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Identification of yeast cloned genes by genetic analysis.

Authors: E, Martín-Rendón; I L, Calderón;

Identification of yeast cloned genes by genetic analysis.

Abstract

Gene cloning in yeast is usually carried out by complementation of recessive mutations. However, the fact that a DNA fragment is able to complement a mutation in a certain gene does not necessarily mean that it contains that gene. The identification of a cloned gene can involve the use of Molecular and/or Classical Genetics techniques. In this paper we describe the strategy to be followed in order to establish the identity of a cloned gene, by using genetic crosses and tetrad analysis. As a practical example of the use of this strategy, we describe the cloning of the THR1 gene which codes for the homoserine kinase in S. cerevisiae. This gene has been isolated from a yeast genomic library by complementation of a thr1 mutation. The complementing DNA fragment has been subcloned and integrated into the yeast genome. By genetic crosses and tetrad analysis it has been demonstrated that integration has occurred at the THR1 locus. Since in this organism integration takes place mainly by homologous recombination, it can be inferred that we have, in fact, cloned the THR1 gene. Biochemical analysis of the transformant that carries multiple copies of the cloned gene confirms this result. It shows that this strain presents a homoserine kinase activity about 60 times higher than that of the wild type.

Related Organizations
Keywords

Microbiological Techniques, Recombination, Genetic, Genes, Fungal, Genetic Complementation Test, Phosphotransferases, DNA, Recombinant, Saccharomyces cerevisiae, Spores, Fungal, Fungal Proteins, Phosphotransferases (Alcohol Group Acceptor), Phenotype, Genetic Techniques, Sequence Homology, Nucleic Acid, Chromosomes, Fungal, Cloning, Molecular, Crosses, Genetic

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citations
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
0
Average
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Average