trans

POST-TRANSCRIPTIONAL REGULATION IN KINETOPLASTIDS 77
chloramphenicol acetyltransferase (CAT) or
luciferase. For quick analyses, constructs have
been transiently transfected into different
life-cycle stages, and the level of reporter gene
expression measured. In order to analyse the
behaviour of mRNAs, however, it is usually necessary
to generate permanent cell lines containing
the reporter constructs, because there
simply isn’t enough RNA made in transient
transfections. The constructs are usually integrated
in the genome of T. brucei, or present on
selectable episomes in T. cruzi and Leishmania.
In each case examined, the sequences responsible
for developmental regulation were found
to be in the 3-untranslated region, and low
RNA abundances were caused by RNA instability.
Genes with regulatory 3-untranslated
regions from T. cruzi include those encoding
amastin, tuzin, GP72, GP85, GP82, and
the genes encoding small and large mucins.
Examples from Leishmania include protein A2
and major surface proteins MSPL, MSPS, and
MSPC. The list of investigated genes is longest
for the African trypanosomes, where we have
strong regulation of aldolase, PGKB, PGKC,
two stage-specific hexose transporters, and
the major surface proteins VSG, EP and GPEET.
Taking PGK as an example again, the 3untranslated
regions of PGKB and PGKC are
completely different (Figure 4.3). A CAT gene
with the PGKB 3-untranslated region is expressed
in procyclics, but gives very little RNA or
protein in bloodstream forms. The reverse is
true for the PGKC 3-untranslated region.
Although the experiments with 3untranslated
regions and RNA degradation all
came to about the same conclusion, the studies
are difficult to compare quantitatively. One
problem is that several of the earlier studies
were done before it was known that correct
polyadenylation relies on the inclusion of the
next downstream splice site. The reporter
constructs were therefore designed on the
assumption that the polyadenylation signal
was in the 3-untranslated region, as in any
other eukaryote. This could result in incorrect
polyadenylation upstream of regulatory
sequences, or inclusion in the mRNA of
extra sequences, downstream of the normal
polyadenylation site, that could influence the
mRNA stability. Ideally studies should include
data showing that the mRNAs produced were
polyadenylated in the correct or expected
position. Other factors that could influence
the expression level are transcription rate and
gene copy number. Thus transcriptional promoters
should if possible be absolutely constitutive,
independent of life-cycle stage. And if
episomal vectors are used, or genes are integrated
in the genome, one has to determine
how many copies of the reporter are present in
each cell line.
To make different experiments comparable
it is extremely useful to include a control
reporter transcript that is expressed constantly
in all life-cycle stages examined. Examples that
have been used include transcripts containing
actin (ACT ) or tubulin (TUB) 3-untranslated
regions. Sometimes, a reporter gene with no
3-untranslated region at all has been used
instead. This is much less satisfactory because
the resulting mRNA may include plasmid
sequences and there is no guarantee that these
will be neutral in the assay. In transient transfection
assays, the inclusion of an unregulated
control is crucial, as transfection efficiencies
of different life-cycle stages are rarely identical.
Once it had been shown that a 3-untranslated
region is responsible for regulation,
the next step has been to attempt to identify
the sequences responsible by deletion or
replacement mutagenesis. This has seldom
been successful. Although it is expected that the
sequences involved should be quite short, usually
several hundred bases have proved essential
for regulation. The major reason for this
MOLECULAR BIOLOGY