Yeast cloning vectors

To increase the amount of DNA covered in each step, three different strategies have been used One approach, the use of vector-host systems likely to allow the direct cloning of DNA fragments larger than those accommodated in cosmid, has led to the recent development of artificial yeast cloning vectors (13). 

The principles that govern the delivery of recombinant DNA in clonable form to a host cell, and its subsequent amplification in the host, are well illustrated by considering three popular cloning vectors commonly used in experiments with E. coli—plasmids, bacteriophages, and bacterial artificial chromosomes—and a vector used to clone large DNA segments in yeast. 

Research work with large genomes and the associated need for high-capacity cloning vectors led to the development of yeast artificial chromosomes (YACS Fig. 9-8). YAC vectors contain all the elements needed to maintain a eukaryotic chromosome in the yeast nucleus a yeast origin of replication, two selectable markers, and specialized sequences (derived from the telomeres and centromere, regions of the chromosome discussed in Chapter 24) needed for stability and... 

Cloning vectors include plasmids, bacteriophages, and, for the longest DNA inserts, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). 

Various bacterial plasmids, bacteriophages, and yeast artificial chromosomes are used as cloning vectors (Brown, 2001). At the heart of the general approach to generating and propagating a recombinant DNA molecule is a set of enzymes which synthesize, modify, cut, and join DNA molecules. 

Alternative cloning vectors have been developed they can be used for genomic fragments of up to 2 mb for the yeast artificial chromosomes (YAC) (Slow et al.,... 

The development of cloning vectors which propagate in eukaryotic hosts, e.g. yeast or cultured animal cells, has in particular eliminated many of the problems associates with the synthesis of eukaryotic proteins. It should be noted that post-translational processing may also vary among different eukaryontes. It is an advantage that shuttle vectors are available that are capable of propagating in both... 

Transient Transfection The simplest of the two expression methods, called transient transfection, employs a vector sIm liar to the yeast shuttle vectors described previously. For use In mammalian cells, plasmid vectors are engineered also to carry an origin of replication derived from a virus that Infects mammalian cells, a strong promoter recognized by mammalian RNA polymerase, and the cloned cDNA encoding the protein to be expressed adjacent to the promoter (Figure... 

However, complementation of the recessive mutation by the wild-type allele carried by one of the plasmid clones In the library allows a transformed mutant cell to grow Into a colony the plasmids bearing the wild-type allele can then be recovered from those cells. Because many of the proteins that regulate the cell cycle are highly conserved, human cDNAs cloned into yeast expression vectors often can complement yeast cell-cycle mutants, leading to the rapid Isolation of human genes encoding cell-cycle control proteins. 

Yeast artificial chromosome (YAC) A genomic cloning vector which is propagated in yeast as an autonomously replicating chromosome. 

Various bacterial plasmids, bacteriophages and yeast artificial chromosomes are used as cloning vectors (Brown, 2(X)1), such as pBR322. 

Yeast. Advances in studies of eukaryotic gene expression systems have been made with lower eukaryotes such as yeast. Protoplasts of yeast may be transformed by DNA carried on a bacterial plasmid at a frequency, indicated by the use of markers on the foreign DNA, of 1 per 10 viable cells. Alternatively a yeast plasmid vector may be used. However, so far, expression of cloned DNA in yeast appears to be non-specific. ... 

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