The demonstration of splice variant forms of cDNA clone 2:2

Amplification by PCR of the entire cDNA clone 2:2 from cDNA had been unsuccessful. However, when primers were designed flanking the open reading frame described above, products were amplified from cDNA made from normal lymphocytes and thymus. The same result was achieved when the PCR was repeated on cDNA made from malignant lymphocytes obtained from patients with B-cell CLL. The PCR programme used was a standard 35 cycle protocol with an annealing temperature of 52®C. The primers used were C12T7(5)- F and C12T3(6)- R. They correspond to two of the primers published by Liu et al. (Liu

et al, 1997), except that restriction sites for EcoR I on the sense primer and Hind III on the antisense primer were added to each oligonucleotide respectively to enable cloning of the PCR products.

C12T7(5)- F

5'- GTCTACGTTGGAGGTAAACA -3' C12T3(6). R

5'- ATGTCCCTATGTTCAGAACA -3’

When the PCR products were resolved by electrophoresis, two products could be clearly seen in all lanes. The two PCR products were amplified from both normal thymus cDNA and cDNA from patients' malignant B lymphocytes (figure 5.10. A). The PCR products from the normal cDNA were cloned into a Bluescript plasmid (KS+) vector and the inserts were sequenced using M l 3 -40 and T3 primers with an ABI 377 automated sequencer. The sequences obtained can be seen in figure 5.10.B. The sequence of the smaller band was the sequence predicted from cDNA clone 2:2. The sequence of the larger band had an additional 53 base pairs at position 347 which was identical to the 53bp 'exon' described in chapter 4 that formed an alternative splice version, revealed by RT PCR using primers F2 and B7 (figure 4.9). The EST w05790 discussed above was 100% homologous to the longer PCR product amplified by these primers i.e. it contained this extra 53bp sequence. It appeared that this 53bp 'exon' formed an alternative splice version of both cDNA clone 1 and cDNA clone 2:2.

1 51 101

GCTTCTTCGG TTGCAGTCCT CTTGCTTCTT GCGCGTGCGT GTAGCGCTTT TGCAAAGCCG CGGAGGTGAA GTGAACTTAG AGGTTGTGGG GCCGAGGGGT

151 CGTCTTATAG CTACCAGCCC ACAGGCATTT AGTCTACGTT GGAGGTAAAC

201

AAATACGGGT CCTGCTTAGG AGAAAAGAAA AACGTCTTAC AGCCAGTGTC

251

TAAACTCCAA ACAACGGAAT GTATCAATGA M R

GACCTTGTAT

P C I

ATGGATACAC W I H GTGCATTTAA AACCGCCCTG CCGGCTTGTA GAGCTTTTGC CGTTCTCCAG 301

V H L K P P C R L V E L L P E S S

CGCTTTACAG GGGTTATCGC ACTTAAGCCT CGGAACAACT TTACCAGTGA 351

A L Q G L S H L S L G T T L P V I

TTCTACCAGA AAGGAATGAA GAACAGAACC TTCAGGAATT GAGTCACAAT

401 L P E R N E E Q N L Q E L

S H N

GCAGACAAAT ATCAAATGGG AGATTGTTGC AAGGAAGAGA TTGATGATAG 451

A D K Y Q M G D C C K E E I D D S

501

TATTTTCTAC I F Y

TAGCCATTGG GAAGATAAAA GGAGACAGAA GATTGAAGCC

551 TTTGCCAGCC ATTCTTTCCC TTTTTGCTTC CAAACTCCTC AACTGGGAAC

601 CTTCATATGT GCAGTATTTA TATTGGATCA TACTGGTGAT TATAAAAGTT

651

CCTAGGAGGC TAGAAGAGCC AACCAACAGA GAAGGGAAAG CAGTCTGTTC

701

TGAACATAGG GACATAAGTT CATTCATGCC AAGTATCTTT CCAGCATGTT 751

TCTCCCATTT AGAATATCTA GCATGTAAGG CCTTTCAATA TTAATATAAG

801

CCCAATATCA GCTCTTTCTC TTTGTATTTC ATCTCTTTCT ACTCTCCTAT 851

TTGTATTTTG TGTTCCTATC AAAGTGTCGT ATCTGGGAGA TGACCTGCCT

901

TATCCTGTTC TATAACAGTT TTGTTTGCTG CTGTGTCTTT AGAACAGTGC

951 CTGGCCACAC

AA

AGTAAGCACT CAATAAATCT TTGATGAATG AAAAAAAAAA

Figure 5.8: cDNA clone 2:2 showing the postulated open reading frame from the ATG to the TAG. The primers used to amplify the coding region in patients are underlined.

MRPCIWIHVHLKPPCRLVELLPFSSALQGLSHLSLGTTLP

MRPCIWIHVHLKPPCRLVELLPFSSALQGLSHLSLGTTLP

MRPCIWIHVHLKPPCRLVELLPFSSALQGLSHLSLGTTLP

VILPERNEEQNLQELSHNADKYQMGDCCKEEIDDSIFY,

VILPERNEEQNLQELSHNADKYQMGDCCKEEIDDSIFY.

VILPERNEEQNLQELSHNADKYQMGDCCKEEL

homology between Leu 1 and cDNA clone 2:2 differences between Leu 1 and cDNA clone 2:2

o V ) (N O

I

I

Figure 5.10.A: Showing PCR products from amplification o f the open reading frame o f cDNA clone 2:2, elctrophoresed on a 1% agarose 0.5 x TBE gel stained with ethidium bromide. All cDNAs, including those from CLL patients A and B, show 2 bands when amplified with the primers C12T7(5)-F and C12T3(6)-R.

1 51 101

G T C T A C G T T G GAGGTAAACA A ATACGGGTC C TG C TTA G G A GAAAAGAAAA A C G T C T T A C A G C C A G TG TC T AA ACTCCA A A CAACGGAATG TA TC A ATG A G

151

A C C T T G T A T A TGGATACACG T G C A T T T A A A A C C G C C C TG C CG G C TTG TA G

2 0 1

A G C T T T T G C C G T TC T C C A G C G C TTTA C A G G G G TTA TC G C A C T T A A G C C T C

2 5 1

GGAACAACTT TA CCA G A TGA GG AC AC CTG A G G TT C A G A T T AAGAAATCTG

301 C C C C A A A 6T C T T A 6 A A C T 6 T G A TT C TA C C A GAAAGGAATG AAGAACAGAA 351 C C TTC A G G A A TTG A G TC A C A ATGCAGACAA A TA TC A A A TG G G A G A TTG TT 4 0 1 GCAAGGAAGA G A TTG A TG A T A G T A T T T T C T A C T A G C C A T T GGGAAGATAA 4 5 1 AAGGAGACAG T C C A A A C TC C AAGATTGAAG TCA A CT C C T T T G C C A G C C A T T C T T T C C C T T T T T G C T

Figure 5.1GB: Sequence o f the larger band, shown in 5.10A. The 53 base pair sequence shown in bold is the sequence spliced out in the smaller band

When the additional sequence was included in the sequence of cDNA clone 2:2, the open reading frame was disrupted as there was a stop codon within this added sequence. This is underlined in figure 5. lO.B. This 53bp sequence is not the intron between the two exons of Leu 1 as they have been shown to lie over 19kb apart at the genomic level (Liu et a l, 1997).