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Molly Schmid - One of the best experts on this subject based on the ideXlab platform.
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characterization of a Temperature Sensitive Mutant of salmonella typhimurium defective in apolipoprotein n acyltransferase
Journal of Biological Chemistry, 1993Co-Authors: Sita D Gupta, Keda Gan, Molly SchmidAbstract:Abstract On screening 440 Temperature-Sensitive (ts) Mutants of Salmonella typhimurium, a Mutant strain SE5312 which accumulated apolipoprotein (ALP) at 42 degrees C was identified. In vitro assay of apolipoprotein N-acyltransferase activity indicated that the Mutant cell envelope contained reduced activity as compared to the wild-type strain. Transduction with a Mud-P22 mapping set placed the ts mutation to 14-17 min region of the S. typhimurium chromosome. P22 transduction using transposon insertions in this region revealed a linkage of the ts mutation to cobD (6%), nag (8%), and corC68 (99%). The ts phenotype was complemented by a 2.3-kilobase EcoRI subclone derived from lambda-phage 170 of Kohara's bank of Escherichia coli. Restriction enzyme analysis of the cloned DNA revealed that this 2.3-kilobase EcoRI fragment included the copper transport (cutE) gene in E. coli. The Mutant strain SE5312 was copper-Sensitive at 30 degrees C, and the complementing clone conferred copper resistance and restored the ALP N-acyltransferase activity in the Mutant cell. Wild-type strain of S. typhimurium harboring this clone exhibited elevated levels of ALP N-acyltransferase activity. These results suggest that the cloned gene encodes the ALP N-acyltransferase. Upon shift to the non-permissive Temperature, the viability of the Mutant cells decreased, and the Mutant cells assumed anomalous morphology. Temperature-resistant revertants could be readily isolated, and a subset of tr revertants contained no detectable lipoprotein. A lpp::Tn10 derivative of the Mutant SE5312 was also Temperature-resistant. These observations suggest that ALP N-acyltransferase is essential for the growth and viability of S. typhimurium, and this requirement is decreased in the absence of major outer membrane lipoprotein.
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isolation and characterization of a Temperature Sensitive Mutant of salmonella typhimurium defective in prolipoprotein modification
Journal of Biological Chemistry, 1993Co-Authors: Keda Gan, Sita D Gupta, Krishnan Sankaran, Molly SchmidAbstract:Abstract A Temperature-Sensitive (ts) Mutant of Salmonella typhimurium that accumulated unmodified murein prolipoprotein at 42 degrees C but not at 30 degrees C was identified. In vivo and in vitro studies of the biosynthesis of Braun's lipoprotein revealed that this Mutant (SE5221) was defective in the glyceryl modification of prolipoprotein. The ts mutation was mapped to 60.6 min of the S. typhimurium chromosome and was linked to argA and cysH. A clone with a 1.4-kilobase S. typhimurium DNA insert that complemented the ts mutation and restored the prolipoprotein modification activity both in vivo and in vitro was isolated. DNA sequencing of the complementing region revealed an open reading frame encoding a protein with 291 amino acids lacking NH2-terminal signal sequence. This open reading frame is immediately 5' to the thyA gene and is allelic to umpA of Escherichia coli. Wild-type strains harboring the cloned gene exhibited elevated levels of prolipoprotein modification activity. At the non-permissive Temperature, the mutation affected both growth and viability, and the Mutant cells exhibited anomalous cell morphology. The ts phenotype was suppressed by the introduction of a lpp::Tn10 mutation. These results suggest that the cloned gene encodes prolipoprotein glyceryl transferase (lgt), and in the wild-type background, this prolipoprotein modification enzyme is essential for the growth and viability of S. typhimurium.
Keda Gan - One of the best experts on this subject based on the ideXlab platform.
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characterization of a Temperature Sensitive Mutant of salmonella typhimurium defective in apolipoprotein n acyltransferase
Journal of Biological Chemistry, 1993Co-Authors: Sita D Gupta, Keda Gan, Molly SchmidAbstract:Abstract On screening 440 Temperature-Sensitive (ts) Mutants of Salmonella typhimurium, a Mutant strain SE5312 which accumulated apolipoprotein (ALP) at 42 degrees C was identified. In vitro assay of apolipoprotein N-acyltransferase activity indicated that the Mutant cell envelope contained reduced activity as compared to the wild-type strain. Transduction with a Mud-P22 mapping set placed the ts mutation to 14-17 min region of the S. typhimurium chromosome. P22 transduction using transposon insertions in this region revealed a linkage of the ts mutation to cobD (6%), nag (8%), and corC68 (99%). The ts phenotype was complemented by a 2.3-kilobase EcoRI subclone derived from lambda-phage 170 of Kohara's bank of Escherichia coli. Restriction enzyme analysis of the cloned DNA revealed that this 2.3-kilobase EcoRI fragment included the copper transport (cutE) gene in E. coli. The Mutant strain SE5312 was copper-Sensitive at 30 degrees C, and the complementing clone conferred copper resistance and restored the ALP N-acyltransferase activity in the Mutant cell. Wild-type strain of S. typhimurium harboring this clone exhibited elevated levels of ALP N-acyltransferase activity. These results suggest that the cloned gene encodes the ALP N-acyltransferase. Upon shift to the non-permissive Temperature, the viability of the Mutant cells decreased, and the Mutant cells assumed anomalous morphology. Temperature-resistant revertants could be readily isolated, and a subset of tr revertants contained no detectable lipoprotein. A lpp::Tn10 derivative of the Mutant SE5312 was also Temperature-resistant. These observations suggest that ALP N-acyltransferase is essential for the growth and viability of S. typhimurium, and this requirement is decreased in the absence of major outer membrane lipoprotein.
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isolation and characterization of a Temperature Sensitive Mutant of salmonella typhimurium defective in prolipoprotein modification
Journal of Biological Chemistry, 1993Co-Authors: Keda Gan, Sita D Gupta, Krishnan Sankaran, Molly SchmidAbstract:Abstract A Temperature-Sensitive (ts) Mutant of Salmonella typhimurium that accumulated unmodified murein prolipoprotein at 42 degrees C but not at 30 degrees C was identified. In vivo and in vitro studies of the biosynthesis of Braun's lipoprotein revealed that this Mutant (SE5221) was defective in the glyceryl modification of prolipoprotein. The ts mutation was mapped to 60.6 min of the S. typhimurium chromosome and was linked to argA and cysH. A clone with a 1.4-kilobase S. typhimurium DNA insert that complemented the ts mutation and restored the prolipoprotein modification activity both in vivo and in vitro was isolated. DNA sequencing of the complementing region revealed an open reading frame encoding a protein with 291 amino acids lacking NH2-terminal signal sequence. This open reading frame is immediately 5' to the thyA gene and is allelic to umpA of Escherichia coli. Wild-type strains harboring the cloned gene exhibited elevated levels of prolipoprotein modification activity. At the non-permissive Temperature, the mutation affected both growth and viability, and the Mutant cells exhibited anomalous cell morphology. The ts phenotype was suppressed by the introduction of a lpp::Tn10 mutation. These results suggest that the cloned gene encodes prolipoprotein glyceryl transferase (lgt), and in the wild-type background, this prolipoprotein modification enzyme is essential for the growth and viability of S. typhimurium.
Sita D Gupta - One of the best experts on this subject based on the ideXlab platform.
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characterization of a Temperature Sensitive Mutant of salmonella typhimurium defective in apolipoprotein n acyltransferase
Journal of Biological Chemistry, 1993Co-Authors: Sita D Gupta, Keda Gan, Molly SchmidAbstract:Abstract On screening 440 Temperature-Sensitive (ts) Mutants of Salmonella typhimurium, a Mutant strain SE5312 which accumulated apolipoprotein (ALP) at 42 degrees C was identified. In vitro assay of apolipoprotein N-acyltransferase activity indicated that the Mutant cell envelope contained reduced activity as compared to the wild-type strain. Transduction with a Mud-P22 mapping set placed the ts mutation to 14-17 min region of the S. typhimurium chromosome. P22 transduction using transposon insertions in this region revealed a linkage of the ts mutation to cobD (6%), nag (8%), and corC68 (99%). The ts phenotype was complemented by a 2.3-kilobase EcoRI subclone derived from lambda-phage 170 of Kohara's bank of Escherichia coli. Restriction enzyme analysis of the cloned DNA revealed that this 2.3-kilobase EcoRI fragment included the copper transport (cutE) gene in E. coli. The Mutant strain SE5312 was copper-Sensitive at 30 degrees C, and the complementing clone conferred copper resistance and restored the ALP N-acyltransferase activity in the Mutant cell. Wild-type strain of S. typhimurium harboring this clone exhibited elevated levels of ALP N-acyltransferase activity. These results suggest that the cloned gene encodes the ALP N-acyltransferase. Upon shift to the non-permissive Temperature, the viability of the Mutant cells decreased, and the Mutant cells assumed anomalous morphology. Temperature-resistant revertants could be readily isolated, and a subset of tr revertants contained no detectable lipoprotein. A lpp::Tn10 derivative of the Mutant SE5312 was also Temperature-resistant. These observations suggest that ALP N-acyltransferase is essential for the growth and viability of S. typhimurium, and this requirement is decreased in the absence of major outer membrane lipoprotein.
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isolation and characterization of a Temperature Sensitive Mutant of salmonella typhimurium defective in prolipoprotein modification
Journal of Biological Chemistry, 1993Co-Authors: Keda Gan, Sita D Gupta, Krishnan Sankaran, Molly SchmidAbstract:Abstract A Temperature-Sensitive (ts) Mutant of Salmonella typhimurium that accumulated unmodified murein prolipoprotein at 42 degrees C but not at 30 degrees C was identified. In vivo and in vitro studies of the biosynthesis of Braun's lipoprotein revealed that this Mutant (SE5221) was defective in the glyceryl modification of prolipoprotein. The ts mutation was mapped to 60.6 min of the S. typhimurium chromosome and was linked to argA and cysH. A clone with a 1.4-kilobase S. typhimurium DNA insert that complemented the ts mutation and restored the prolipoprotein modification activity both in vivo and in vitro was isolated. DNA sequencing of the complementing region revealed an open reading frame encoding a protein with 291 amino acids lacking NH2-terminal signal sequence. This open reading frame is immediately 5' to the thyA gene and is allelic to umpA of Escherichia coli. Wild-type strains harboring the cloned gene exhibited elevated levels of prolipoprotein modification activity. At the non-permissive Temperature, the mutation affected both growth and viability, and the Mutant cells exhibited anomalous cell morphology. The ts phenotype was suppressed by the introduction of a lpp::Tn10 mutation. These results suggest that the cloned gene encodes prolipoprotein glyceryl transferase (lgt), and in the wild-type background, this prolipoprotein modification enzyme is essential for the growth and viability of S. typhimurium.
Jacomine Krijnse Locker - One of the best experts on this subject based on the ideXlab platform.
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characterization of ts 16 a Temperature Sensitive Mutant of vaccinia virus
Journal of Virology, 1995Co-Authors: Maria Ericsson, Sally Cudmore, S Shuman, R C Condit, Gareth Griffiths, Jacomine Krijnse LockerAbstract:We have characterized a Temperature-Sensitive Mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive Temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive Temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these Mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive Temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive Temperature appeared to have a protein pattern identical to that of wild-type IMV, in the Mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the Mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive Temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive Temperature. Finally, our data suggest that at the nonpermissive Temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.
Juan Ortin - One of the best experts on this subject based on the ideXlab platform.
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genetic analysis of influenza virus ns1 gene a Temperature Sensitive Mutant shows defective formation of virus particles
Journal of Virology, 2005Co-Authors: Urtzi Garaigorta, Ana Falcon, Juan OrtinAbstract:To perform a genetic analysis of the influenza A virus NS1 gene, a library of NS1 Mutants was generated by PCR-mediated mutagenesis. A collection of Mutant ribonucleic proteins containing the nonstructural genes was generated from the library that were rescued for an infectious virus Mutant library by a novel RNP competition virus rescue procedure. Several Temperature-Sensitive (ts) Mutant viruses were obtained by screening of the Mutant library, and the sequences of their NS1 genes were determined. Most of the mutations identified led to amino acid exchanges and concentrated in the N-terminal region of the protein, but some of them occurred in the C-terminal region. Mutant 11C contained three mutations that led to amino acid exchanges, V18A, R44K, and S195P, all of which were required for the ts phenotype, and was characterized further. Several steps in the infection were slightly altered: (i) M1, M2, NS1, and neuraminidase (NA) accumulations were reduced and (ii) NS1 protein was retained in the nucleus in a Temperature-independent manner, but these modifications could not justify the strong virus titer reduction at restrictive Temperature. The most dramatic phenotype was the almost complete absence of virus particles in the culture medium, in spite of normal accumulation and nucleocytoplasmic export of virus RNPs. The function affected in the 11C Mutant was required late in the infection, as documented by shift-up and shift-down experiments. The defect in virion production was not due to reduced NA expression, as virus yield could not be rescued by exogenous neuraminidase treatment. All together, the analysis of 11C Mutant phenotype may indicate a role for NS1 protein in a late event in virus morphogenesis.
