Efectos de la contaminación en la bahía interior del lago Titicaca:

Methodologies based around the screening of cDNA libraries allow the isolation of those cDNAs corresponding to mRNAs expressed in the cell line being screened. However, if expression of the coding sequence is restricted to a limited num ber of tissue types, or a specific stage in developm ent, or if the m RNA species has low stability or very low abundance, then such approaches m ay be unsuccessful. In such cases analysis of the genomic DNA directly is required. This can be achieved by the in vitro isolation o f coding seq u en ces. A num ber of m ethods have been d e scrib e d w h ich utilise vectors that contain both donor and acceptor splice sites in the vector to screen for both acceptor and donor sites flanking coding sequences in genomic DNA fragm ents. These include exon trap clo n in g (A uch and R e th , 1990) and exon a m p lific a tio n (Buckler et al., 1991). The exon amplification method has been the most widely used. Its vector utilises the HIV t a t intron to replace an intron from the rabbit p globin gene, in the vector p S P L l. Essentially genomic DNA is shotgun cloned into the vector, upon transfection into mammalian cells, the reporter gene with inserted genomic fragm ent is transcribed using an SV40 early prom oter. W hen a fragm ent containing an entire exon with flanking intron sequence is present, the exon is retained in mature poly A+ RNA. R NA b ased PC R am p lificatio n using p rim ers specific fo r the splicing plasmid exons is then used to detect the presence of the exon. A vector has recently been described (sC 0 G H 2 ) that extends the size range of insert that can be analysed in one experiment up to that of a cosmid. It was shown that clones of 25 to 40 kb could be successfully analysed with up to seven exons correctly spliced (Datson et al., 1995).

1.8.6 ESTs: Sequencing and Mapping

D espite the ingenuity of the methods described above and their c o n trib u tio n to the id en tificatio n of genes involved in h u m an d ise a se , they will soon be largely red u n d a n t b ecause o f the estab lish m en t of a genome wide gene m ap. W hat m akes this project possible is rapid, single-pass sequencing of cDNA libraries p re p a r e d fro m d iv erse h u m an tis s u e s . T he r e la tiv e ly sh o rt fr a g m e n ts o f se q u e n c e g e n e ra te d h a v e b e c o m e k n o w n as e x p re ssed sequence tags (ESTs) (A dam s et al., 1992). The 3 ’ u n tra n s la te d region of a gene u su ally show s little se q u e n c e co n servation betw een human and rodent h o m ologues, it fo llo w s that STSs derived from the 3 ’UTR usually allow amplification of a specific product even where a highly conserved gene fam ily may be involved. This means that STSs can be defined that am plify unique human products out of a rodent chrom osomal background. The 3 ’ UTR is also rarely interrupted by introns hence product

sizes are p redictable and assays m ore reliable (W ilcox et al.,

1 9 9 1 ).

A set of strategies has evolved to localise ESTs to specific genetic intervals. Somatic cell hybrids are invariably used to assign ESTs to sin g le c h ro m o s o m e s, su b s e q u e n t s u b lo c a lis a tio n has b een achieved by a variety of methods. Panels of somatic cell hybrids containing defined fragments of single human chrom osom es allow localisation to broad cytogenetic regions (Patel et al., 1995). The 3 ’ UTR frequently contains polym orphic elem ents, which have been ex p lo ited to genetically localise ESTs against fram e w o rk m aps

using the CEPH reference pedigrees (K han et al., 1992). The

developm ent of genome wide STS based physical maps has also

facilitated the mapping of ESTs via the identification of clones in the CEPH YAC library that can be linked to STSs from the genetic maps (Berry et al., 1995).

An adjunct to mapping ESTs exists in the availability of a CpG island library (Cross et al., 1994). STSs generated from sequenced clones in the library have some advantages over ESTs. Firstly, because CpG islands are normally at the 5 ’ end of genes full length clones can easily be identified from them. It is also likely that some genes not represented in cDNA libraries will be found in the CpG island library. At the present time about 20,000 single pass se q u en c in g reads have been m ade from the lib rary and are present in the EMBL database.

1.8.6.1 Radiation Hybrid Maps

The approaches described above to regionally localise transcribed sequences though quite successful, have been largely superseded by the construction of high density radiation maps of the whole genom e (W alter et al., 1994; Hudson, et al., 1995). In radiation hybrid m apping, human fibroblasts are irradiated with X-rays to f r a g m e n t t h e ir c h ro m o s o m e s . T h e s e are th e n fu s e d w ith thymidine kinase deficient hamster cells to yield hybrid cells with random fragm ents of hum an DNA e ith er integrated into m ouse chrom osomes or as “new ” hum an chrom osomes. Distances between m arkers are expressed in cR (n rad) where 1 cR corresponds to a 1% freq u en cy of breakage betw een two m arkers. At least two d i f f e r e n t p a n e l s h a v e b e e n c o n s t r u c t e d an d ty p e d f o r m icrosatellite markers to anchor subsequent gene locations on the genetic map (Hudson et al., 1995).

1.8.7 Computational Gene Identification

W here the complete or a substantial part of the genomic sequence is know n for a region, computational analysis provides a useful a lte rn a tiv e to e x p erim en tal m eth o d s to d e te rm in e its c o d in g potential along with inform ation relating to its overall structure and evolutionary history.

E sse n tia lly there are two approaches to id en tify in g p o ten tially in te r e s tin g re g io n s o f g e n o m ic D N A . E ith e r h o m o lo g y is d e m o n stra te d to existing proteins or DNA sequences or som e intrinsic property of the sequence is analysed, such as patterns in c o d o n p r e f e r e n c e (P ic k e tt, 1982). T ru e c o m p u ta tio n a l gene identification lies outside the scope of this thesis, but a b rief introduction to the BLAST program is given below because of its ubiquity and utility in sequence analysis.

1.8.7.1 The BLAST Family of Programs

The exponential increase in the amount of DNA sequence data has driv en the d e v elo p m e n t of tools fo r its a n a ly sis. P a ra m o u n t am ongst these have been m ethods to provide rapid searches of existing databases with new sequences to identify homologies. The basic com ponent of a homology based approach is the concept of dynam ic program m ing. A m atrix com parison is m ade betw een a pair of sequences, as illustrated in figure 1.8, applying a scoring schem e to the alignm ents indicates their degree of related n ess.

The most widely used program is known as BLAST (Altschul et aL,

1990). V a ria n ts of the p ro g ram can deal w ith all p o ssib le c o m b in a tio n s of database and query as show n in table 1.3. BLASTX is especially useful for detecting protein coding potential

in raw DNA sequence, because of the increase in sensitivity due to the redundancy in the genetic code as com pared to com paring D N A s e q u e n c e s , b e tw e e n w h ic h th ere m ay be s ig n if ic a n t divergence despite coding for similar peptides.