CONCLUSIONES Y RECOMENDACIONES

Since the initial mapping of the disease, positional cloning efforts using Y AC clones were initiated to facilitate the search for the disease gene causing DHRD and related phenotypes (as discussed above) as well as providing candidates for other diseases mapping to the region. This work has been further discussed in Chapter 4.

CHAPTER 4

Construction of a YAC and a partial PAC contig across the DHRD critical region

4.0 Introduction

A physical mapping approach is applied to a study, once the disease has been linked by genetic linkage analysis to a relatively small genetic interval, to which fiirther cloning strategies can be applied. To prevent gross time consumption in construction of large contigs, the advisable maximum genetic distance across which a construction o f a contig is suitable is approximately 1 centi Morgan (cM), which in molecular distance approximates to 1 mega-base (Mb). Unfortunately, narrow refinements o f a cM do not always occur and the constructions o f YAC contigs are initiated across intervals greater than a mega base.

Initially, Doyne honeycomb retinal dystrophy (DHRD) was genetically reduced by linkage analysis to chromosome 2p 16-21, in the genetic interval D2S2316 and D2S378 of approximately 5 cM (Gregory et a i, 1996). This locus was later refined to a 4 cM interval between the microsatellite markers D2S2739 and D2S378 (Evans et al, 1997). Since this interval could not be genetically refined further at that point in time, due to lack of availability o f microsatellite markers in the region, a physical map using YACs was initiated across the DHRD locus. Recently, recombination events found within individuals from an additional branch o f the Doyne’s family have further refined the DHRD locus between markers D2S2352 and D2S2251 (Kermani et a l, 1999), an estimated physical distance of approximately 3 Mb.

4.1 Physical mapping

Physical mapping involves the assembly o f overlapping clones known as a contig that would faithfully represent the genomic region of interest in a linear order. Physical mapping of a disease should allow direct access to the genomic segment that would include the disease gene. Following the construction o f a complete contig, the genomic region can be characterised in detail for the identification of the disease gene. Incidentally, the disease region can be used to map pre-characterised genes to the locus or used in the isolation of novel transcripts using various techniques like exon trapping/exon amplification (Duyk et a l, 1990; Buckler et a l, 1991). An alternative method is to use cDNA selection (Parimoo et a l, 1991) to isolate genes in the critical region. This strategy

has been called the positional cloning approach as it allows the isolation o f a disease gene based on its map position.

Physical maps arrayed with yeast artificial chromosomes (YACs) provide long range-continuous coverage which are aligned by microsatellite markers, sequence tagged sites (STS) and expressed sequence tags (EST) at appropriate distances.

4.1,1 Yeast Artificial Chromosomes (YACs)

The development of YACs has increased the genomic cloning capacity by 5-10 fold over cosmids, which contain inserts o f approximately 40 kb compared to YACs which contain inserts of up to 1 Mb (Burke et a l, 1987; Anand et a l 1989, Albertson et at. 1990, Earih et a l 1991). In general^ large fragments o f the human genome are cleaved by restriction enzymes and then ligated between two vector arms, each o f which ends in telomere sequences and contain centromeres (CEN), replication origin (ARS) and selectable markers to stabilise the YACs in the yeast host. The original vectors contained a suppressor tRNA (which, because it was split allowed the recognition o f YAC colonies) and pBR322 sequences to facilitate mapping and recovery o f one end o f the insert as a plasmid in E. coli. The vector sequences total approximately 10 kb and the inserts are usually 300 to 500 kb. Owing to their large size, contigs of up to several megabases can be assembled, facilitating the construction o f overlapping clone/STS maps over large regions o f chromosomes.

The development of Pulse Field Gel Electrophoresis (PFGE) allows a modified electrophoretic apparatus to separate DNA molecules as large as 10 Mb enabling intact yeast chromosomes to be separated and thus inserts can be sized (Schwartz and Cantor, 1984). YACs introduced into mammalian cells have the ability to encode normal enzymatic products (D’Urso et a l, 1990, Huxley et a l, 1991). Therefore, YACs can be important tools for research in gene expression and regulation with potential clinical applications as well as useful for the construction of YAC contigs.

The main disadvantage of YACs (Kouprina et a l 1994; Monaco & Larin 1994) is that it can often contain chimeric inserts which implies that the insert is composed of two or more fragments that were derived from noncontiguous regions o f the genome. Chimerism can be verified by FISH analysis. Approximately 10-60 % o f YAC clones in existing libraries can represent chimeric DNA sequences. Chimeric clones may result from a co-ligation of two different restriction fragments prior to transformation. Unstable

important drawback in construction of a contig. Deletions vary in size from 20 to 260 kb which can be generated both during the transformation process and mitotic growth transformants (Kouprina et a l, 1993). Loss of entire YACs can also occur during mitotic growth. Use of recombination deficient strain can reduce frequency o f deletion. Co­ cloning events are also a common drawback, where two or more YACs are cloned into the same yeast cell. Difficulties in purifying YAC inserts from the yeast background and poor insert DNA yields are other limitations with YACs. However, the easy availability of YAC libraries and their large insert size capacity is helpful for providing long range continuous coverage of disease regions and for this region YACs were our initial choice for the construction of a contig across the DHRD interval.