Co-translational trimerization of the reovirus cell attachment protein.

The reovirus cell attachment protein, sigma1, is a trimer with a 'lollipop' structure. Recent findings indicate that the N-terminal fibrous tail and the C-terminal globular head each possess a distinct trimerization domain. The region responsible for N-terminal trimerization (formation of a triple alpha-helical coiled-coil) is located at the N-terminal one-third of sigma1. In this study, we investigated the temporality and ATP requirement of this trimerization event in the context of sigma1 biogenesis. In vitro co-synthesis of the full-length (FL) and a C-terminally truncated (d44) sigma1 protein revealed a preference for homotrimer over heterotrimer formation, suggesting that assembly at the N-terminus occurs co-translationally. This was corroborated by the observation that polysome-associated sigma1 chains were trimeric as well as monomeric. Truncated proteins (d234 and d294) with C-terminal deletions exceeding half the length of sigma1 were found to trimerize post-translationally. This trimerization did not require ATP since it proceeded normally in the presence of apyrase. In contrast, formation of stable FL sigma1 trimers was inhibited by apyrase treatment. Collectively, our data suggest that assembly of nascent sigma1 chains at the N-terminus is intrinsically ATP independent, and occurs co-translationally when the ribosomes have traversed past the midpoint of the mRNA.     

DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV-crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40-infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell-cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.     

Influence of compaction from wheel traffic and tillage on arbuscular mycorrhizae infection and nutrient uptake by Zea mays

Interactive effects of seven years of compaction due to wheel traffic and tillage on root density, formation of arbuscular mycorrhizae, above-ground biomass, nutrient uptake and yield of corn (Zea mays L.) were measured on a coastal plain soil in eastern Alabama, USA. Tillage and soil compaction treatments initiated in 1987 were: 1) soil compaction from tractor traffic with conventional tillage (C,CT), 2) no soil compaction from tractor traffic with conventional tillage (NC,CT), 3) soil compaction from tractor traffic with no-tillage (C,NT), and, 4) no soil compaction from tractor traffic with no-tillage (NC,NT). The study was arranged as a split plot design with compaction from wheel traffic as main plots and tillage as subplots. The experiment had four replications. In May (49 days after planting) and June, (79 days after planting), root biomass and root biomass infected with arbuscular mycorrhizae was higher in treatments that received the NC,NT treatment than the other three treatments. In June and July (109 days after planting), corn plants that received C,CT treatment had less above-ground biomass, root biomass and root biomass infected with mycorrhizae than the other three treatments. Within compacted treatments, plants that received no-tillage had greater root biomass and root biomass infected with mycorrhizae in May and June than plants that received conventional tillage. Corn plants in no-tillage treatments had higher root biomass and root biomass infected with mycorrhizae than those in conventional tillage. After 7 years of treatment on a sandy Southeastern soil, the interactive effects of tillage and compaction from wheel traffic reduced root biomass and root biomass infected with mycorrhizae but did not affect plant nutrient concentration and yield. ei]J H Graham     

Cell-Cell Recognition during Fertilization in a Red Alga, Antithamnion sparsum (Ceramiaceae, Rhodophyta)

A gamete recognition mechanism in Antithamnion sparsum Tokidais proposed based on experiments using various lectins and carbohydrates.Spermatial binding to trichogynes is inhibited by pre-incubationof spermatia with concanavalin A (ConA) and/or L-fucose, whiletrichogyne receptors are blocked by the complementary carbohydrate-methyl D-mannose and/or the lectin Ulex europaeus agglutinin(UeA1). Binding inhibition (40–50%) was observed with10–50 mM carbohydrates and 25–50 µg ml^-1 lectins.The inhibitory effects of ConA and UeA1 is partially reversed(to 80–90% of controls) by addition of -methyl D-mannoseand L-fucose, respectively. Lectin binding to spermatial surfaceswas visualized by Fluorescein isothiocyanate (FITC) conjugatedConA, whereas carbohydrate receptors along the trichogyne andspermatium were localized with -mannosylated-FITC-albumin andL-fucosylated-FITC-albumin, respectively. These results suggestthat gamete recognition in Antithamnion sparsum is mediatedby a double-docking recognition system consisting of spermatiapossessing surface L-fucose receptors and -methyl D-mannosemoiety, and trichogynes possessing the complementary receptors. (Received December 5, 1995; Accepted April 22, 1996)     

Adenylosuccinate synthetase: site of action of hydantocidin, a microbial phytotoxin.

The site of action of hydantocidin was probed using Arabidopsis thaliana plants growing on agar plates. Herbicidal effects were reversed when the agar medium was supplemented with AMP, but not IMP or GMP, suggesting that hydantocidin blocked the two-step conversion of IMP to AMP in the de novo purine biosynthesis pathway. Hydantocidin itself did not inhibit adenylosuccinate synthetase or adenylosuccinate lyase isolated from Zea mays. However, a phosphorylated derivative of hydantocidin, N-acetyl-5'-phosphohydantocidin, was a potent inhibitor of the synthetase but not of the lyase. These results identify the site of action of hydantocidin and establish adenylosuccinate synthetase as an herbicide target of commercial potential.     

Diauxic Growth in Rice Suspension Cells Grown on Mixed Carbon Sources of Acetate and Glucose

Diauxic growth was observed in rice (Oryza sativa L.) suspension cells growing on acetate (10 mM) and glucose (10 mM). Cells used acetate during the first growth phase and the acetate level in the medium was rapidly decreased, whereas the level of glucose remained essentially unchanged. After acetate was depleted from the medium, cells started to use glucose, forming the second growth phase. It appears that uptake of [14C]glucose was repressed during the first growth phase and became active during the second growth phase. In contrast, uptake of [14C]acetate occurred actively throughout the diauxic growth. By further demonstrating the specific induction of isocitrate lyase (EC 4.1.3.1), a glyoxylate cycle enzyme, and hexokinase (EC 2.7.1.1), a glycolysis enzyme, during the first and second growth phases, respectively, it was clearly shown that rice cells use acetate first and do not use both carbon sources simultaneously. This kind of diauxic growth pattern has been observed in bacteria. To our knowledege, this study is the first report demonstrating the presence of diauxic growth in plant cells.     

C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells

Summary The potential of tobacco BY-2 suspension-cultured cells for examining in vivo targeting and import of proteins into plant peroxisomes was shown recently in our laboratory. In the current study, the necessity and sufficiency of putative C-terminal targeting signals on cottonseed malate synthase and bacterial chloramphenicol acetyl-transferase (CAT) were examined in BY-2 cells. Cotton suspension cells also were evaluated as another in vivo peroxisome targeting system. Ultrastructural views of BY-2 cells showed that the peroxisomes were relatively small (0.1-0.3 m diameter), a characteristic of so-called unspecialized peroxisomes, Peroxisomes in cotton and tobacco cells were identified with anti-cottonseed catalase IgGs as distinct immunofluorescent particles clearly distinguishable from abundant immunofluorescent mitochondria and plastids, marked with antibodies to -ATPase and stearoyl-ACP 9 desaturase, respectively. The C-terminal ser-lys-leu (SKL) motif is a well-established peroxisome targeting signal (PTS 1) for mammals and yeasts, but not for plants. Antiserum raised against SKL peptides recognized proteins only in peroxisomes in cotton and tobacco cells. The necessity of SKL-COOH for targeting of proteins to plant peroxisomes had not been demonstrated; we showed that SKL-COOH was necessary for directing cottonseed malate synthase to BY-2 peroxisomes. KSRM-COOH, a conservative modification of SKL-COOH, was shown by others to be sufficient for redirecting CAT in stably-transformed Arabidopsis plants to the leaf peroxisomes. Here we show with the same CAT constructs (e.g., pMON316CAT-KSRM) that KSRM is sufficient for targeting transiently-expressed passenger proteins to unspecialized BY-2 peroxisomes. These results provide new direct evidence for the necessity of SKL-COOH (a type 1 PTS) and sufficiency of a conservative modification of the PTS 1 (KSRM-COOH) for in vivo, heterologous targeting of proteins to plant peroxisomes.Abbreviations CAT chloramphenicol acetyltransferase - CHO cells Chinese hamster ovary cells - DAB 3,3-diaminobenzidine - GUS -glucuronidase - ICL isocitrate lyase - KSRM lysine-serine-arginine-methionine - MS malate synthase - PBS phosphate-buffered saline - PTS peroxisome targeting signal - SKL serine-lysine-leucine - tobacco BY-2 Bright Yellow-2 Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Identification of quantitative trait loci for plant height, lodging, and maturity in a soybean population segregating for growth habit

The use of molecular markers to identify quantitative trait loci (QTLs) has the potential to enhance the efficiency of trait selection in plant breeding. The purpose of the present study was to identify additional QTLs for plant height, lodging, and maturity in a soybean, Glycine max (L.) Merr., population segregating for growth habit. In this study, 153 restriction fragment length polymorphisms (RFLP) and one morphological marker (Dt1) were used to identify QTLs associated with plant height, lodging, and maturity in 111 F₂-derived lines from a cross of PI 97100 and Coker 237. The F₂-derived lines and two parents were grown at Athens, Ga., and Blackville, S.C., in 1994 and evaluated for phenotypic traits. The genetic linkage map of these 143 loci covered about 1600 cM and converged into 23 linkage groups. Eleven markers remained unlinked. Using interval-mapping analysis for linked markers and single-factor analysis of variance (ANOVA), loci were tested for association with phenotypic data taken at each location as well as mean values over the two locations. In the combined analysis over locations, the major locus associated with plant height was identified as Dt1 on linkage group (LG) L. The Dt1 locus was also associated with lodging. This locus explained 67.7% of the total variation for plant height, and 56.4% for lodging. In addition, two QTLs for plant height (K007 on LG H and A516b on LG N) and one QTL for lodging (cr517 on LG J) were identified. For maturity, two independent QTLs were identified in intervals between R051 and N100, and between B032 and CpTI, on LG K. These QTLs explained 31.2% and 26.2% of the total variation for maturity, respectively. The same QTLs were identified for all traits at each location. This consistency of QTLs may be related to a few QTLs with large effects conditioning plant height, lodging, and maturity in this population.