in vitro, and shown to interact with hr5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IE1 translated in vitro revealed that the mobility of the protein was heterogeneous. This pattern was altered by translation in the presence of an oligonucleotide corresponding to the IE1 specific binding site but was not affected by translation in the presence of a nonspecific DNA. These results suggest that binding of IE1 to DNA causes a conformational change in the protein that alters the accessibility of IE1 to protein kinases.

The Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in the nuclei of infected cells and encodes several proteins required for viral DNA replication. As a first step in the functional characterization of viral replication proteins, we purified a single-stranded DNA-binding protein (SSB) from AcNPV-infected insect cells. Nuclear extracts were chromatographed on single-stranded DNA agarose columns. An abundant protein with an apparent molecular weight of 43,000 was eluted from the columns at 0.9 to 1.0 M NaCl. This protein was not evident in extracts prepared from control cells, suggesting that the SSB was encoded by the virus. SSB bound to single-stranded DNA in solution, and binding was nonspecific with respect to base sequence, as single-stranded vector DNA competed as efficiently as single-stranded DNA containing the AcNPV origin of DNA replication. Competition binding experiments indicated that SSB showed a preference for single-stranded DNA over double-stranded DNA. To determine whether SSB was encoded by the lef-3 gene of AcNPV, the lef-3 open reading frame was cloned under the control of the bacteriophage T7 promoter. Immunochemical analyses indicated that LEF-3 produced in bacteria or in rabbit reticulocyte lysates specifically reacted with antiserum produced by immunization with purified SSB. Immunoblot analyses of infected cell extracts revealed that SSB/LEF-3 was detected by 4 h postinfection and accumulated through 48 h postinfection.

An in vitro transcription system based on cytidine-free cassette was developed for the late 39k gene and the very late polyhedrin gene of Autographa californica nuclear polyhedrosis virus (AcNPV). Optimization of transcription conditions revealed that a preincubation step was not required for transcription of late and very late promoters, although preincubation was essential for efficient transcription from an early promoter. The 39k and polyhedrin constructs were actively transcribed by nuclear extracts prepared from AcNPV-infected Spodoptera frugiperda cells at 12 or 36 h postinfection but not by nuclear extracts prepared from uninfected or infected cells harvested during the early phase of infection. Transcription from the very late polyhedrin promoter was fivefold higher than that from the 39k late promoter with the nuclear extract prepared at 36 h postinfection. The 36-h extract was fractionated by phosphocellulose chromatography. Transcription activity eluted in two fractions, at 0.3 and 0.5 M KCl. Both the 39k and polyhedrin constructs were transcribed by these fractions; however, the patterns of late and very late transcription were distinctly different. With the 0.3 M fraction, incorporation into the 39k transcript was approximately 10-fold higher than incorporation into the polyhedrin transcript. Alternatively, with the 0.5 M fraction, transcription of the polyhedrin construct was twofold higher than transcription of the 39k construct. These results indicate that this in vitro system will be useful for purification and identification of factors that discriminate between late and very late promoters.

The baculovirus Autographa californica multicapsid nuclear polyhedrosis virus expresses two immediate-early genes from the HindIII-G region (map units 90.4 to 96.8) of the genome. During the early phase of infection, nonspliced 1.9-kb and spliced 2.1-kb transcripts are expressed which encode the IE1 and IE0 (spliced IE1) gene products, respectively. These two gene products differ only in that IE0 contains an additional 54 amino acids at the amino terminus. RNA analysis of these two genes during infection revealed that they were differentially expressed. IE1 was expressed early and late, whereas IE0 was expressed only early in infection. The regulation of these two immediate-early genes was analyzed by transient expression assays. The IE1 gene product stimulated expression of IE1 promoter-directed expression but down-regulated expression from the IE0 promoter. The IE0 gene product also transactivated the IE1 promoter but did not affect expression from its own promoter. Unlike IE1, which transactivates the delayed early 39K gene in the presence and absence of the homologous region (hr) enhancers, IE0 transactivated the 39K promoter only in the presence of cis-linked hr5 enhancer. The results of this study in conjunction with previous results suggest that the IE1 gene encodes a multifunctional gene product that may be involved in (i) repression of immediate-early gene expression, (ii) continued expression of its own gene product during infection, and (iii) transactivation of the delayed early and late classes of genes.

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.

The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1.

Autographa californica multicapsid nuclear polyhedrosis virus-infected insect cells express a viral immediate-early transcriptional regulatory protein, IE1, that has been shown by transient-expression assays to stimulate the expression of certain baculovirus delayed-early (DE) promoters and to inhibit the expression of other immediate-early (IE) genes. It is believed that certain DE promoters are activated, in part, by direct interactions between IE1 and enhancer elements located in regions adjacent to these genes. We have used transient cotransfection and DNA-binding assays to examine the function of mutant forms of IE1. Our results indirectly show that IE1 has at least two separable domains that are essential for its role in the modulation of baculovirus gene expression. A domain rich in acidic residues and essential for transactivation is located within the N-terminal 145 amino acids of the polypeptide. A second domain, located in the C-terminal 437 amino acids of IE1, is required for inhibitory and DNA-binding activities. Several nontransactivating IE1 mutants trans-dominantly interfered with wild-type IE1 transactivation of enhancer-linked DE genes. trans-dominant interference was expressed only by IE1 mutants that retained the N-terminal putative acidic activation domain, suggesting that this region may be involved in associations with a factor(s) essential for activation of enhancer-linked genes.

The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle.

Autographa californica nuclear polyhedrosis virus (AcMNPV) is a double-stranded DNA virus that expresses several immediate-early genes under the control of different promoters. The expression of one of these transcription units, IE-N, is shown here, by a transient expression assay, to be regulated by both cis- and trans-acting viral elements. The steady-state levels of IE-N mRNA were very abundant soon after infection but were nearly undetectable during the late phase of the viral life cycle. Analysis of the transient expression of a reporter construct driven by the IE-N promoter (IE-NCAT) was conducted to define viral elements which regulate IE-N gene expression. Viral enhancer hr1 and two immediate-early genes, IE-1 and IE-N, were shown to affect relative levels of reporter enzyme activity produced by IE-NCAT. The hr1 enhancer stimulated the expression of IE-NCAT, independent of orientation and position relative to the promoter and in the absence of any trans-acting viral factors. Regulation of IE-NCAT expression by the IE-1 and IE-N genes required less than 290 bp of promoter sequences upstream of the site of transcription initiation and was not dependent upon the hr1 enhancer. Coexpression of the IE-N gene had an autostimulatory effect upon IE-NCAT activity, whereas coexpression of the IE-1 gene reduced levels of reporter activity. The levels of reporter activity measured upon coexpression of either immediate-early gene with IE-NCAT linked to the hr1 enhancer appear to be the combined result of both cis- and trans-regulatory elements influencing expression from IE-NCAT. These results suggest that IE-N gene expression in baculovirus infection may be influenced by the concerted activity of three AcMNPV regulatory elements.

Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase alpha-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase alpha-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase alpha-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase alpha-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase alpha-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase alpha-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase alpha-primase had been purified. However, the human DNA polymerase alpha-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.

Several biochemical parameters, including that of polyamine content, accompanying the growth of the cyanobacterium Anacystis nidulans were studied. At all stages of growth under autotrophic conditions, the organisms were found to be rich in spermidine and lacking in spermine, as is typical of procaryotic organisms. The cells were quite low in putrescine, and no unusual polyamine was observed to be present as a major component. Conjugated polyamines were not detected in the cultures. At maximal culture density, the levels of spermidine, DNA, RNA, protein, and chlorophyll were also maximal. Shortly after the inception of the stationary phase, the spermidine content of the cells was the first parameter observed to decrease in cultures which were shortly to become yellow. Spermidine lost from the cells was not recovered in the medium in a free or conjugated form. This indication of degradation of spermidine was studied by the addition of polyamines to growing cultures. Exogenous spermidine and spermine were found to be metabolized rapidly by the organisms, of which diaminopropane was one product. Putrescine was found to be markedly toxic, whereas spermidine, some other triamines, and spermine were much less toxic.

Analyzing 25-hydroxyvitaminD (25OHD) serum levels and common carotid intima-media thickness (IMT) relationship, and evaluating the association of FokI and BsmI vitamin D receptor (VDR) gene polymorphisms with IMT in essential hypertensive patients were our study aims.

Our aims were to analyze the relationship between 25-hydroxyvitamin D (25OHD) plasma levels and clinical and ambulatory blood pressure (BP) values, and to identify any possible association between hypertension and FokI and BsmI vitamin D receptor (VDR) gene polymorphisms in essential hypertensive patients.