scholarly journals In vivo construction of linear vectors based on killer plasmids from Kluyveromyces lactis: selection of a nuclear gene results in attachment of telomeres.

1989 ◽  
Vol 9 (9) ◽  
pp. 3931-3937 ◽  
Author(s):  
J Kämper ◽  
F Meinhardt ◽  
N Gunge ◽  
K Esser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Nuclear Gene ◽  
Leu2 Gene ◽  
In Vivo Recombination ◽  
Hybrid Vectors ◽  
Hybrid Molecules ◽  
Specific Sequences ◽  
Selection Of

Linear vectors based on plasmids pGKL1 and pGKL2 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae and selected for integration of the nuclear LEU2 gene. The linear hybrid molecules obtained had no proteins attached to their 5' ends, as is found for native pGKL plasmids. However, telomere-specific sequences were added to the ends of pGKL1. In contrast to the cytoplasmically localized pGKL plasmids, the newly obtained linear hybrid vectors probably replicate within the nucleus and provide evidence that the nuclear LEU2 gene cannot be expressed in the cytoplasm.

In vivo construction of linear vectors based on killer plasmids from Kluyveromyces lactis: selection of a nuclear gene results in attachment of telomeres

1989 ◽  
Vol 9 (9) ◽  
pp. 3931-3937
Author(s):  
J Kämper ◽  
F Meinhardt ◽  
N Gunge ◽  
K Esser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Nuclear Gene ◽  
Leu2 Gene ◽  
In Vivo Recombination ◽  
Hybrid Vectors ◽  
Hybrid Molecules ◽  
Specific Sequences ◽  
Selection Of

Linear vectors based on plasmids pGKL1 and pGKL2 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae and selected for integration of the nuclear LEU2 gene. The linear hybrid molecules obtained had no proteins attached to their 5' ends, as is found for native pGKL plasmids. However, telomere-specific sequences were added to the ends of pGKL1. In contrast to the cytoplasmically localized pGKL plasmids, the newly obtained linear hybrid vectors probably replicate within the nucleus and provide evidence that the nuclear LEU2 gene cannot be expressed in the cytoplasm.

scholarly journals Generation of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae by PCR mutagenesis and in vivo recombination: characteristics of the mutant strains imply that CBP1 is involved in stabilization and processing of cytochrome b pre-mRNA.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 981-991 ◽  
Author(s):  
R R Staples ◽  
C L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
In Vivo Recombination ◽  
Mutant Strains ◽  
Precursor Rna

Abstract Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.

Cloning of the LEU2 gene of Saccharomyces cerevisiae by in vivo recombination

1989 ◽  
Vol 152 (3) ◽  
pp. 263-268 ◽  
Author(s):  
R. Valinger ◽  
G. Braus ◽  
P. Niederberger ◽  
M. K�nzler ◽  
G. Paravicini ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Leu2 Gene ◽  
In Vivo Recombination

Effects on mRNA splicing of mutations in the 3' region of the Saccharomyces cerevisiae actin intron

1987 ◽  
Vol 7 (1) ◽  
pp. 225-230 ◽  
Author(s):  
L A Fouser ◽  
J D Friesen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Point Mutations ◽  
Mrna Splicing ◽  
Polypyrimidine Tract ◽  
Context Change ◽  
Sequence Context ◽  
Primary Transcript ◽  
In Vivo Analysis ◽  
Specific Sequences

Point mutations, deletions, and a sequence context change were introduced at positions 3' to the internal conserved TACTAAC sequence of the Saccharomyces cerevisiae actin intron. In vivo analysis of yeast mRNA splicing suggests that, in contrast to the importance of the polypyrimidine tract in metazoan introns, specific sequences in this region are not required for efficient excision of a yeast intron. However, a double point mutation near the 3' junction (GG/AC) does severely inhibit splicing. Although this mutagenesis of the 3' junction, as well as deletion of most nucleotides between the TACTAAC and the 3' junction, caused only a slight accumulation of primary transcript, the observed accumulation of lariat intermediate by these mutants demonstrates the significance of this region for a step(s) in the splicing process after lariat formation.

A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids

1986 ◽  
Vol 6 (11) ◽  
pp. 4053-4059
Author(s):  
Y Kikuchi ◽  
A Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Complementation Group ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Initiation Of Replication ◽  
Low Levels ◽  
Stable Maintenance

We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

scholarly journals A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids.

1986 ◽  
Vol 6 (11) ◽  
pp. 4053-4059 ◽  
Author(s):  
Y Kikuchi ◽  
A Toh-e
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Complementation Group ◽  
Chromosomal Replication ◽  
Autonomously Replicating Sequence ◽  
Initiation Of Replication ◽  
Low Levels ◽  
Stable Maintenance

We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

scholarly journals Annotated expression and activity data for murine recombinase alleles and transgenes: the CrePortal resource

2021 ◽  
Author(s):  
Michelle N. Perry ◽  
Constance M. Smith ◽  
Hiroaki Onda ◽  
Martin Ringwald ◽  
Stephen A. Murray ◽  
...  
Keyword(s):  
Mouse Genome Informatics ◽  
Activity Data ◽  
In Vivo Recombination ◽  
Recombination System ◽  
Informatics Infrastructure ◽  
Direct Data ◽  
Spatio Temporal ◽  
Selection Of ◽  
Expression And Activity

AbstractRecombinase alleles and transgenes can be used to facilitate spatio-temporal specificity of gene disruption or transgene expression. However, the versatility of this in vivo recombination system relies on having detailed and accurate characterization of recombinase expression and activity to enable selection of the appropriate allele or transgene. The CrePortal (http://www.informatics.jax.org/home/recombinase) leverages the informatics infrastructure of Mouse Genome Informatics to integrate data from the scientific literature, direct data submissions from the scientific community at-large, and from major projects developing new recombinase lines and characterizing recombinase expression and specificity patterns. Searching the CrePortal by recombinase activity or specific recombinase gene driver provides users with a recombinase alleles and transgenes activity tissue summary and matrix comparison of gene expression and recombinase activity with links to generation details, a recombinase activity grid, and associated phenotype annotations. Future improvements will add cell type-based activity annotations. The CrePortal provides a comprehensive presentation of recombinase allele and transgene data to assist researchers in selection of the recombinase allele or transgene based on where and when recombination is desired.

scholarly journals Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

2003 ◽  
Vol 69 (8) ◽  
pp. 4448-4454 ◽  
Author(s):  
So-Young Kim ◽  
Jung-Hoon Sohn ◽  
Jung-Hoon Bae ◽  
Yu-Ryang Pyun ◽  
Michael O. Agaphonov ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Gene Dosage ◽  
Hot Spot ◽  
Hansenula Polymorpha ◽  
Autonomously Replicating Sequence ◽  
Library Construction ◽  
In Vivo Recombination ◽  
Recombination System ◽  
Selection Of

ABSTRACT A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.

scholarly journals Role of the H-2 complex in induction of T helper cells in vivo. II Negative selection of discrete subgroups of T cells restricted by I-A and I-A/E determinants.

1981 ◽  
Vol 153 (4) ◽  
pp. 823-831 ◽  
Author(s):  
J Sprent ◽  
B Alpert
Keyword(s):  
T Cells ◽  
T Helper Cells ◽  
Negative Selection ◽  
T Helper ◽  
Discrete Subgroups ◽  
Hybrid Molecules ◽  
Donor T Cells ◽  
Selection Of

Previous studies have shown that negative selection of T cells to sheep erythrocytes (SRC) after adoptive transfer to irradiated mice requires a sharing of H-2 determinants between the donor T cells and the selection hosts. This paper examines which part of the H-2 complex controls selection. The results show that, in the case of T cells of the H-2k haplotype, complete selection occurs with donor host matching limited to the I-A through I-E subregions of the H-2 complex. Selection to SRC was partial in I-A compatible, I-E incompatible hosts, minimal or not detectable in I-A incompatible, I-E compatible hosts, but near-complete in hosts matched at both the I-A and I-E subregions. Consecutive selection in hosts matched solely at (a) the I-A subregion and (b) the I-E subregion led to incomplete selection. From these and other findings it is argued that H-2k T cells comprise a mixture of T cells restricted by I-A and I-A/E hybrid molecules.

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