Yeast artificial chromosomes.

In the past, contig assembly was limited to small regions that rarely exceeded 200 kb and was restricted from extending further by the presence o f large uncloned “gaps”. This was mainly due to the small cloning capacity of the available vectors such as lambda bacteriophage and cosmids. Extension from a contig is usually performed by “chromosome walking”. This is where terminal clone sequences are isolated to generate an STS or single copy probe to use in isolating new clones that stretch out further from the contig. This approach although successful was laborious and time consuming when using small clones, as the distance covered was limited by their sizes, so other methods were employed. A physical map o f yeast chromosome V

Introduction

(Olson et a l 1986) and most of the C. elegans genome (Coulson et a l 1986) was completed by detecting overlaps in lambda and cosmid clones using restriction digestion “fingerprinting”. Another method, chromosome jumping libraries help identify clones at some distance from the clone of interest, but do not supply sequences in between them (Poustka et a l 1987). These methods and vectors have been superseded by the yeast cloning strategy for physical mapping. Yeast artificial chromosomes (YACs, Burke et a l 1987) are capable of cloning fragments of up to 2 Mb of DNA and have allowed the construction o f long range contigs by greatly reducing the number of cloned fragments which are required to obtain coverage of a particular genomic region. These exist as single copy clones and have been found to be generally stable when growing in the Saccharomyces cerevisiae host (Little et al

1992; Green and Olson 1990a). YAC libraries are usually constructed by ligating partial or complete restriction digested total genomic DNA, or DNA from monochromosomal hybrid panels for chromosome specific libraries, into the YAC vector. The genomic DNA is usually size fractionated to a desirable size before cloning. These fragments are transformed into yeast spheroplasts and are maintained during the host’s growth and replication.

The YAC vector is constructed out of a combination of yeast-derived and pBR322-derived sequences (Schlessinger 1990). When restricted with Bam HI and

E g o RI, it releases an unessential stuffer fragment and two “arms” between which the

insert DNA is ligated. The standard features of YACs include inverted telomeric sequences (TEL) on both arms, an autonomously replicating sequence {ARS) and a

centromeric sequence {CEN) for the maintenance of the clone in the yeast host cell. Selectable markers URA3 and TRP\ are also incorporated. Ligation into the cloning

site interrupts the sup4 gene, a colour selection marker used for discriminating

recombinants from non-recombinants (section 2.1.3). The most commonly employed YAC vector is pYAC4, which has been used in the construction of the most widely available human genomic YAC libraries (section 3.1.3). Modifications have been incorporated in some vectors to extend the use o f YACs to other applications. For example, sequences have been incorporated to permit rescue of insert ends to generate end clones for chromosome walking. Some vectors have included T3 and T7 promoters to synthesise RNA probes from insert ends. Also selectable markers have

Introduction

been included in some vectors to allow transfer into mammalian hosts.

Several problems are associated with this cloning system which can hinder physical mapping efforts and its use in further experiments. Firstly, the isolation of pure cloned YAC material is difficult as it co-exists in the host as an additional chromosome. One procedure is to isolate it from preparative PFGE separated samples, but the YAC must not co-migrate with the host’s chromosomes. However the yield obtained is low as the YAC clone exists in the host as a single copy and there is almost always contamination from degraded yeast chromosomes. Another method is to subclone the entire yeast genome and identify the human specific clones by hybridisation, a time consuming task. Secondly, with the advantage o f large insert sizes arises the problem of chimerism, where a clone contains fragments which are derived from different regions of the genome but are joined together. Chimeric clones are thought to derive from co-ligation of fragments during cloning, or by homologous recombination between two clones which have co-transformed (Green et a l 1991). Approximately 10% of YACs are believed to carry two independent clones because of co-transformation (Schlessinger 1990). In addition the instability of some clones can result in internal deletions. Unlike chimeric clones, these rearrangements are more dangerous as they are less likely to be detectable by STS content mapping or FISH.

Nevertheless, YACs have had an immense impact on physical mapping efforts. When YACs were later incorporated into the C. elegans cloning effort, the

investigators found that YACs have the ability to clone regions o f the genome that could not be retrieved from cosmid libraries (Coulson et al. 1986). In addition the S.

cerevisiae host presents another advantage. The yeast homologous recombination

activity can be exploited on overlapping YACs to produce a single YAC clone, which contains an entire gene and its regulatory elements, by recombination. This was accomplished for the cystic fibrosis disease causing gene, CFTR (Green and Olson

1990a).

More recently developed cloning systems for physical mapping include the PI clones (Sternberg 1990), bacterial artificial chromosomes (BACs; Shizuya et al. 1992),

and the PI derived cloning system (PACs; loannou et al. 1994).]

Introduction