10^ phage clones of a stage 12-15 chick embryo cDNA library in XzapII, were
screened with the Idd clone KM4, and three positives were isolated. The chick cDNA positive clones C2, C3 and C5 were rescued in pBS and found to contain inserts of 2kb, 3 kb and 5kb respectively. Each clone was end sequenced using the sequencing primers T3 and T7, and the sequence obtained was compared to the human and murine Idd nucleotide and peptide sequences. C2, C3 and C5 all had one identical end, that was found to contain the leader peptide sequence and first LDLR-like binding domain (figure 3. It). At the amino acid level, Cidd was found to be 75% identical to the murine Idd gene, with two gaps (figure 3. Iv). The human IDD gene is 74% identical to this chick clone, at the protein level. Interestingly there are no gaps between the human and chick predicted proteins.
The other end of each chick cDNA clone contained different nucleotide sequences to each other, that were not homologous to any part of the human or murine Idd genes. This suggests that each chick clone contained partial Cidd sequences, that had become co-ligated to other cDNA sequences during construction of the cDNA library. For this reason, the chick clones were not sequenced entirely.
c g c t t c g c t c c c c g c g c c t t t g t g g c g c t g c g c c g g g g g a g g a t g a a c g g a g g a t a a a t g M g t g c c c a a a g c g g a c a g c g g c a c c t t c c t c c t c c t c t t c c t c c t g g t g c t g a g c a t c a c c 6 1 + + + + + + 120 V P K A D S G T F L L L F L L V L S I T g a g c c g c t g c g g c c a g a g c t c c g c t g c a c c c c a g g g c a g t t t g c g t g t c g g a g t g g t a c c E P L R P E L R C T P G Q F A C R S G T a t c c a g t g c a t c c t t c c c a a c t g g c a g t g c g a t g g a t g g c c c a c c t g t g a g g a t g a g a g c I Q C I L P N W Q C D G W P T C E D E S g a t g a g g t t g a c t g t c c a g g a t t g a c a g g g g a c c a g c g c a c t t a c c a t g g g a a g g a g a a t D E V D C P G L T G D Q R T Y H G K E N gtggactccaggtataatcgagggagaggag agacgacc V D S R Y N R G R G E T T
F ig u r e 3 .lu . N ucleotide sequence an d p red ic ted p ro tein sequ en ce o f the p a r tia l ch ick cDNA clone C5 (Cidd).
BESTFIT of: Cidd.pep from: 1 to: 94 to :Tdd.pep from: 1 to: 548
Percent Similarity: 82.609 Percent Identity: 75.000
1 M V P K A D S G TFLLLFLLVLSITEPLRPELRCT PGQFACRSGTIQCILPNWQ 50 l l l l l l l | . | | | l l l l l | . : | | | | | | | | | | . | | | | | | : : | | | l l l II 1 M V P K A D S G AFLLLFLLVLTVTEPLRPELRCN PGQFACHGGTIQCIPLPWQ 50 51 CDGWPTCEDESDEVDCPGLTGDQRTYHGKEN VDSRYNRGRGETT 94 M M M M M I M M =11 = . 1- 1 l l l - l l I - h l h - - 51 CDGWPTCEDKSDEADCP.VTGEARPY.GKET VDLRQGRARGGDP 92
F igure 3 .lv . B estfit an alysis betw een the chick C idd an d the m urine Idd p e p tid e seq u en ces. The peptide sequence of C id d is shown on the top
line of each alignment in red type. Identical peptides are marked with a vertical line. Peptides with very similar chemical characteristics, and slightly similar chemical characteristics are indicated by two dots and one dot respectively. Gaps within the alignment are indicated by red type dots.
3.2 Mutation Screening of HIRA.
No TUPLE mutations have been detected in non-deleted DGS patients after extensive mutation screening (U. Atif unpublished results). HIRA, as previously discussed, represents a more abundant splice variant of TUPLE with an additional 207 internal residues (621 bp), plus an extra 44 N-terminal residues (132bp) (Lamour
et al 1995). These DNA stretches may represent hotspots for DNA mutation in the non-deleted DGS patients, however, initial analysis of the additional 5’ nucleotides has also detected no mutations (R.Wadey, unpublished results). In this study the remaining variant sequences were examined.
Nested PCR reactions were performed to amplify the 621bp fragment fi'om HIRA for mutation detection, as shown in figure 3.2a. cDNA derived from ectopically
transcribed RNA from fifteen patients, diagnosed to fall within the criteria associated with the CATCH22 phenotype, were used for this analysis. The second round PCR products from each nested PCR (primers Fl/R l amplified product C5-621A and primers F2/R2 amplified product C5-621B) were analysed by SSCP.
F 2 ►— - - - R2 (F2/R2 338bp) (F1/R1 356bp) F I --- ► — _ _ _ _ _ _ _ ^ ---- R1 C5T7F3 — ► — — — — — — — — ^ C5T7R3 C5T3F4 ► _ _ _ _ _ _ _ _ - ^ ' C5T3R4 1621 bp HIRA 2001 bp 2622bp
Figure 3,2 a, A schematic diagram o f HIRA showing the 621 bp region screened fo r mutations. Two rounds of PCR amplification using nested primer pairs
(C5T3F4/C5T3R4 : F l/R l) and (C5T7F3/C5T7R3 : F2/R2), resulted in DNA products C5-621A and C5-621B respectively.