Autonomía

Six cosm ids were chosen for another round of exon trapping (see table 3.2). As previous experim ents had shown that m ore exons are found when cosm ids are trapped singly rather than in pools (Church et a l 1994) and that exon trapping experim ents using B am H I and P stI as subcloning enzym es give a different but overlapping set of trapped products (W oodw ard 1995), cosmids were trapped individually w ith each of these enzym es separately. At the time, these cosmids (m arked in red in appendix II) cam e from regions of the contig not yet covered by other exon trapping experim ents (although som e regions have since been exon trapped by our collaborators K w iatkow ski et a l) . These included one cosm id know n to contain the D B H gene to act as a positive control to check that the technique was w orking. C osm id D N A was prepared by a m axiprep m ethod and FISH was perform ed (R. Ekong) to check that cosm ids m apped betw een breakpoints S D l and 9T12 in 9q34 (see figure 1.3). Five of the cosm ids mapped as expected, but 81E3 did not m ap betw een S D l and 9T12, prom pting the rearrangem ent o f this part of the cosm id contig. E xon trapping was continued with the other five cosmids. U nfortunately 183D9 is now no longer in the contig near D 9 S 1 14, but as it does m ap in 9q34 between S D l and 9T12 it was included in the exon trapping experim ents.

C osm id Region of contig containing cosm id

183D9 betw een S D l and 9T12, position unknow n (previously near D 9 S 1 14)

240D 2 near CEL gene

176A1 near CEL gene

A B 0 .1 7 contains A BO gene

254C11 contains part o f D B H gene

81E3 proxim al to S D l (see fig. 1.3). Previously thought to be near SU R F genes

Table 3.2 C osm ids used for exon trapping

C osm ids w ere digested w ith B am H I or P stI and subcloned into pSPLB, an exon trapping vector. The ligation m ixture was transform ed into com petent JM lO l cells and D N A from overnight cultures w ere prepared. This DNA was digested with the enzym e used for cloning and run alongside the original cosm id digest to show that m ost restriction fragm ents had been successfully cloned (see figure 3.4).

A portion o f the m iniprep was used to transfect C O S-7 cells and two to three days later, RN A was extracted from these cells. The RNA was used for RT-PCR w ith prim ers SA2 (first strand synthesis) follow ed by dU SA 4 and dU SD 2 (see figure 2.1) in the presence o f the restriction enzym e B stX I to reduce vector-vector splice products (see figure 3.5). PCR products larger than 177bp (the size o f vector-vector splice products) w ere cut out of an agarose gel and cloned into pA M P 10. The pA M P 10 ligation was transform ed into com petent JM lO l cells, blue/w hite selection was perform ed and glycerol stocks were m ade from several recom binant colonies. These glycerol stocks could then be used for PC R w ith prim ers K S F l and K S R l to check the size o f the exon trapped (see figure 3.6). From each cosm id digest, exons o f unique size w ere chosen for sequencing with the M l 3 R everse prim er (binds in pA M PlO ). A total o f 44 products w ere chosen, of w hich 40 have been sequenced (see table 3.3 and appendix V).

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Figure 3.4 Subcloned cosmids in pSPL3

B a m H I (upper panel) and PstI (lower panel) digests o f cosm ids (b) alongside

digests o f subcloned pSPL3/cosmid D N A (a). The gels show that most but not all

cosm id fragments were successfully subcloned. M; Ikb ladder

Figure 3.5 RT-PCR amplification of exon trapped products

RNA was extracted from COS-7 cells which had been transfected with the subcloned

cosm ids or water (mock transfection). The RNA was used for RT-PCR (with SA2 as

first strand primer and dUSD2 and dUSA4 as PCR primers) with (+B) and without

(-B) the restriction enzym e BstXI. The gels show that BstXI reduces the intensity o f

the 177bp vector-only product and increases the efficiency o f amplification o f larger

fragments containing exons. M; D N A size marker (50, 100, 200, 300, 400, 500, 750,

1000, 1500 and 2000bp fragments).

\S3D9 PstI exon trapping products I I ^ T) 25ACW PstI exon trapping products 1 2 3 176A1 PstI exon trapping products -I r 5 6 8 1 6 7

Figure 3.6 PCR amplification of individual exon trapping products

An example o f PCR with primers K S F l/K S R l on individual recombinant colonies to

check the size o f the trapped exon. PCR products were initially exam ined by

electrophoresis on 2% agarose gels (upper panel). Where exons appeared the same size

by agarose gel electrophoresis, PCR products were examined on 10% acrylamide gels

with silver staining (lower panel). The selected exons in the upper panel were all

different sizes so were all sequenced, but in the lower panel som e exon trapping

products appeared to be o f identical size so only one representative product was

sequenced. M: pGEM size markers

size (bp)

183D9 PstI A 194 78bp same as 183pste, 116bp HIV artefact

B cloning vector artefact

C 116 same as 183bamg

D 132 none

E 78 none

F 66 none

BamHI A 287 gene A

B 262 same as 183pstc, 183bamg and 183bamc

C 210 same as 183bamb, 183bamg and 183pstc

D - not sequenced

E 160 none

F 116 HIV artefact

G 117 same as 183pstc, 183bamb and 183bamc

240D2 PstI A - no insert

(near CEL) B 116 HIV artefact

C 116 HIV artefact

D 115 ESTs

BamHI A 187 MERIO repeat - same as 240bamc and 240bamd

B 214 MERIO

C 142 MERIO

D 138 MERIO

E 115 HIV artefact

176A1 PstI A 148 none

(near CEL) B 117 HIV artefact

C 116 same as 183 exons - mix up

D 110 RALGDS exon 2

E 85 RALGDS exon 9

F - not sequenced

BamHI A 157 92bp RALGDS exon 16 (- strand) + 65bp HIV artefact

B 116 HIV artefact

C 85 RALGDS exon 9

A B O A1 PstI A - not sequenced

B 118 HIV artefact

C 87 none

BamHI A - no insert!

B 117 HIV artefact

C 64 none

254C 11 PstI A 174 DBH exon 6 (- strand)

(DBH) B 167 DBH exon 6 C 116 HIV artefact D 117 HIV artefact E - not sequenced BamHI A 169 DBH exon 6 B 116 HIV artefact

Table 3.3 Exon trapping results

Cosmid 183D9

T hirteen exon trapping products were isolated from cosm id 183D9, o f w hich 12 have been sequenced. Only one of these products is entirely a vector derived artefact, although about half of another product is a vector derived artefact (183d9psta). Several o f the rem aining eleven and a half exon trapping products overlap with each other. A fter elim inating redundant products, six unique exons rem ain (183d9pste, 183d9bam b, 183d9pstd, 183d9pstf, 183d9bama and 183d9bame). One o f these exons, 183d9bama, is alm ost identical to bases 158-439 of gene A (the same part o f the gene as another trapped exon D 9S114bam a, see chapter 4) which maps near D 9S114. It is possible that this cosm id overlaps cosm ids at the distal end o f the contig (the cosm id was at one point thought to be in this region o f the c o n tig ). O ther explanations are that there is a related gene w hich also maps in interval 2, or that there has been som e cross contam ination during the exon trapping process. The other five exon trapping products do not have hom ology to anything in the public databases (9.97).

Cosmid 240D2

Exon trapping results from this cosm id are disappointing. O f nine exon trapping products, three are vector-derived artefacts and another one is a cloning artefact. A further four are part o f a repeat unit called M ERIO (Kaplan et al. 1991, M erm er et al.

1987). It is possible that this m edium reiteration frequency repeat, of w hich there are estim ated to be 4000-8000 copies in the hum an genom e, contains cryptic splice sites w hich can direct splicing in the exon trapping construct. The final exon m atches the sequence of cosm id 240D2 near the 3 ’ end of a predicted grow th factor inhibition gene and som e ESTs w hich are likely to be part of this gene (see chapter 5).

Cosmid 176A1

E ight uniquely sized exon trapping products were obtained from this cosm id, o f w hich seven have been sequenced. Three exons are from the RA LG DS gene in the correct orientation (exons 2 and 9 of the gene) and half o f 176albam a is part of exon 16 of R A LG D S in the reverse orientation. Tw o o f these exon trapping products are entirely

vector-derived artefacts, as well as the other half o f 176albam a. The rem aining exon, 17 6 A lp sta does have hom ology to any sequences in the database. It w ould be expected to m atch a cosm id sequence in the database as cosm ids overlapping the w hole o f 176A1 have been sequenced, so is therefore likely to be an artefact.

Cosmid AB0.17

Six exon trapping products were obtained from cosm id ABO. 17. Five have been sequenced, of w hich two represent vector-derived artefacts. O ne seems to have no insert on sequencing. The two rem aining exon trapping products have little hom ology to anything except the sequence o f cosm id ABO. 17. It is surprising that no exons o f the A BO gene w ere trapped - the sequence of the cosm id shows that it contains all 5 of the internal, trappable exons of ABO (see chapter 5). It is possible that the cosm id that exon trapping was perform ed from had an internal deletion - a m olecular com bing experim ent using D N A grown from the same glycerol stock show ed a very short cosm id of about 20kb, but when grown from another stock o f the cosm id a longer signal was obtained, consistent with the 38.4kb sequence obtained from this cosm id (R. Ekong, personal com m unication). It is interesting to note that m ost o f the internal exons o f the A B O gene are particularly small and were not detected by the G RAIL2 gene finding program (see chapter 5). All five of the internal exons lie on a very large B am H I

restriction fragm ent (over 16kb) which may be difficult to trap exons from. It is difficult to determ ine w hether this fragm ent was properly subcloned (see figure 3.4).

Cosmid 254C11

Seven supposedly unique exon trapping products w ere generated from this cosm id, of w hich six have been sequenced. Three represent pSPL3-derived artefacts, and three contain sequence from exon 6 o f the D B H gene. It is not know n how m uch o f the gene is in this cosm id, but it is unlikely to contain all 12 exons. O ne o f these exon trapping products contains the exon in the reverse orientation to that expected. This has been observed previously (Church et al. 1994) but the m echanism o f exon trapping is not know n for these cases.