A method for determining overall protein fold from NMR distance restraints

Summary We describe a simple method for determining the overall fold of a polypeptide chain from NOE-derived distance restraints. The method uses a reduced representation consisting of two particles per residue, and a force field containing pseudo-bond and pseudo-angle terms, an electrostatic term, but no van der Waals or hard shell repulsive terms. The method is fast and robust, requiring relatively few distance restraints to approximate the correct fold, and the correct mirror image is readily determined. The method is easily implemented using commercially available molecular modeling software.     

Use of disease progress curves to study the effects of the biocontrol agent Sporidesmium sclerotivorum on lettuce drop

At the end of the spring 1987 growing season, the mycoparasite Sporidesmium sclerotivorum was applied at 0, 0.2, 2 or 20 kg ha^‐1 to lettuce plants infected with Sclerotinia minor. Disease incidence was monitored in the same plots for five subsequent crops (three fall and two spring crops) without additional application of either pathogen or mycoparasite. Logistic growth curves were fitted to the data to describe disease progression over time for each inoculum level within each of the five crops. Within each crop, increasing the quantity of mycoparasite inoculum resulted in positive horizontal displacement of the curve with respect to time. As quantities of inoculum of S. sclerotivorum increased, inflection points of the disease progress curves increased at a decreasing rate. Thus, additional mycoparasite inoculum resulted in ever‐smaller increases in inflection point, and after a certain threshold level of mycoparasite inoculum (< 0.2 kg ha^‐1), increases in inflection point did not result in meaningful increases in harvestable lettuce. Maximum rates of disease increase were not different among the treatments within each crop, but were different between crops. Maximum rates of disease increase averaged 3.4, 3.4, 2.1, 3.6 and 1.5% day^‐1 for the fall 1987, spring 1988, fall 1988, spring 1989, and fall 1989, respectively. At all inoculum levels, the fall epidemics began later after planting than the spring epidemics.     

Cloning and sequence analyses of cDNAs encoding vasotocin and isotocin precursors of chum salmon,Oncorhynchus keta: evolutionary relationships of neurohypophysial hormone precursors

Summary The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.Abbreviations AVP vasopressin - C carboxyl - h human - IT isotocin - MT mesotocin - N amino - OXT oxytocin - S chum salmon - SDS sodium dodecyl sulfate - t toad - VT vasotocin     

Molecular cloning,characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.     

Aspects of the life history of Cercopithifilaria johnstoni (Nematoda:Filarioidea)

and 1988. Aspects of the life history of Cercopithifilaria johnstoni (Nematoda:Filarioidea). International Journal for Parasitology 18: 1087–1092. Cercopithifilaria johnstoni (Nematoda:Filarioidea) occurs in the subcutaneous connective tissues of a spectrum of native murid and marsupial hosts in Eastern Australia. Life cycle studies revealed that: (i) microfilaria occur in lymphatic capillaries and extravascular connective tissue of the dermis (= ‘skin-inhabiting’), (ii) ixodid ticks, particularly Ixodes trichosuri, are intermediate hosts in nature, (iii) development from microfilariae to infective third-stage larva occurs only while the tick is off the host, that is, during ecdysis from larva to nymph or from nymph to adult. Transmission of C. johnstoni in a wild population of bush rats (Rattus fuscipes) occurred in summer and winter, and was associated with peaks in the number of larval and/or nymphal stages of ticks on rats. C. johnstoni was transmitted experimentally to bandicoots (Isoodon macrourus, Perameles nasuta), bush rats and laboratory rats (R. norvegicus), indirectly by subcutaneous inoculation of third-stage larvae and directly by tick feeding. The prepatent period was approximately 3 months and the longest duration of microfilariae in the ‘ skin’ was more than 25 months. Dermal and ocular lesions were observed in R. fuscipes. The host-parasite relationship has the potential for development as an inexpensive and practical model for human onchocerciasis.     

The number of nucleotides required to determine the branching order of three species,with special reference to the human-chimpanzee-gorilla divergence

Summary A mathematical theory for computing the probabilities of various nucleotide configurations among related species is developed, and the probability of obtaining the correct tree (topology) from nucleotide sequence data is evaluated using models of evolutionary trees that are close to the tree of mitochondrial DNAs from human, chimpanzee, gorilla, orangutan, and gibbon. Special attention is given to the number of nucleotides required to resolve the branching order among the three most closely related organisms (human, chimpanzee, and gorilla). If the extent of DNA divergence is close to that obtained by Brown et al. for mitochondrial DNA and if sequence data are available only for the three most closely related organisms, the number of nucleotides (m^*) required to obtain the correct tree with a probability of 95% is about 4700. If sequence data for two outgroup species (orangutan and gibbon) are available, m^* becomes about 2600–2700 when the transformed distance, distance-Wagner, maximum parsimony, or compatibility method is used. In the unweighted pair-group method, m^* is not affected by the availability of data from outgroup species. When these five different tree-making methods, as well as Fitch and Margoliash's method, are applied to the mitochondrial DNA data (1834 bp) obtained by Brown et al. and by Hixson and Brown, they all give the same phylogenetic tree, in which human and chimpanzee are most closely related. However, the trees considered here are gene trees, and to obtain the correct species tree, sequence data for several independent loci must be used.     

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

Summary The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.Journal article no. 1223 of the New Mexico Agricultural Experiment Station     

雄激素对大鼠储精囊分泌蛋白mRNA的调节作用

本文报道了应用DNA重组和分子克隆技术研究雄激素与大鼠储精囊分泌蛋白基因的相互作用。大鼠储精囊总mRNA在逆转录酶作用下合成dsc-DNA,并克隆于pBR322/X1776中。经原位杂交法筛选含有互补于雄激素调节的mRNA的三个克隆株,其中二株经信使选择杂交翻译法证实它们是编码54K和16.6K道尔顿的分泌蛋白质的基因。同时应用后者的cDNA作为探针进一步研究雄激素对处于不同生理态状下的大鼠储精囊mRNA水平的影响。     

Trypanosoma brucei: analysis of relapsing populations in sensitive and resistant breeds of cattle

The clone DiTat 1.1 of Trypanosoma brucei brucei was injected into four bovids, and clones obtained from successive waves of parasitemia were used to study the expressed variant-specific surface glycoprotein repertoire. Twenty-four clones were obtained which could be classified into 12 different variable antigen types, in addition to the clone injected, using agglutination or immunofluorescence with monospecific antisera. The variable surface glycoproteins of the 25 clones were extracted using the detergent octyl-beta-D-glucopyranoside in the presence of the protease inhibitor, N-cbz-L-phenylalaninechloromethylketone. The molecular weights varied from 52,000 to 69,000 and the pI from 5.0 to 8.8. The virulence of 14 clones representing 13 variable antigen types was ascertained in mice. The mean survival time ranged from 20.5 to 43.0 days. Clones isolated from early peaks of parasitemia in the bovid were the most virulent while clones derived from later peaks were less virulent. It seems that organisms of diminishing virulence appear in bovids, leading to self-cure of the disease. All clones were sensitive to human serum in a blood infectivity inhibition test. Antibody against all virulent clones appeared in 20 cattle (10 Zebus, 10 Baoulés) which had been injected with T. brucei DiTat 1.1. There was no evidence for parasites of high or low virulence being preferentially expressed in resistant or sensitive hosts.