Defensin gene family in <Emphasis Type="Italic">Medicago truncatula:</Emphasis> structure,expression and induction by signal molecules

A large gene family encoding the putative cysteine-rich defensins was discovered in Medicago truncatula. Sixteen members of the family were identified by screening a cloned seed defensin from M. sativa (Gao et al. 2000) against the Institute for Genomic Research’s (TIGR) M. truncatula gene index (MtGI version 7). Based on the comparison of their amino acid sequences, M. truncatula defensins fell arbitrarily into three classes displaying extensive sequence divergence outside of the eight canonical cysteine residues. The presence of Class II defensins is reported for the first time in a legume plant. In silico as well as Northern blot and RT-PCR analyses indicated these genes were expressed in a variety of tissues including leaves, flowers, developing pods, mature seed and roots. The expression of these genes was differentially induced in response to a variety of biotic and abiotic stimuli. For the first time, a defensin gene (TC77480) was shown to be induced in roots in response to infection by the mycorrhizal fungus, Glomus versiforme. Northern blot analysis indicated that the tissue-specific expression patterns of the cloned Def1 and Def2 genes differed substantially between M. truncatula and M. sativa. Furthermore, the induction profiles of the Def1 and Def2 genes in response to the signaling molecules methyl jasmonate, ethylene and salicylic acid differed markedly between these two legumes.     

Characterization of antigenic proteins from Tritrichomonas foetus recognized by antibodies in rabbit serum, bovine serum and bovine cervicovaginal mucus

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract and is recognized as 1 of the more common infectious agents causing decreased reproductive efficiency in beef cattle. Infections result in reproductive failure and produce considerable economic loss. Vaccination of heifers with vaccines containing T. foetus induces elevated serological responses to many T. foetus antigens, decreases the rate and/or length of infection with T. foetus, and decreases fetal loss caused by infection. Because T. foetus infections are usually limited to lumen and mucosal surfaces of the reproductive tract, it has been assumed that protection from infection and abortion is partially mediated by immunoglobulins in the uterus and vagina. The objective of this study was to identify and characterize specific antigens of T. foetus that show promise for use in a recombinant vaccine that will generate a protective mucosal immune response in cattle. Surface proteins were identified by using polyclonal rabbit anti-trichomonal sera eluted from paraformaldehyde-fixed cells. Analyses of these proteins, utilizing mucosal antibodies from vaccinated and convalescent cows, have identified proteins involved in generating a local immune response. Western immunoblot analysis indicates that these proteins are well conserved and are excellent candidates for incorporation into a recombinant vaccine.     

Investigations of the incidence of bovine trichomoniasis in nevada and of the efficacy of immunizing cattle with vaccines containing Tritrichomonas foetus

Trichomonas cultures taken from 2389 bulls showed that approximately 4.7% of them were infected. Correlation of these data with the ranches from which diagnostic samples were obtained indicated that in the period of 1984 through 1987 26.7 to 44.1% of ranches had at least one infected bull. Thirty-four 18-month-old Holstein heifers were assigned to one of three groups, controls n = 12 animals, soluble vaccine n = 11 animals, and whole vaccine n = 11 animals to determine the effect of Tritrichomonas foetus vaccines on the reproductive performance of T . foetus infected animals. Heifers were bred with T . foetus infected bulls beginning two weeks after the second T . foetus vaccination. All immunized animals developed antibody titers of at least 1:1000 following vaccination. In addition, all control and immunized animals became infected with T . foetus . However, the duration of infection was approximately two weeks shorter in immunized animals. Approximately 42% (5 of 12) of control heifers remained infected with T . foetus for the duration of the experiment, while only 18% (2 of 11) of each of the vaccine groups remained infected for the duration of the experiment. Finally, 27% (3 of 11) of heifers in each of the vaccine groups were pregnant at slaughter, while none of the control heifers were pregnant at slaughter. Therefore, both vaccine formulations appeared to protect heifers (P<0.05) from fetal loss due to trichomoniasis.     

Variation amongEucalyptus species in resistance to eucalyptus longhorned borer in Southern California

Eucalyptus L'Héritier (Myrtaceae: Leptospermoideae) species are native to the Austro-Malaysian region, but have been widely planted in temperate and subtropical regions around the world. In most regions whereEucalyptus have been imported, the Eucalyptus Longhorned Borer (Phoracantha semipunctata F.) (Coleoptera: Cerambycidae) has been accidently introduced. Larvae of the beetle bore through the bark and mine along the cambium of stressed trees, usually killing their host. We report here the relative susceptibilities of 12Eucalyptus species in two mixed-species plantations in California, USA. These trees were stressed by water deficit resulting from a prolonged drought.Eucalyptus species that appeared resistant to the borer includedE. camaldulensis Dehnhardt,E. cladocalyx F. Muller,e. sideroxylon A. Cunn. ex Woolls, andE. trabutii (anE. camaldulensis hybrid). Species that were more susceptible to attack wereE. diversicolor F. Mueller,E. globulus LaBillardière,E. grandis Hill ex Maiden,E. nitens (Deane & Maiden),E. saligna Sm., andE. viminalis LaBillardière. Survival of trees was influenced by fine-scale moisture variation resulting from slope and irrigation effects. Resistance characteristics of theseEucalyptus species did not correlate with taxonomic relatedness or bark characteristics, but did correspond to drought tolerance traits in their native habitat.Eucalyptus species that were resistant to attack byP. semipunctata were those that are most tolerant of drought in Australia.     

CONTROL OF TREHALASE SYNTHESIS IN NEUROSPORA CRASSA

When an aconidial strain (STL6A) of Neurospora crassa is grown on carbon sources such as glucose, maltose, sucrose, etc., trehalase activity per unit weight of mycelium is very low. By contrast, media containing arabinose, glutamic acid, glycine, etc., which support growth only poorly, produce mycelium with very high trehalase activity. Retarding growth limiting the supply of a necessary nutrient, altering the pH and temperature, or adding toxic substances, however, does not derepress trehalase activity. Repression and derepression of trehalase was found to be reversible through the transfer of cultures to appropriate media. It is likely that the increase in trehalase activity results from de novo synthesis because labeled enzyme can be isolated from acrylamide gels after isolation from medium containing C¹⁴-labeled leucine and after purification by other means. These experiments are interpreted in terms of catabolite repression which may be correlated with events during growth and conidiation.     

Assembly of High-Resolution Restriction Maps Based on Multiple Complete Digests of a Redundant Set of Overlapping Clones

An approach to restriction-site mapping and contig building that uses fragment-size data from multiple complete digests of a set of clones that oversample a genomic region is presented. Maps containing both fragment-length data and clone-end data are maintained for each restriction enzyme. Synchroniza- tion between the maps for the different enzymes is achieved by requiring the clone-end maps for all enzymes to be compatible. Basic concepts that under- lie multiple-complete-digest mapping—including the match/merge approach to map incorporation, extension vs assimilation, ambiguity, and clone-end compatibility—are presented. An initial application of multiple-complete-digest mapping to real data on a set of cosmid clones suggests that this mapping method has exceptional power to produce accurate maps that are well suited to the needs of large-scale DNA-sequencing projects.     

THE USE OF M.99 FOR THE IMMOBILISATION OF THE DEFASSA WATERBUCK, (KOBUS DEFASSA PENRICEI)

Waterbuck were darted with the new "Hypodart" fired from the crossbow, and with the "Cap-Chur" gun.
Eleven waterbuck were successfully immobilised with M.99 alone. The remaining 37 received M.99 in conjunction with an ataractic, acetylpromazine maleate. Hyoscine hydrobromide, a parasympatholytic drug, never formed part of the immobilising dose and was given to only five of the immobilised animals. Not only is this drug unnecessary; its use is a positive disadvantage and was responsible for the death of one waterbuck. Three other waterbuck died, two of these probably from heat stroke, a consequence of using acepromzine on a hot day. The remaining animal damaged a leg when trying to get up and was killed by lion five days later.
This paper gives justification for combining M.99 and acepromazine in the approximate ratio of 1:1 for the immobilisation of this species, and suggests that the losses experienced by other workers through torticollis may well have been the result of the acepromazine.     

P69-T Global Analysis of Phosphotyrosine Sites in Src-Transformed Cells: Improved Methods for Sample Preparation and Analysis

Interest in tyrosine phosphorylation patterns has been sparked since increases in phosphotyrosine have been associated with malignancy and tumor formation. We have employed several methods to investigate the phosphotyrosine profile of Src-transformed cells, including an extensive study using the methods published by Rush et al.¹ In order to streamline the Rush et al. protocol, we have generated peptides by running the lysate approximately 1.5 cm into a 10% bis-tris polyacrylamide gel (with reduction and alkylation prior to the gel) followed by in-gel digestion. Phosphotyrosine-containing peptides from tryptic digestions were enriched using either the pY100 antibody or the 4G10 antibody. Phosphotyrosine-containing peptides were eluted off the beads as described by the Rush et al. protocol or with the addition of an acetonitrile elution of the beads. Samples were analyzed on a Thermo LTQ by reverse-phase chromatography using data-dependent analysis. The results were searched using the Sequest algorithm and the mouse subset of the Uniref100 database with concatenated reverse database searching for false-positive estimation. Results were filtered in a two-step process based on multiple Sequest scores, with increased stringency for novel sites. Results from multiple biological replicates were compared with those that we obtained using the Rush et al. protocol, and they produced comparable results. Peptides accepted from multiple replicates resulted in a false-positive rate of less than 3%. The addition of an acetonitrile wash increased the elution of peptides from the beads and increased identifications. This procedure saved 1–2 d for sample preparation with equivalent or improved results. We are currently exploring digestion of whole-cell lysates in trifluoroethanol to further streamline this protocol, and the use of the LTQ-Orbitrap to more confidently identify the sites.