Capsid processing requirements for abrogation of Fv1 and Ref1 restriction

Murine leukemia virus is restricted in mouse cells lines by a host factor known as Fv1 and in human cell lines by Ref1. Genetic evidence indicates that these restriction factors target the virus capsid (CA) protein. Restriction can be overcome by adding virus at a high multiplicity of infection, indicating that the restriction factors can be saturated. Cells preexposed to restricted virus will allow infection by a second virus which would normally be restricted. This phenomenon is known as abrogation; it provides us with a tool with which to study the interaction of virus with restriction factors. We tested the abilities of several Gag processing mutants to abrogate restriction. Our results show that CA must be cleaved from both p12 and nucleocapsid in order for the incoming virion to interact with the restriction factor. Endogenous expression of properly processed CA, however, failed to abrogate restriction. These results suggest that as well as being processed, CA must also be properly assembled in the form of a condensed viral core in order to interact with Fv1 and Ref1. This polymeric structure may contain restriction factor binding sites not present in monomeric CA.     

Identification of the regions of Fv1 necessary for murine leukemia virus restriction

The Fv1 gene restricts murine leukemia virus replication via an interaction with the viral capsid protein. To study this interaction, a number of mutations, including a series of N-terminal and C-terminal deletions, internal deletions, and a number of single-amino-acid substitutions, were introduced into the n and b alleles of the Fv1 gene and the effects of these changes on virus restriction were measured. A significant fraction of the Fv1 protein was not required for restriction; however, retention of an intact major homology region as well as of domains toward the N and C termini was essential. Binding specificity appeared to be a combinatorial property of a number of residues within the C-terminal portion of Fv1.     

Retroviral capsid determinants of Fv1 NB and NR tropism

The specificity determinants for susceptibility to resistance by the Fv1 n and b alleles map to amino acid 110 of the murine leukemia virus CA protein. To study the interaction between Fv1 and CA, we examined changes in CA resulting in the loss of susceptibility to Fv1 resistance in naturally occurring NB- and NR-tropic viruses. A variety of amino acid changes affecting Fv1 tropism were identified, at CA positions 82, 92 to 95, 105, 114, and 117, and they all were mapped to the apparent exterior of virion-associated CA. These amino acids may form a binding surface for Fv1.     

Use of colicin-based genetic tools for studying bacterial protein transport

Transport of proteins across the envelope of Gram-negative bacteria is a very challenging domain of investigation, which involves membrane-embedded proteinaceous complexes at which specific targeting occurs. These transporters (translocon or secreton) have been studied both with genetics and biochemistry. In this review we report recent developments that should help to identify novel interactions that exist within these complexes, and to decipher the signals that specifically direct transported proteins to the cognate system. These developments are exclusively based on the re-routing of colicins to these molecular machineries. The re-routing induces a lethal situation in the case of efficient or inefficient transport, depending on the system, thus creating a genetic tool for selection of mutations that correct or generate a transport default.     

Use of restriction fragment length polymorphisms for genetic counseling: Population genetic considerations

Two-locus population genetic models are analyzed to evaluate the utility of restriction fragment length polymorphisms for purposes of genetic counseling. It is shown that the linkage disequilibrium between a neutral marker and a tightly linked overdominant mutant will increase rapidly as the mutant moves to its polymorphic equilibrium. The linkage disequilibrium decays for deleterious recessive mutants. Two measures involving the linkage disequilibrium are investigated to determine how much information the transmission of the neutral marker provides about the transmission of the selected gene. In certain kinds of matings, where the parental two-locus genotypes and linkage phases are known, it is possible to determine whether or not a progeny is homozygous for the selected gene on the basis of the fetal genotype at the marker locus. A quantity of primary interest is the fraction of matings between individuals heterozygous for the selected gene in which exact diagnosis can be made in this way. The expected proportion of such matings, taken over all two-locus matings involving heterozygotes at the selected locus, is calculated as a function of the gene frequencies at the two loci and the linkage disequilibrium between them. This expected value is maximized when the linkage disequilibrium is at its maximum in absolute value. Fewer than half of all matings are informative if the linkage disequilibrium is small in magnitude or if the gene frequencies at the two loci are quite different. Consideration is also given to various conditional measures of association that may be useful when the parental two-locus genotypes are unknown. The results suggest that the utility of tightly linked neutral marker genes in predicting the transmission of a selected gene is generally less when selection acts against a recessive gene than for overdominant selection.     

Characterization of an amino-terminal dimerization domain from retroviral restriction factor Fv1

The Fv1 protein is an endogenous factor in mice that confers resistance to infection by certain classes of murine leukemia virus, a phenomenon referred to as restriction. The mechanism of restriction is not understood, and the low endogenous level of Fv1 in cells has prevented any biochemical or biophysical analysis of the protein. We have now purified recombinant Fv1(n) protein from a baculovirus system and demonstrate that Fv1 exists in a multimeric form. Furthermore, we have mapped the position of two domains within the protein using limited proteolysis. Biophysical characterization of the N-terminal domain reveals that it comprises a highly helical and extended dimeric structure. Based on these biochemical and biophysical data, we propose a model for the arrangement of domains in Fv1 and suggest that dimerization of the N-terminal domain is necessary for Fv1 function to allow the protein to interact with multiple capsid protomers in retroviral cores.     

Use of transposons for studying the genetic organization of Vibrio cholerae non 01]

The nonagglutinating vibrions having Tn-elements inserted into the chromosome were obtained as a result of conjugal transfer of vector plasmids carrying the different transposons (Tn9, Tn10, Tn601, Tn5-Mob) and of the consequent isolation of plasmid-free clones of Vibrio cholerae non OI. Identification of auxotrophic mutations induced by the transposons inserted into the bacterial genome made possible the construction of the primary chromosomal map of Vibrio cholerae non OI. The efficient donor strains of Vibrio cholerae non OI were constructed by introducing the transposon Tn5-Mob and the helper plasmid RP-4. The donors are capable of oriented conjugal transfer of chromosome.     

Use of a transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation

Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250-300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat-shock (45 degrees C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.     

抗肝癌单链抗体的活性检测

将抗肝癌单克隆抗体 ＨＡｂ２５的轻、重链可变区基因通过Ｌｉｎｋｅｒ连接起来,构建成抗肝癌单链抗体ｓｃＦｖ２５,然后亚克隆入含 Ｅ－ｔａｇ检测标签的 ｐＥＴ１５ｂ表达载体进行融合表达。表达产物（ｓｃＦｖ２５－Ｅ－ｔａｇ）采用 ＳＡＢＣ法对肝癌细胞涂片进行染色,利用鼠抗Ｅ－ｔａｇ抗体间接检测初步纯化的ｓｃＦｖ２５的活性,同时采用竞争结合实验检测ｓｃＦｖ２５的亲和活性。ｓｃＦｖ２５能够与靶细胞特异性结合,阳性部位主要定位于细胞膜上;ｓｃＦｖ２５可封闭５３％的亲本抗体的亲和活性。ｓｃＦｖ２５较好地保持了亲本抗体的特异性和亲和活性。     

Genetic map of the region surrounding the retrovirus restriction locus,Fv1, on mouse Chromosome 4

The Friend virus susceptibility-1 (Fvl) gene maps to mouse Chromosome (Chr) 4 close to a cluster of four endogenous murine leukemia viruses (MLVs). To investigate the feasibility of cloning Fvl by a positional approach, we have performed an extensive genetic analysis of this region of Chr 4. We have typed 368 backeross mice for the four proviruses, Nppa, Lck, and D4Smh6b. Recombinant animals were screened in a hierarchical fashion with a variety of other markers, including Fvl and the isozyme marker Gpd1. A detailed genetic map of the region surrounding Fvl was derived. Three markers, Xmv9, Nppa, and Iap3rc11, were identified that showed no recombination with Fvl. By combining backcross and recombinant inbred strain data, we estimated that Xmv9 and Nppa must lie within 0.6 cM of one another and Fvl.     

Use of a cryoultramicrotomy method for the purpose of studying chlamydial ultrastructure]

The method of cryoultramicrotomy was adapted for the study of the ultrastructure of HeLa and McCoy cells in monolayer cultures infected with Chlamydia, obligatory intracellular procaryotic parasites, the causative agents of ornithosis (strain Loth) and paratrachoma (strain LB 1). The cryosections were obtained by the fixation of the monolayer with 2.5% glutaraldehyde, by the gradual infiltration of precipitated cells with sucrose (0.6--1.2--1.8--2.3 M) prior to freezing in liquid nitrogen, and by the treatment of sections with 1% aqueous methyl cellulose solution before drying. This method ensured good preservation of both Chlamydia, in intracytoplasmic inclusions and host cells, as well as regular reproducibility of the results. Ultrathin sections showed a considerable polymorphism in the vegetative forms of Chlamydia, which was probably due to the structure of their cell walls. Chlamydia, were found to form small vesicle-like structures in the cavities of inclusions. The cell walls and granules inside the elementary bodies of the causative agent of ornithosis were stained with the use of phosphotungstic acid--HCl, pH 0.5.     

A modified direct phosphate assay for studying ATPases

A simple, rapid assay for purified ATPases is presented, based upon the formation of phosphomolybdate and its extraction into butyl acetate. The inclusion of imidazole makes the assay more sensitive and reproducible apparently because of the formation of an imidazole-phosphomolybdate complex. Protein (100 micrograms), Hepes buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (0.1 M) and nucleotides (1 mM) were all shown to cause interference. The interference by nucleotides could be counteracted by using more molybdate. Butyl acetate was shown to extract virtually all of the phosphomolybdate almost instantaneously upon vortex mixing.     

Host specificity determinants as a genetic continuum

Host specificity is an important concept that underlies the interaction of all clinically and agriculturally relevant microbes with their hosts. Changes in the host specificity of animal pathogens, in particular, are often of greatest concern due to their immediate and unexpected impact on human health. Host switching or host jumps can often be traced to modification of key microbial pathogenicity factors that facilitate the formation of particular host associations. An increase in the number of genome-level studies has begun revealing that almost any type of change, from the simplest to the most complex, can potentially impact host specificity. This review highlights examples of host specificity determinants of viruses, bacteria and fungi, and presents them from within a genetic continuum that spans from the single residue through to entire genomic islands.