Población

The 2.5kb band on the N orthern blot makes it clear that cD N A 32603 is not a full length clone for gene D. A fter the experiences with chim eric clones, the Stratagene foetal brain cD N A library was not used again. Instead, adult and foetal kidney cDNA libraries were used. These libraries, provided by D r A.-M. Frischauf, w ere in a bacteriophage vector, Xgt22, so were not as convenient to w ork with as libraries in a plasm id based or excisable vector. However, these libraries had previously been found to have several clones with large inserts and problems w ith chimeric clones had not been reported. Each library had been split into eight aliquots, each with 80 - 100 000 independent clones, and the cDNAs had been directionally cloned, using S a il at the 5 ’ end and N o tl

at the 3 ’ end. The first four aliquots o f the adult kidney library were chosen for screening, because expression in adult kidney had already been shown by N orthern blot analysis. 400000 pfu were screened from each fraction o f the library using a pool of probes (cD NA 32603 insert, cD N A 46195 insert - see gene E below, and exon trapping products A BO lV pstC and 4D D lbam B , which had not yet been incorporated into larger transcriptional units). 17 strong duplicate positive signals w ere obtained (9 from aliquot 1, 5 from aliquot 3 and 3 from aliquot 4) (see figure 4.15a and 4.15b) as w ell as 9 w eaker duplicate signals after a long exposure (4 from aliquot 1, 3 from aliquot 3 and 2 from aliquot 4). Positive plaques were picked and subjected to secondary screening with the pool o f probes, follow ed by tertiary screening using the four probes individually (see figure 4.15c and 4.15d). All of the strong signals from the first round o f screening showed signals at this stage, but none o f the w eaker primary signals cam e through the secondary screening. At the tertiary screening, all isolates except one had positive hybridisation signals with cD N A 32603. The other plaque, (A1.9, th e ninth signal from aliquot 1) did not hybridise to any o f the four probes. This has rem ained

uninvestigated, although plating it at higher density and rescreening m ight reveal that it

is p o sitive for one o f the probes in the pool.

The phage supernatant from the 16 plaques p ositive for c D N A 3 2 6 0 3 w ere subjected to

PCR using vector primers ( I g t l 1 F/Xgt 1 IR - these primers w ere d esig n ed for an older

vector ^ g tl 1 from w hich A,gt22 is derived) to determ ine the insert sizes. M ost clon es

gave an am plification product (sizes are show n in table 4 .7 ), but som e failed. An

op tim istic v iew o f the failure to am plify is that the insert w as too large to am plify w ell.

From size From size From size

aliquot 1 (kb) aliquot 3 (kb) aliquot 4 (kb)

A l . l 1.8 A3.1 2.9 A4.1 1.2 A L 2 1.8 A 3 .2 A 4 .2 0.6 A 1.3 1.8 713.3 1.8 AÆ3 0 .6 A 1 .4 1.8 )AT4 1.8 A 1 .5 1.8 7V3.5 1.8 A 1 .6 A 1.7 1.8 A L 8

T able 4 .7 G ene D c D N A s obtained by screening part o f an adult kidney library

T he clo n e with the largest known insert o f know n size, A 3 .1 , and a clo n e o f unknown

insert size, A 3 .2 , w ere chosen for D N A preparation. R estriction d igests o f the D N A

sh o w ed that both have an insert o f approxim ately 2.9kb. T he insert o f A 3 .2 was

su b clon ed into pBluescript and partially sequenced by primer w alk in g starting from

both ends o f the vector. Sequence from the 3 ’ end o f the c D N A m atched the 3 ’

seq u en ce o f cD N A 3 2 6 0 3 , and sequence from the 5 ’ end w as exten ded by primer

w alk in g until it reached the 5 ’ end o f cD N A 3 26 03 (see figure 4 .1 6 ).

T w * L'ft I I: <

•A I.2

•A 1.4

A1.5

•A1.6

AI.1

• A1.3

A1.7

i

Figure 4.15a (above) and 4.15b (below) Library screen (primary signals) to obtain gene D cDNAs

D uplicate lifts taken from aliquot 1 of an adult kidney cDNA library were each hybridised to a mixture of four probes (cDNA 32603, cDNA 46195 and exon trapping products ABO Pst C and 4DD1 Bam B). A liquots 2, 3 and 4 were also screened resulting in several positive cDNAs (data not shown). Duplicate positive signals are numbered in the upper panel and the markings used to align the duplicate lifts can be

0-3 • • •6* • ' . * •* V c : • • f A c - A - : • . • • ' cDNA 32603 (gene D) exon trap product 4DD1 Bam B cDNA 46195 (gene E)

exon trap product ABO Pst C

Figure 4.15c (above) and 4.15d (below) Library screen (secondary and tertiary) to obtain gene D cDNAs

Secondary lifts (above) were hybridised to the same mixture of probes as primary lifts (two representative results shown). Tertiary filters were divided into four parts each of which was hybridised to one of the probes from the pool separately (see diagram).