trans

POST-TRANSCRIPTIONAL REGULATION IN KINETOPLASTIDS 81
shock protein (HSP) genes of kinetoplastids
were easy to find through high sequence conservation
with other HSP genes, and seemed
to be ideal candidates for identification of
inducible RNA polymerase II promoters. The
response to heat shock ought to be biologically
relevant, as parasites in the mammalian
host can cause fever, and the insect forms are
subjected to both variations in environmental
temperature within the insect and a temperature
rise of up to 10°C upon injection into a
mammalian host. Heat shock treatments of
these magnitudes have therefore been used to
analyse HSP expression in kinetoplastids: for
example, a switch from 37°C to 42°C to mimic
fever, and elevations from 25°C or 27°C to 35°C
to mimic the transfer of insect forms to the
skin of a mammalian host. The first candidates
to be investigated were the HSP70 genes
of T. brucei and Leishmania. These are arranged
as polycistronically transcribed tandem repeats,
and the levels of RNA were indeed mildly elevated
upon heat shock. It was therefore hoped
that there would be a promoter between each
gene or, failing that, one promoter upstream
of the cluster. Rather poor sequence similarities
with the controlling promoter elements
from higher eukaryotes were pointed out with
enthusiasm. But in the end – as in all other
searches for RNA polymerase II promoters
upstream of protein-coding genes – it became
clear that no promoters were there.
Meanwhile, additional HSP genes were
found. Expression of mitochondrial HSP60
has been little investigated (although it is an
excellent mitochondrial marker protein and
shows developmental regulation in T. brucei)
but Leishmanial HSP85 has been analysed
extensively. There is evidence from T. brucei
that mRNA trans-splicing is disrupted by heat
shock in bloodstream trypanosomes, but the
HSP70 mRNAs seemed little affected. Both
HSP70 and HSP85 (sometimes also called
HSP83) mRNAs may be more stable at high
temperatures than other RNAs, and there
is evidence from L. amazonensis that HSP83
genes are also preferentially translated during
heat shock. In L. infantum, there are six HSP70
genes arranged as a tandem repeat. Nearly
all the RNA comes from gene 6, but only
expression from gene 1–5 mRNAs was increased
by heat shock. In all cases investigated, the
sequences responsible have been shown to lie
in the 3-untranslated region. But it is most
unlikely that the regulation of HSP70 or HSP83
mRNA levels or translation has any biological
relevance, as no-one has ever shown any
changes in total HSP70 or HSP83 protein
levels as a consequence of heat shock. HSP70
and HSP83 each make up between 2% and 3%
of the total protein in unstressed Leishmania
promastigotes, which is presumably sufficient
to protect against physiological stresses.
The most interesting HSP found so far is
HSP100 from L. major and L. donovani. This
is upregulated during heat shock of promastigotes,
and during differentiation of promastigotes
into amastigotes; gene replacement
experiments indicate that it plays a role in parasite
virulence.
Degradation and translation of
the EP mRNAs
The EP and GPEET genes illustrate several
aspects of gene regulation in trypanosomes
(Figure 4.4). Regulation of EP transcription was
studied by inserting a CAT reporter gene, bearing
a 3-untranslated region that gave constitutive
expression, into the EP locus (Figure 4.5A).
Expression of the reporter was roughly 10-fold
higher in procyclic forms than in bloodstream
forms, indicating that transcription was regulated
10-fold. But EP RNA is extremely abundant
in procyclics, and virtually undetectable
in bloodstream forms, so the transcriptional
MOLECULAR BIOLOGY