Clase Estructura

fram e B la st? 2 .0 .8 results 1 43 A 1 6 d l2 174* gb/A F027156 (2e-50), g b /H S M A N lB 0 9 (3 e-2 1 ), gb/M D U 3458 (3e-21), gb/M D U 3457 (3e-21), g b /H S M A N lB 0 7 (le -2 0 ), g b /M M A 1 2 M I B 0 7 (le -ll) + 1= 76 aa -2= 39 aa - 2 = ?

143A 16e5 194 . gb/A F027156 (3e-92), gb /H S M A N lB 07 (7e-56), gb/M D U 3457 (4e-51), gb/M D U 3458 (4e-51), gb/M M A 12M IB07 (9e-37), g b /H S M A N lB 0 9 (le -2 6 ), g b /H S M A N lB 0 9 (le -0 4 ) -2= 24 aa -3= 71 aa -2 = ? -3 = g i/3 1 2 7 0 4 7 ( le -1 9 ), gi/3025749 ( le - 1 9 ) , sp /M A N X _M ou se (6e-19), pir/A 54407 (6 e-1 9 ), pir/B54408 (7 e-1 0 ), sp/M A 12_R abbit (7e-10), sp/M A 12_H um an (3e-09) B143D 4n3 180* ref/N M 00207 ( 5 e - l l ) , gb/U 12390 ( 5 e - l l ) , gb/A F062916 ( 5 e - l l ) 7 _? 208B1 liV 126* gb/U 12390 (10-51), em b/AJ276255 (8e-34), em b/AJ276256 (3e-33), em b/AJ276253 (3e-33) ? ?

5.4.2.1 Clones 143A 16dl2 and 143A16e5

Both the exon-trapped sequences from clone 143A 16dl2 and 143A 16e5, displayed sim ilarities, at the nucleotide level, to the non-redundant database en try for Homo

sapiens and M us m usculus a l ,2-m annosidase IB mRNA. The hom ology is seen across the length o f the exon-trapped sequence, respectively, w ith 174 out o f 189 and 196 out o f 200 nucleotides identical to the database entry. The difference in size and positive identities may be caused by an artefact o f the low er quality o f the sequence derived from exon-trapped clone 143A 16dl2, Open reading fram e analysis identified an O RF in the negative fram e (as expected from the directional nature o f the exon trapping and final cloning) w ith significant hom ology to entries w ithin the non-redundant protein databases (using B last?). This exon-trapped sequence displayed hom ology to the protein sequence o f H. sapiens a 1,2-m annosidase IB.

Further analysis o f the sequences o f both exon-trapped clones revealed th a t the trapped “exon” is in fact the result o f the concom itant splicing and cloning events o f two different exons into the pSPLSB vector. Therefore, these “exon”-trapped clones (1 43 A 1 6d l2 and 143A16e5) correspond both to exon 4 and 5 o f the a l ,2-m annosidase IB gene.

5.4.2.2 Clone B143D4n3

The sequence derived from this clone showed high sim ilarity to a H. S apiens dual specificity phosphatase. However, the same stretch o f sequence (nucleotides 1-80) show ed hom ology to the pS portl vector. This cloned also show ed unspecific hybridisation pattern and w as dism issed from further analysis.

5.4.2.3 Clone 2 0 8 B lli7

The sequence o f this clone show ed high sim ilarity to the cloning vector p S p o rtl and to 3 partial m RN A clones in the database. All these 3 clones’ entries in the database m apped to the breast cancer susceptibility locus. H owever, due to the h ig h sim ilarity to entries representing the vector sequence and the absence o f any ORF this exon-trapped clone w as not subm itted to further analysis.

5.5 Results - cDNA selection

5.5.1 V alidation of the cDNA selection technique

BAC clone B74L23 within the Ip 13.1 contig was found to contain the only gene in the region, V7LSB (Rivas et al, 1995; R uegg et al, 1995; Brintnell et al, 1997). This clone w as therefore used to validate the cD N A technique (Lovett et al, 1991; Parim oo et al.

1991) prior to analysis o f the entire contig.

Selection o f cDNAs was carried out as described in Chapter 2 (section 2.3.2; see also Figure 5.2) using hum an bone marrow and breast mRNAs. The m R N A was reverse transcribed and converted to double stranded DNA, creating two cDNA pools - one from breast and another from bone m arrow mRNA. Specific linkers w ere then ligated in order to provide each cD N A within the pool with a characterised stretch o f sequence to enable subsequent PC R am plification with specific primers. Each cDNA pool w as then purified and repetitive sequences were suppressed. Simultaneously, dot-blots o f all the 10 clones in the minim al tiling path were produced (DNA immobilised on a solid support). Hybridisation o f the cDNA population to the immobilised DNA was carried out overnight at 65°C, followed by high stringency washes. The homologous cDNAs w ere eluted in TE at 50°C and another step o f PCR was executed. In total, two rounds o f enrichm ent were perfoim ed.

Secondary P C R products w ere sub-cloned using the CLONEAM P® pA M P 10 System (Life T echnologies) according to the m anufacturer’s specifications and inoculated on m edia containing IPTG and X-gal for colour screening. W hite colonies (containing insert) w ere picked into a 384 well m icrotitre dish and incubated o vernight at 37°C. N ylon m em branes w ere spotted and processed as described (section 2.2.1). The m icrotitre dishes w ere then stored at -80°C in 20% glycerol for subsequent analysis. D N A from aliquots retrieved after each step o f enrichment (no enrichment, round, and 2”^ round o f enrichment), was resolved on a 1.5% agarose gel and Soutliem -blotted (section 2.2.7). A PC R probe originated from the V7-LSB gene was radioactively labelled and hybridised to the blot. Figure 5.7 illustrates the successful enriclim ent o f V7-LSB cDNAs following one round o f selection from the bone m arrow cDNA library. M axim um enrichm ent was observed after the second round o f PCR (lane 3).

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