Development of a new host vector system in mycobacteria

The hybrid plasmid pYT72/pYT92 constructed from an Escherichia coli plasmid pACYC177 and mycobacterial plasmid pMSC262 isolated from Mycobacterium scroflaceum strain W262 transformed both E. coli and BCG. Phage-sensitive mutants S-10 and S-20 isolated from BCG Tokyo strain showed higher frequency of transformation than the wild-type strain. Frequency of transformation was dependent on age of the culture and the electroporation condition used. Several deletion mutants were generated from pYT72/92 to determine the minimum region for the replication in the mycobacteria. A 2.3-kb fragment of pMSC262 was found to contain an essential region. Using this fragment and pACYC177, a small shuttle vector pYT937 containing two drug-resistance markers, kanamycin- and ampicillin-resistance, was constructed. pYT937 contains AatII, BamHI, BbvII, GsuI, HincII, PstI, ScaI and XbaI cloning sites.     

Cloning and characterization of a gene encoding a new thermostable hemolysin from Vibrio parahaemolyticus

A new thermostable hemolysin (delta-VPH) gene was cloned from a Kanagawa-negative Vibrio parahaemolyticus strain into vector pBR322 in Escherichia coli K12. The nucleotide and amino acid sequences had no homology with those of the thermostable direct hemolysin (TDH) which causes the Kanagawa phenomenon, and of the thermolabile hemolysin (TLH) of V. parahaemolyticus. The gene was present in all V. parahaemolyticus strains tested and also in one strain of V. damsela.     

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc²155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc²155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc²155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.     

A host-vector system for molecular study of the intracellular growth of Mycobacterium tuberculosis in phagocytic cells

The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M . smegmatis strain mc²155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli - mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated.
In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M . tuberculosis in phagocytic cells; this system may be useful as a screening tool for M . tuberculosis genes.     

Differing responses of Nijmegen breakage syndrome and ataxia telangiectasia cells to ionizing radiation

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. Originally thought to be a variant of ataxia telangiectasia (AT), the cellular phenotype of NBS has been described as almost indistinguishable from that of AT. Since the gene involved in NBS has been cloned and its functions studied, we sought to further characterize its cellular phenotype by examining the response of density-inhibited, confluent cultures of human diploid fibroblasts to irradiation in the G(0)/G(1) phase of the cell cycle. Both NBS and AT cells were markedly sensitive to the cytotoxic effects of radiation. NBS cells, however, were proficient in recovery from potentially lethal damage and exhibited a pronounced radiation-induced G(1)-phase arrest. Irradiated AT cells showed no potentially lethal damage and no G(1)-phase arrest. Both cell types were hypersensitive to the induction of chromosomal aberrations, whereas the distribution of aberrations in irradiated NBS cells was similar to that of normal controls, AT cells showed a high frequency of chromatid-type aberrations. TP53 and CDKN1A (also known as p21(Waf1)) expression was attenuated in irradiated NBS cells, but maximal induction occurred 2 h postirradiation, as was observed in normal controls. The similarities and differences in cellular phenotype between irradiated NBS and AT cells are discussed in terms of the functional properties of the signaling pathways downstream of AT involving the NBS1 and TP53 proteins.     

Quantitative Measurements of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> Distribution in Rice Using Fluorescent-Labeling

The rice host sensor, XA21, confers robust resistance to most strains of Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of bacterial blight disease. Using in planta fluorescence imaging of Xoo strain PXO99Az expressing a green fluorescent protein (Xoo-gfp) we show that XA21 restricts Xoo spread at the point of infection. This noninvasive and quantitative method to measure spatial distribution of Xoo populations in planta facilitates detailed assessment of plant disease resistance.     

Radiation sensitivity of primary fibroblasts from hereditary retinoblastoma family members and some apparently normal controls: colony formation ability during continuous low-dose-rate gamma irradiation

We previously described an enhanced sensitivity for cell killing and G(1)-phase cell cycle arrest after acute gamma irradiation in primary fibroblast strains derived from 14 hereditary-type retinoblastoma family members (both affected RB1(+/-) probands and unaffected RB1(+/+) parents) as well as distinctive gene expression profiles in unirradiated cultures by microarray analyses. In the present study, we measured the colony formation ability of these cells after exposure to continuous low-dose-rate (0.5-8.4 cGy/h) (137)Cs gamma radiation for a 2-week growth period. Fibroblasts from all RB family members (irrespective of RB1 genotype) and from 5 of 18 apparently normal Coriell cell bank controls were significantly more radiosensitive than the remaining apparently normal controls. The average dose rates required to reduce relative survival to 10% and 1% were approximately 3.1 and 4.7 cGy/h for the Coriell control strains with normal radiosensitivity and approximately 1.4 and 2.5 cGy/h for the radiosensitive RB family member and remaining apparently normal Coriell control strains. The finding that a significant proportion of fibroblast strains derived from apparently normal individuals are sensitive to chronic low-dose-rate irradiation indicates such individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes that may likewise predispose them or their children to cancer.     

Establishment and characterization of a human scirrhus type gastric cancer cell line, GCIY, producing CA19-9]

A human gastric cancer cell line, designated GCIY, was established from ascites of a patient with scirrhus type gastric cancer. Doubling time of this cell line was 55 hours. The karyotype indicated that these cells were human cells and the chromosome number varied widely, the mode being 57. GCIY cells were of moderate size and showed monolayer arrangement. They had a large nucleus and a clear nucleolus. Papanicolaou staining showed that they had the characteristics of adenomatous epithelia. They could be subcutaneously transplanted into nude mice, and they histologically resembled the original tumor. They were immunohistochemically recognized by anti-CA19-9 antibody and weakly by anti-CA125, CEA and alpha FP antibodies. Cultured medium also contained CA19-9, CA125, CEA and alpha FP.     

Electrotropism of Nicotiana Pollen Tubes

Electrotropism of tobacco pollen tubes towards the anode wasanalysed. The threshold and saturation values for the electrotropismwere less than 50 mV mm^–1 and 200 mV mm^–1, respectively.The tropic response gradually increased with increasing durationto exposure, but no further increase in the tropic responsewas observed when exposure of the electric field was terminated.Pollen tubes growing towards the cathode had a tendency to burstin a strong electric field. These results suggest that an externallyapplied electric field acts as a motive force for electrotropismbut not as a trigger and that endogenous currents play a rolein tip growth of pollen tubes. Possible mechanisms responsiblefor the electrotropism of pollen tubes are discussed. (Received July 9, 1993; Accepted September 18, 1993)