TÉCNICAS E INSTRUMENTO DE RECOLECCIÓN DE DATOS

3 0 5 4

1 6 3 5

1 0 1 8

5 1 6 /

Fig 3 .2 .2

Alu-W/

1 7A, 1 4 A 4 ,

The hybrids g e n e r a t e a discrete s e t of Alu products ranging in size from 0.3 -3 kb. Hybrid 14A4 is a subclone of hybrid 14A. It retains only t h e m o s t stable of th e th re e human f ra g m e n ts retained in t h e pa rental irradiation hybrid line. The Alu PCR fingerprints of t h e s e tw o hybrids have a large number of visible amplification p r o d u c ts in com mon (lane 3 and 4) as ex p e c te d from their c o -r ete n tio n of an identical f r a g m e n t g e n e r a t e d from c h ro m o s o m e 9q. Hybrids 17B a nd 19B retain 2 co-migrating visible p r o d u c t s in c o m m o n of b e t w e e n 1.6 and 2 .0 kb. Hybrids 17B and 20A retain 3 visible p r o d u c t s in com mon of approximately 1.3, 1.0 and 0 .6 kb. Visible Alu p ro d u cts in common bet ween hybrids are arrowed.

chromosome spreads (fig 3.2.3). Both hybrids gave signals solely on the distal long-arm of chromosome 9. This is c o n s is te n t with th e marker retention studies of both hybrids (Table 3.1.1). The larger signal obtained using 12C Alu-PCR p ro d u c ts re fle c ts t h e retention of a larger se gment of the distal region of chromo some 9q in this hybrid in comparison to hybrid 17B.

3 . 2 . 4 Isolation o f human cosmid clones from t h e TSCl interval using Alu-PCR products from the hybrid 1 7B

Radiolabelled Alu-PCR products g enera te d from hybrid 17B were hybridised t o lysed colony filters of a human cosmid DNA library (sec tions 2.7.4, 2.8) in duplicate (fig 3 .2 .4 ) t o identify cosmid clones contained within th e limits of t h e human DNA p r e s e n t in t h e hybrid line 17B. Several positive hybridising signals were identified and subsequently purified (fig 3.2.5).

In total 4 cosmids, c17B2, c17B6, c17B7 and c1 7 B 1 2 were isolated and mapped to chromosome 9q34 by FISH of total cosmid (section 2.2.2.2) DNA to human metaph ase chromosom e spreads. Fig 3.2.6 and fig 3.2.7 shows the FISH analysis of cosmids c17B6 and C17B12. The three cosmids c17B6, c17B7, and c17B12 were sublocalised t o discrete regions of the ch ro m o so m e 9q region co ntaine d in th e 17B hybrid by probing with t h e A lu p ro d u cts derived from t h e s e cosmids onto the Alu-PCR fingerprint of the entire hybrid panel (fig 3.2.1).

The Alu products derived from the cosmids c l 7BG and c17B7 t e s t e d positive for th e hybrids 6C, 12C, 17B, and 19B and were weakly positive for hybrid 14A and the parent line 6 4 0 6 3 a 1 2 (fig 3.2.8). This suggests from the pattern of marker retention in t h e s e hybrids t h a t these 2 cosmids map to the region proximal to D9S67 and D9S14 and distal to D9S66 (see fig 3.1.14). The Alu product derived from cosmid cl 78 1 2 t e s t e d positive for hybrids 6C, 17B, 20A and 20B and was weakly positive for hybrids 12C, 13A, 13B

(A ) (B)

Fig 3 . 2 . 3 Localisation o f h u m a n f r a g m e n t s p r e s e n t in ir ra dia tio n hybrids 12C and 1 7B on hum an c h r o m o s o m e 9 using A lu e l e m e n t - m e d i a t e d PCR p r o d u c t s a s p r o b e f o r c h r o m o s o m e in situ p a i n t i n g .

Biotlnylated Alu-PCR products were hybridised u nder conditions t h a t s u p p r e s s signals from repetitive DNA s e q u e n c e s t o human m e t a p h a s e c h r o m o s o m e s p re a d s . Hybridisation sig na ls w e r e d e t e c t e d using f l u o r e s c e i n c o n j u g a t e d avidin ( g r e e n ) . C hrom osom es were co u n tersta in ed with propidium iodide (red). The t w o fluorochrom es were s im u lta n e o u s ly vis ualise d using c o n f o c a l s c a n n e r la se r m ic ro s c o p y . Arrows i n d i c a t e t h e fluorescent signals on chromosome 9 (A) Hybridisation of 12C Alu- p r o d u cts t o t h e distal long-arm of chromosome 9, 9 q 3 3 - 3 4 ; (B) Hybridisation of 17B A/u-pro ducts t o t h e distal long-arm of chromosome 9, 9q34.

Fig 3 . 2 . 4 Primary screening of a human cosmid library using >^/u-IV PCR products derived from th e irradiation hybrid line 17B.

Lysed colony filters were probed in duplicate, replica 1 and 2 with radiolabelled Alu-W PCR products. Colony areas giving duplicate positive signals (arrowed) were picked and secondary scre en s carried out (fig 3.2.5).

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f A

< L

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H

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Fig 3 .2 .5 Secondary screening of th e primary colonies

isolated after probing with

Alu-W/

hybrid 17B onto a human cosmid library.

Between 50 and 2 0 0 colonies were rescreened in duplicate with the Alu-W/ PCR products. The number of positives identified in secondary screenings varied considerably eg secondary 1 and 2. These were independent of the actual number of rescre en ed colonies and most likely reflected the size of the region picked to isolate t h e primary signals. A number of positively hybridising colonies were picked, DNA isolated and their 9 q 3 4 localisation confirmed by FISH.

3 . 2 . 6 F l u o r e s c e n t in sit u h y b r id is a tio n o f b i o t i n y a l a t e d h u m a n c o s m i d c l o n e c 1 7 B 6 o n t o h u m a n m e t a p h a s e c h r o m o s o m e s p r e a d s 3 . 2 . 7 F l u o r e s c e n t in s i t u h y b r i d i s a t i o n o f b i o t l n y l a t e d h u m a n c o s m i d c l o n e c l 7 B 1 2 o n t o h u m a n m e t a p h a s e c h r o m o s o m e s p r e a d s

The whole cosmid was used as probe u n d e r c o n d i t i o n s t h a t s u p p r e s s signals from repetitive DNA s e q u e n c e s . Hybridisation signals were d e t e c t e d using fluorescein conju gated avidin (green). C h ro m o s o m es were c o u n t e r s ta i n e d with propidium iodide (red). The tw o fluoro chro m es were s im u l t a n e o u s l y v is u a lis e d using confocal scanning laser microscopy. Arrows indicate t h e signals on chromosome 9.

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m_{0) 0)}ta

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Fig 3 . 2 . 8 Southern analysis of an A lu IV p r o d u c t derived f r o m C 17 B 7 o n t o an A l u f i n g e r p r i n t of t h e e n t i r e irradiation hybrid panel (fig 3 .2 .1 ).

Hybrids 6C, 12C, 14A, 17B, 1 9B and 6 4 0 6 3 a ! 2 g a v e p o s i t i v e signals. All remaining hybrids g a v e no visible hybridising signal.

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Fig 3 . 2 . 9 Southern analysis of an A lu IV product derived f ro m C17B12 was used as p r o b e fo r s o u t h e r n a n a l y s i s o n t o an A lu IV fingerprint of th e entire hybrid panel (fig 3 . 2 . 1 ) .

Hybrids 5A, 6C, 12C, ISA, 13B, 14A, 17B, 20A, 20B and 6 4 0 6 3 a ! 2 g a v e po si tiv e hybridising signals

and 6 4 0 6 3 a 1 2 (fig 3.2.9). This places c17B12 distal t o t h e ABO locus and proximal to the D9S14-D9S67 loci. The identification of overlapping regions between c17B12 and a cosmid clone retaining th e D9S66 locus, placing c17B12 adjacent to the D9S66 (Hornigold, p e rso n a l c o m m u n ic a tio n ) is c o n s i s t e n t with t h e m a p p i n g information derived from southern analysis of the hybrid lines (fig 3.2.9, fig 3.1.14).

In general the intensity of the hybridisation signal s e e m s to be inversely proportional to the number of Alu products g e n e r a t e d by a given irradiation hybrid line, for example the intensity of th e hybridisation signal in hybrid 17B is greater than t h a t observed for hybrid 12C although th e human fragments retained in both hybrid lines are completely stable (fig 3.2.8; fig 3.2.9). This is c o n s i s t e n t with th e retention of a smaller am ount of human DNA in t h e former. The signal s t r e n g t h will also decline with f r a g m e n t instability as this r e d u c e s th e am ount of s t a r t i n g t e m p l a t e available for amplification. The w e a k er hybridisatio n signal observed for some hybrids, for example hybrid 13A and 13B in fig 3.2.9 in c o n trast to th e parent hybrid 6 4 0 6 3 a 1 2 which retains a larger proportion of human material s u p p o r t s this s u g g e s t i o n (Table 3.1.3). No hybridisation signal was ever observed in th e human lane which reflec ts th e large number of A lu p r o d u c t s g e n e r a t e d from th e human genome as a whole and th e obvious reduction in the amount of product generated for any particular Alu amplification.

3 .2 .5 Cloning o f A/ci-IV products from th e i r r a d i a t i o n hybrid lines

There is preferential amplification of so me Alu p ro d u c ts over o th e rs as obse rved by th e differential ethidium b r o m i d e staining intensities of products under transillumination (fig 3.2.2). This s u g g e sts t h a t th e use of pooled Alu amplification products from any hybrid will invariably consist of a large proportion of the

b e t t e r amplified sequences. The presence of an EcoRI site in th e Alu IV primer allows the efficient cloning of Alu-PCR p r o d u c t s g e n e r a t e d from any particular hybrid (section 2.9). Using this a p p ro a c h Alu IV PCR products from a pool of Alu IV p r o d u c t s derived from the hybrid lines 17A, 17B, 19B and 20A have been cloned into the vector bluescript (fig 3.2.2, fig 3.2.10). Vector DNA was digested with EcoRI (section 2.4.1), dephosphorylated (section 2.4.2) and ligated to EcoRI digested Alu IV products. Recombinant clones were identified af te r transformation of J M I O I s ( s ec tio n 2.2.3)

Preliminary examination of a small number of t h e s e c l o n e s have sugg ested t h a t certain Alu products are preferentially cloned, for example clones 1, 2 and 5 and 6, 9, 12 and 15 are identical in size ( 3 . 2 . 1 0 ) suggesting t h a t the y are th e sa me clone. Other clones, for example clones 1 and 10 g e n e r a t e a nu m be r of individual EcoRI products afte r digestion. This m o st p r o b a b l y reflect co-ligation ev e nts during the ligation procedure.

The pattern of retention of a number of th e s e products has been determined by southern analysis of th ese Alu products to the A lu IV fingerprint of the entire hybrid panel (fig 3.2.1). The s e g re g a tio n of t h e s e Alu IV products in t h e hybrid panel was consistant with their localisation to the chromosome 9 q 3 4 region. One ex ception , clone 8, was found t o hybridise t o t h e A lu fingerprint of hybrid 17A and more weakly to hybrids 19B, 20A and 20B (fig 3.2.11). This was not consistent with marker retention ana ly se s which failed to d e t e c t overlap b e t w e e n t h e human material retained in hybrids 17A and 20A (fig 3.1.14). The finding t h a t hybrid 20A has retained some human material from t h e centromeric region (section 3.3) and the retention of th e D9S48 marker in hybrids 17A and 19B sugge sts t h a t this Alu product is probably derived from the centromeric region. This s u g g e s t s t h a t hybrid 20A retains more human material th an d e t e r m in e d by marker retention analysis.

c l o n e i o n 12 13 14 15

Fig 3 . 2 . 1 0 EcoRI digestion of cloned

Alu

1 \ig of plasmid DNA from 15 cloned Alu PCR products in the vector bluescript and a vector control were digested with EcoRI and electrophoresed on a 0.8% agarose gel (lanes 1-16). Several Alu products were preferentially cloned while other products may reflect co-ligation events during the cloning process, see t e x t for more details.

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