The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Daniel Schlieper - One of the best experts on this subject based on the ideXlab platform.
-
a positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal mutant of thecrpgene ofescherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Harald Sobek, Manfred Schmidt, Brigitte Von Wilckenbergmann, Benno MullerhillAbstract:Abstract We have constructed a cloning vector with a tight positive selection for recombinant clones inEscherichia coli.The positive selection pressure results from a Lethal Mutation within theE. coligene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this “suicide” vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
-
A positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal mutant of the crp gene of Escherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Brigitte Von Wilcken-bergmann, Harald Sobek, Manfred Schmidt, Benno Müller-hillAbstract:We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a Lethal Mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this 'suicide' vector, pCAP(s), is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
Breck Byers - One of the best experts on this subject based on the ideXlab platform.
-
ndc1 a nuclear periphery component required for yeast spindle pole body duplication
Journal of Cell Biology, 1993Co-Authors: Mark Winey, Martin Andrew Hoyt, Liliane Goetsch, David Botstein, C. Chan, Breck ByersAbstract:The spindle pole body (SPB) of Saccharomyces cerevisiae serves as the centrosome in this organism, undergoing duplication early in the cell cycle to generate the two poles of the mitotic spindle. The conditional Lethal Mutation ndc1-1 has previously been shown to cause asymmetric segregation, wherein all the chromosomes go to one pole of the mitotic spindle (Thomas, J. H., and D. Botstein. 1986. Cell. 44:65-76). Examination by electron microscopy of mutant cells subjected to the nonpermissive temperature reveals a defect in SPB duplication. Although duplication is seen to occur, the nascent SPB fails to undergo insertion into the nuclear envelope. The parental SPB remains functional, organizing a monopolar spindle to which all the chromosomes are presumably attached. Order-of-function experiments reveal that the NDC1 function is required in G1 after alpha-factor arrest but before the arrest caused by cdc34. Molecular analysis shows that the NDC1 gene is essential and that it encodes a 656 amino acid protein (74 kD) with six or seven putative transmembrane domains. This evidence for membrane association is further supported by immunofluorescent localization of the NDC1 product to the vicinity of the nuclear envelope. These findings suggest that the NDC1 protein acts within the nuclear envelope to mediate insertion of the nascent SPB.
Yasuhiro Anraku - One of the best experts on this subject based on the ideXlab platform.
-
Mutations in yeast calmodulin cause defects in spindle pole body functions and nuclear integrity
Journal of Cell Biology, 1992Co-Authors: Aiko Hirata, Yoshikazu Ohya, Yasuhiro AnrakuAbstract:Yeast calmodulin (CaM) is required for the progression of nuclear division (Ohya, Y. and Y. Anraku. 1989. Curr. Genet. 15:113-120), although the precise mechanism and physiological role of CaM in this process are unclear. In this paper we have characterized the phenotype caused by a temperature-sensitive Lethal Mutation (cmdl-101) in the yeast CaM. The cmdl-101 Mutation expresses a carboxyl-terminal half of the yeast CaM (Met72-Cys147) under the control of an inducible GAL1 promoter. Incubation of the cmdl-101 cells at a nonpermissive temperature causes a severe defect in chromosome segregation. The rate of chromosome loss in the cmdl-101 mutant is higher than wild-type cell even at permissive temperature. The primary visible defect observed by immunofluorescence and electron microscopic analyses is that the organization of spindle microtubules is abnormal in the cmdl-101 cells grown at nonpermissive temperature. Majority of budded cells arrested at the high temperature contain only one spindle pole body (SPB), which forms monopolar spindle, whereas the budded cells of the same strain incubated at permissive temperature all contain two SPBs. Using the freeze-substituted fixation method, we found that the integrity of the nuclear morphology of the cmdl-101 mutant cell is significantly disturbed. The nucleus in wild-type cells is round with smooth contours of nuclear envelope. However, the nuclear envelope in the mutant cells appears to be very flexible and forms irregular projections and invaginations that are never seen in wild-type cells. The deformation of the nuclear becomes much more severe as the incubation at nonpermissive temperature continues. The single SPB frequently localizes on the projections or the invaginations of the nuclear envelope. These observations suggest that CaM is required for the functions of SPB and spindle, and the integrity of nucleus.
Benno Mullerhill - One of the best experts on this subject based on the ideXlab platform.
-
a positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal mutant of thecrpgene ofescherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Harald Sobek, Manfred Schmidt, Brigitte Von Wilckenbergmann, Benno MullerhillAbstract:Abstract We have constructed a cloning vector with a tight positive selection for recombinant clones inEscherichia coli.The positive selection pressure results from a Lethal Mutation within theE. coligene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this “suicide” vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.
Benno Müller-hill - One of the best experts on this subject based on the ideXlab platform.
-
A positive selection vector for cloning of long polymerase chain reaction fragments based on a Lethal mutant of the crp gene of Escherichia coli
Analytical Biochemistry, 1998Co-Authors: Daniel Schlieper, Brigitte Von Wilcken-bergmann, Harald Sobek, Manfred Schmidt, Benno Müller-hillAbstract:We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a Lethal Mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this 'suicide' vector, pCAP(s), is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the Lethal gene.