Genetic and structural analysis of a virulence determinant in polyomavirus VP1.

The LID strain of polyomavirus differs from other laboratory strains in causing a rapidly lethal infection of newborn C3H/Bi mice. This virulent behavior of LID was attenuated by dilution, yet at sublethal doses LID was able to induce tumors at a high frequency, like its parent virus PTA. By constructing and assaying LID-PTA recombinant viruses and by DNA sequencing, the determinant of virulence in LID was mapped to the major viral capsid protein, VP1. The VP1s of LID and PTA differed at two positions: at 185, LID has phenylalanine and PTA has tyrosine, and at 296, LID has alanine and PTA has valine. Results obtained with viruses constructed by site-directed mutagenesis showed that alanine at position 296 is sufficient to confer a fully virulent phenotype regardless of which amino acid is at position 185. However, with valine at position 296, an effect of phenylalanine at position 185 is apparent, as this virus possesses an intermediate level of virulence. A crystal structure of polyomavirus complexed with 3'-sialyl lactose previously indicated van der Waals contacts between the side chain of valine 296 and the sialic acid ring (T. Stehle, Y. Yan, T. L. Benjamin, and S. C. Harrison, Nature [London] 369:160-163, 1994). When this interaction was modeled with alanine, these contacts were greatly reduced. Direct confirmation that the substitutions in VP1 affected receptor binding was obtained by studying virus hemagglutination behavior. The ensemble of results are discussed in terms of the idea that a lower affinity of the virus for its receptor can result in more rapid spread and increased pathogenicity.     

Genotypic selection of simian immunodeficiency virus in macaque infants infected transplacentally.

To understand viral and host factors that contribute to transplacental transmission of human immunodeficiency virus, we developed an animal model using pregnant female macaques infected with simian immunodeficiency virus (SIV). Pregnant females were inoculated intravenously during midgestation with either a well-characterized primary isolate of SIV (SIV/DeltaB670) or a combination of SIV/DeltaB670 and the macrophage-tropic molecular clone SIV/17E-Fr. The viral genetic diversity in five infected female macaques and their in utero-infected infants was analyzed. All of the mothers harbored a genetically diverse virus population at parturition, whereas a single genotype from the maternal quasispecies was identified in the infants at birth. Only one of two variants was found in the infants: SIV/17E-Fr (two cases) or a genotype contained within the SIV/DeltaB670 quasispecies (three cases). The macrophage-tropic properties of both transmitted genotypes were suggested by productive replication in primary rhesus macrophage cultures in vitro and the clonal presence in central nervous system tissue of infected monkeys with encephalitis. These observations provide compelling evidence for both genotypic and phenotypic selection in transplacental transmission of SIV and suggest a critical role for macrophages in fetal infection in utero.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3 Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

Evidence that predator satiation may restrict the spatial spread of a tussock moth (Orgyia vetusta) outbreak

Western tussock moths (Orgyia vetusta Bdv., Lymantriidae) infest one stand of bush lupine (Lupinus arboreus Sims, Fabaceae) heavily and several other stands very lightly at the Bodega Marine Reserve (Sonoma Co., Calif., USA). We found that the disappearance rates of experimentally placed larvae and pupae were consistently lower in the outbreak area than in non-outbreak areas. For pupae but not larvae, this difference was removed by using tanglefoot to repel nonflying predators. However, the major nonflying predator of pupae, the ant Formica lasioides, was no more abundant in non-outbreak areas than in the outbreak area. We found inverse density-dependence in the rate of attack by F. lasioides on experimental pupae, suggesting this generalist predator is satiated within the outbreak area, but preys more heavily on the moth where the moth is sparse.     

Leaf anatomy and water loss of in vitro PEG-treated ‘Valiant’ grape

Polyethylene glycol was used to induce water stress of micropropagated Valiant grape. Reduced growth and slow rooting were observed in treated plantlets with 2, 4 and 6% polyethylene glycol as compared to control plantlets with no polyethylene glycol in the rooting medium. At high concentrations of 4 and 6%, leaves exhibited wilting and necrosis. At the 2% level, plantlets recovered and grew satisfactorily. Detached leaves of treated plantlets with 2% polyethylene glycol lost less water than controls when exposed to low humidity for 4 hours. Leaf anatomy of plantlets treated with 2% polyethylene glycol, control (in vitro plantlets) and greenhouse-grown plants were compared under light microscopy. Leaves from control plantlets contained larger mesophyll cells, lacked normal palisade layer formation, had greater intercellular pore spaces and fewer chloroplasts. Leaves from polyethylene glycol-treated plantlets, however, had smaller mesophyll cells, a more defined palisade layer, reduced intercellular pore space and the greatest number of chloroplasts. These results suggest that an osmoticum such as polyethylene glycol may be used to induce more normal leaf anatomy and reduced water loss in micropropagated Valiant grapes.Abbreviations BA 6-benzylaminopurine - FAA formalin-acetol-alcohol - MS Murashige & Skoog (1962) medium - MW molecular weight - NAA napthaleneacetic acid - PEG polyethylene glycol - TBA tertiary butyl alcohol     

A Genetic Analysis of the 63e-64a Genomic Region of Drosophila Melanogaster: Identification of Mutations in a Replication Factor C Subunit

We have performed and F(2) genetic screen to identify lethal mutations within the 63E-64A genomic region. We have isolated 122 mutations in 20 different complementation groups. Of these groups, 16 are represented by multiple alleles. We have also established that the Rop and Ras2 genes are located within the 63E-64A genomic domain at 64A10,11. We have sequenced 10.2 kb of DNA surrounding this gene pair and find that in addition to Rop and Ras2 there is another gene located within this DNA sequence. The gene product, which we have named Rfc40, shows 68% identity to the 40-kDa subunit of replication factor C. We find that the members of one complementation group (13 alleles) derived from our screen correspond to mutations in the Rop gene, whereas the members of another (five alleles) correspond to mutations in the Rfc40 gene. In addition we have isolated 11 new mutant alleles of the disembodied gene.     

Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation

Carotenoids are dietary antioxidants transported with plasma lipoproteins, primarily low-density lipoprotein (LDL). In this study in vitro methods were used to increase the amounts of specific, individual carotenoids in LDL. By addition of carotenoid to isolated LDL or to serum, followed by (re)isolation of the lipoproteins, samples of LDL were enriched 4- to 150-fold with lutein, 2- to 15-fold with lycopene, or 3- to 25-fold with β-carotene. Enrichment with specific carotenoids was achieved without affecting the electrophoretic mobility of the lipoprotein, its cholesterol to protein ratio, or the levels of other cartenoids or -tocopherol. The distributions among lipoproteins of carotenoid added to serum were similar, but not identical, to the distributions of the endogenous carotenoids. In particular, for added lutein, a greater proportion was found in HDL, and for added β-carotene, more was found in very low-density lipoprotein (VLDL). We then studied the effect of enriching LDL with specific carotenoids on its susceptibility to oxidation by copper ions. Lutein, β-cryptoxanthin, lycopene, and β-carotene, the four major plasma carotenoids, and -tocopherol were destroyed before the formation of lipid peroxidation products. The rates of destruction of the individual carotenoids differed; lycopene was destroyed most rapidly and lutein most slowly. Upon oxidation of β-carotene-enriched LDL, the rates of destruction of β-carotene, lycopene, and lutein were slowed and the lag times before the initiation of lipid peroxidation increased from 19 to 65 min. Neither effect was observed in LDL enriched with lutein or lycopene. Thus, β-carotene was unique among the carotenoids studied in having a small, but significant effect on LDL oxidation in vitro.     

In vitro acclimatization of date palm (Phoenix dactylifera L.) plantlets: A quantitative comparison of epicuticular leaf wax as a function of polyethylene glycol treatment

Wax deposits on leaf surfaces ofin vitro-grown plantlets,in vitro plantlets treated with polyethylene glycol and greenhouse-grown seedlings from five cultivars of date palm (Phoenix dactylifera L.) were extracted and quantified. Significant variations among treatments and cultivars were obtained. Greenhouse-grown plants had the greatest wax deposits followed by the acclimatized plantlets.In vitro plantlets had an average of 15% of the wax of greenhouse plants. Cultivar and plant age differences had a significant effect on the quantity of wax deposits. Greenhouse seedlings of Majhool, Deglet Noor and Khadraoui (cultivars grown under irrigation) had less wax accumulation than Zahidi and Sayer, dryland cultivars.The increase in wax deposition as a result of polyethylene glycol treatment, explains in part, the decreased water loss observed in acclimatized plantlets when transferredex vitro. Abbreviations EW epicuticular wax - NAA naphthalene acetic acid - PEG polyethylene glycol