View PDF Version - RePub - Erasmus Universiteit Rotterdam

cDNA sequence analysis
Sequence analysis was performed in both orientations on different selected overlapping MNI
cDNA clones. Sequencing this MNI transcript was not an easy task, as the first exon (4713
nucleotides) has a high overall GC content (69%). The numerous sequence artifacts
encountered were dissolved by sequencing other independent cDNA clones, subcloned
fragments andlor by using shifted internal primers with and without the interchange of dATP
and dGTP for 7-deaza dATP and 7-deaza dGTP in the sequencing reactions. The nucleotide
sequence which we have analyzed at the moment this chapter was written did not predict a
single contiguous ORF. Therefore, we used the GRAIL program to deduce the potential
protein coding regions from the nucleotide sequence. The program predicts two putative
ORFs with a reasonable length with the quality score excellent protein coding region. The
amino acid sequences of both ORFs between the stop codons are depicted in figure 6. The
most 5' ORF (Fig. 6A) contains 6 methionine codons, of these the ones at positions 53 and
88 show the closest match with the Kozak consensus sequence for translation initiation sites
(Kozak, 1987). Upstream of this ORF 3 additional ATGs are situated, all followed by stop
codons (data not shown). The 3' ORF (Fig. 6B) contains 8 methionine codons, of these the
ones at positions 88, 275, 548 and 651 fit best with the Kozak consensus sequence.
The MNI nucleotide sequence revealed two trinucleotide (CAG) repeat sequences
located between both ORFs. These repeats were investigated for tumor associated length
variation in our series of meningioma DNAs and control DNAs lIsing radioactive peR with
primers flanking the repeats and separation on sequence gels (Verkerk et aI., 1991). The
most 5' CAG repeat was not polymorphic in any of the 114 DNA samples analyzed,
including 89 meningiomas. However, the other CAG repeat was polymorphic and detected
three alleles differing six or nine nucleotides from the smallest allele respectively. The
frequencies of the alleles, as estimated from 92 unrelated DNAs of Caucasian origin, were
5% for the smallest, 73% for the middle one and 22% for the largest fragment. In 63
meningioma DNA samples all three alleles were recognized, but no increase in length was
observed (data not shown).
134