Læknablaðið - 15.09.1993, Blaðsíða 43

LÆKNABLAÐIÐ 1993; 79: 293-4
293
NÝR DOKTOR í LÆKNISFRÆÐI
JÓN JÓHANNES JÓNSSON
Þann 31. júlí 1992 varði Jón Jóhannes
Jónsson, læknir, doktorsritgerð við
Minnesotaháskóla í Bandaríkjunum. Ritgerðin
nefnist á frummálinu »Expression of the
Human Purine Nucleoside Phosphorylase
Gene«. Fer ágrip hér á eftir.
The T cell immunodeficiency associated
with purine nucleoside phosphorylase (PNP)
deficiency is considered a prototype disease
for gene therapy. This thesis describes
identification and characterization of key
regulatory elements of the human PNP
gene and the subsequent engineering of
abbreviated PNP genes for retroviral-
mediated transduction. The sequence of the
PNP promoter was characterized and the
transcriptional start site identified. Minigenes
containing various complements of tentative
PNP regulatory elements were constructed and
their expression tested in NIH 3T3 fibroblasts.
A 91 bp minimal promoter was defined and
all elements necessary for optimal expression
were included in the first 217 bp 5’ of the
transcriptional start site. Introns were required
for expression of the human PNP gene.
Intron-1 had the greatest effect on expression
which could be explained by an enhancer
located between 424 to 577 bp within the
first intron. These results presumably explain
poor expression of retroviral vectors with
the PNP coding sequence regulated by
internal heterologous promoters. A full length
retroviral vector genome appears to abrogate
the intron-requirement for PNP expression
but the long terminal repeat is an unreliable
promoter in hematopoietic progenitors
undergoing differentiation.
To comply with the intron-requirement
for PNP expression five retroviral vectors
containing various PNP minigenes in
the reverse orientation were constructed,
packaged, and tested by infecting fibroblasts.
Two types of deletions were repeatedly
present in proviruses after one round of
replication:
i) Deletions flanked by direct repeats with
one copy of the repeat remaining in truncated
proviruses. These deletions presumably
resulted from reverse transcriptase slippage
on RNA template during (-) DNA strand
synthesis.
ii) Deletions due to fortuitous splice sites in
the PNP complementary strand. Two 5’ splice
sites and three 3’ splice sites were identified
in a 3.0 kb PNP minigene. The fortuitous 5’
splice sites but not the 3’ splice sites could
be predicted by searching for consensus
sequences of mammalian splice sites in the
PNP complementary strand. Elimination or
modification of PNP sequences associated
with deletions in retroviral vectors in the 5’