Lethal Transposition of Mud Phages in Rec(-) Strains of Salmonella Typhimurium

Under several circumstances, the frequency with which Mud prophages form lysogens is apparently reduced in rec strains of Salmonella typhimurium. Lysogen formation by a MudI genome (37 kb) injected by a Mu virion is unaffected by a host rec mutation. However when the same MudI phage is injected by a phage P22 virion, lysogeny is reduced in a recA or recB mutant host. A host rec mutation reduces the lysogenization of mini-Mu phages injected by either Mu or P22 virions. When lysogen frequency is reduced by a host rec mutation, the surviving lysogens show an increased probability of carrying a deletion adjacent to the Mud insertion site. We propose that the rec effects seen are due to a failure of conservative Mu transposition. Replicative Mud transposition from a linear fragment causes a break in the host chromosome with a Mu prophage at both broken ends. These breaks are lethal unless repaired; repair can be achieved by Rec functions acting on the repeated Mu sequences or by secondary transposition events. In a normal Mu infection, the initial transposition from the injected fragment is conservative and does not break the chromosome. To account for the conditions under which rec effects are seen, we propose that conservative transposition of Mu depends on a protein that must be injected with the DNA. This protein can be injected by Mu but not by P22 virions. Injection or function of the protein may depend on its association with a particular Mu DNA sequence that is present and properly positioned in Mu capsids containing full-sized Mu or MudI genomes; this sequence may be lacking or abnormally positioned in the mini-Mud phages tested.     

Use of Phaseolus vulgaris leukoagglutinating lectin in histochemical and blotting techniques: a comparison of digoxigenin- and biotin-labelled lectins

An increase in the number of 1,6 branches of the trimannosyl core of asparagine-linked oligosaccharides has been shown to be directly correlated with the metastatic potential of cultured tumour cells. The Phaseolus vulgaris leukoagglutinating lectin (PHA-L) binds to 1,6 branches of tri- and tetra-antennary oligosaccharides. We have applied digoxigenin- and biotin-conjugated PHA-L to establish a non-radioactive detection system for 1,6 branches, which can be used in lectin blotting as well as light and electron microscopic cytochemistry. For this purpose the HCT116 human colon carcinoma cell line and colon carcinoma tissue were investigated. Digoxigenin-conjugated PHA-L in conjunction with alkaline phosphatase-conjugated anti-digoxigenin antibodies was superior to biotin-conjugated PHA-L in lectin blotting with respect to sensitivity and specificity. Similarly, the digoxigenin conjugated PHA-L in conjunction with gold-labelled anti-digoxigenin antibodies resulted in more intense specific staining and lower background compared to biotin-conjugated PHA-L visualized with a streptavidin immunogold complex. The specificity of lectin binding in blotting and cytochemical studies was demonstrated by the absence of staining when the lectin was omitted or preabsorbed with glycoprotein, and following pretreatment of the cellular homogenates or tissue sections by N-glycosidase F. Our results demonstrate that digoxigenin-conjugated PHA-L provides high sensitivity and specificity for histochemical and blotting techniques and is amenable for quantification. The technique should have applications in tumour research.     

Basis for selective incorporation of glycoproteins into the influenza virus envelope.

The ability of mutant or chimeric A/Japan hemagglutinins (HAs) to compete for space in the envelope of A/WSN influenza viruses was investigated with monkey kidney fibroblasts that were infected with recombinant simian virus 40 vectors expressing the Japan proteins and superinfected with A/WSN influenza virus. Wild-type Japan HA assembled into virions as well as WSN HA did. Japan HA lacking its cytoplasmic sequences, HAtail-, was incorporated into influenza virions at half the efficiency of wild-type Japan HA. Chimeric HAs containing the 11 cytoplasmic amino acids of the herpes simplex virus type 1gC glycoprotein or the 29 cytoplasmic amino acids of the vesicular stomatitis virus G protein were incorporated into virions at less than 1% the efficiency of HAtail-. Thus, the cytoplasmic domain of HA was not required for the selection process; however, foreign cytoplasmic sequences, even short ones, were excluded. A chimeric HA having the gC transmembrane domain and the HA cytoplasmic domain (HgCH) was incorporated at 4% the efficiency of HAtail-. When expressed from simian virus 40 recombinants in this system, vesicular stomatitis virus G protein with or without (Gtail-) its cytoplasmic domain was essentially excluded from influenza virions. Taken together, these data indicate that the HA transmembrane domain is required for incorporation of HA into influenza virions. The slightly more efficient incorporation of HgCH than G or Gtail- could indicate that the region important for assembling HA into virions extends into part of the cytoplasmic domain.     

Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration.

Retroviral integrases mediate site-specific endonuclease and transesterification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition processes is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Moloney murine leukemia virus (M-MuLV) integrase (IN). Modifications in the target DNA portion of the disintegration substrate decreased enzymatic activity, while substitution of the highly conserved CA in the viral long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleimide (NEM) modification of the cysteine residues of M-MuLV IN as well as by mutations. Both integration activities, 3' processing, and strand transfer, were completely inhibited by NEM modification of M-MuLV IN, while disintegration activity was only partially sensitive. However, structural analogs of the disintegration substrates that were modified in the target DNA and had the conserved CA removed were not active with NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys region of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest that the domains of M-MuLV IN that are required for the forward integration reaction substrate differ from those required for the reverse disintegration reaction substrate.     

Distribution of NCAM-180 and polysialic acid in the developing tectum mesencephali of the frog Discoglossus pictus and the salamander Pleurodeles waltl

The 180 kDa component of the neural cell adhesion molecule (NCAM-180), total NCAM (NCAM-total) and the polysialic acid modification of NCAM (PSA) show similar temporal and spatial regulation in the developing tecta of Pleurodeles waltl (salamander) and Discoglossus pictus (frog). Whereas NCAM-total is found throughout the tectal tissue on neurons and glia, NCAM-180 is only found on nonproliferating neurons and in fiber layers. PSA is expressed by a subset of NCAM-180-positive cells. Western blots show that there is little polysialylated NCAM-140 in the developing amphibian tectum. Regions unstained for PSA and NCAM-180 correspond precisely to the growth zones of the tectum. NCAM-180 and PSA are not present in tecta of early larvae. Staining intensity is strongest at midlarval stages for both antigens. At metamorphosis, PSA is strongly downregulated, whereas NCAM-180 is downregulated in juvenile animals. Both antigens are still present in fiber layers of adult animals. In dissociated tissue culture of the frog tectum, NCAM-180 is not present on astrocytes, but on neuronal cells. Expression is enhanced at cell contact sites, suggesting that NCAM-180 is involved in cell contact stabilization. This study shows that general features of temporal and spatial regulation of NCAM isoforms and PSA are highly conserved in frog and salamander tecta, despite large differences in the rate of cell migration and the degree of lamination in these homologous brain regions.     

Histidine Mutants Requiring Adenine: Selection of Mutants with Reduced hisG Expression in SALMONELLA TYPHIMURIUM

A method is described for the selection of Salmonella typhimurium mutants with reduced levels of hisG enzyme activity. This method is based on the fact that the hisG enzyme catalyzes the consumption of ATP in the first step of histidine biosynthesis. Normally, this reaction is closely regulated, both by feedback inhibition and by repression of the operon. However, conditions can be set up that result in the uncontrolled use of adenine in histidine biosynthesis. Cells grown under these conditions become phenotypic adenine auxotrophs. Some revertant clones that no longer require adenine contain mutations in hisG, hisE, or the his-control region. The hisG mutations are of all types (nonsense, frameshift, missense, deletion and leaky types), and they map throughout the hisG gene.     

Comparison of titration results of diphtheric antitoxic antibodies obtained by means of Jensen's method and the methods of tissue cultures and haemagglutination.

In 33 human sera the determination of diphtheric antitoxic antibodies was performed in a double blind test using Jensen's method, the method of tissue cultures and the haemagglutination method. In the method of tissue cultures the antibody levels in the sera were determinated in the first and second experiment with the precision of +/- half dilution of the geometrical progression. In Jensen's method, the difference between the first and second measurements slightly exceeded +/- 1 dilution. In the haemagglutination method the error considerably exceeded the binary step dilution. In most cases, the determination fluctuated up to seven times the actual value. Differences among the mean values of examination results obtained by Jensen's method and the method of the tissue cultures are statistically insignificant. The differences between the haemagglutination method and both the other methods are statistically significant.