Sequence and S1 nuclease mapping of the 5' region of the dihydrofolate reductase-thymidylate synthase gene of Leishmania major.

Abstract

The 5' structure of mRNA transcribed from the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of the protozoan parasite Leishmania major has been characterized. S1 nuclease mapping identifies a heterogenous 5' structure which is not affected by growth phase or developmental stage. The DNA sequence of the 5' region of the DHFR-TS gene does not reveal homology with other trypanosomatid genes, eukaryotic consensus genetic elements, or the mammalian DHFR promoter element. This latter finding is especially significant as we show that the 5' region of the E. coli DHFR gene exhibits homology to the mammalian DHFR promoter element, despite their greater evolutionary distance.

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Volume Sequence and Si nuclease mapping of the 5' region of the dihydrofolate reductase-thymidylate
Volume 15 Number 8 1987
15 Number 8 1987
Nucleic Acids Research
Nucleic Acids Research
-Sequence and S1 nuclease mapping of the 5'regionof the dihydrofolate reductase-thymidylate
synthase gene of Leishmania major
Geoffrey M.Kapler1.2, Kang Zhangt and Stephen M.Beverleyl*
'Department of Pharmacology and 2Department of Genetics, Harvard Medical School, Boston, MA
02115, USA
Received October 28, 1986; Revised and Accepted March 12, 1987
Accession no. Y00124
ABSTRACT
The
reductase-thymidylate synthase (DHFR-TS) gene of the protozoan
parasite Leishmania major has
mapping
iden ifii
affected
by growth phase or developmental
of the
5' region of the DHFR-TS gene does not reveal
with other trypanosomatid genes, eukaryotic consensus genetic
elements, or the mammalian DHFR promoter element.
finding
is especially significant as we show that the 5' region
of the
E.
coli DHFR gene exhibits homology
promoter eTiment,
despite their greater evolutionary distance.
5' structure of mRNA
transcribed from the dihydrofolate
been characterized. S1 nuclease
a heferogenous
5'
structure
stage. The DNA sequence
which
is
not
homology
This latter
to the mammalian DHFR
INTRODUCTION
Protozoan
number of novel molecular phenomena (1,2).
unusual structure of cellular mRNAs, which exhibit a bipartite
structure
in which
a common
35 nucleotide extension
the
5' end (3,4). This sequence, the "mini-exon" (5) or "spliced
leader"
(6), is encoded by
a separate genetic locus from the body
of
the
mRNAs
(7,8),
and
is
added
unknown.
Another feature
of trypanosomatid
general
absence of "consensus" genetic elements implicated
various
aspects of gene expression
in
1).
To approach
some of
the questions associated with gene
expression
in
trypanosomatids,
ubiquitous
genes which have been studied
provide useful comparative
insights.
The
enzymes
dihydrofolate
reductase
synthase
(TS)
have
been extensively
because they catalyze critical
steps
precursors
forDNA
synthesis
parasites
of the family Trypanosomatidae exhibit
a
Among these
is the
is
found
on
by
a mechanism(s)
genes
which
been
is
hasthe
in
in other eukaryotes (reviewed
we
reasoned
that
examination
of
in other species would
(DHFR)
and
thymidylate
many species,
studied
in the de novo synthesis
are
important
in
of
and
targets
for
©I RL Press Limited, Oxford, England.
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Nucleic Acids Research
chemotherapy (9,10).
genes encoding these enzymes
regulated at
steps
(11-17).
transfection methodology, allowing localization of cis-acting
transcriptional
elements
such
Structural
studies
indicate
heterogeneity
ends of DHFR and TS mRNAs (15,18,20,21,22),
the different mouse DHFR mRNAs being invariant thoughout the cell
cycle (23).
DHFR and TS thus represent paradigms for the study of
gene
structure and expression.
In teishmania
and
all
protists
bifunctional polypeptide encoded by
others have recently reported the DNA sequence of the protein
coding portion of the DHFR-TS gene (27,28), which consists of a
5' DHFR domain followed by the TS domain. These studies have been
aided by the availability of lines which have amplified the DHFR-
TS
gene
ona
30kb
extra-chromosomal
consequently
overproduce
the DHFR-TS mRNA
paper we report on the
5' structure of the Leishmania DHFR-TS
gene and mRNA.
In mammal ian cell s the expression of the
is growth
phase
transcriptional
and
These genes
have
been examined
andcell
cycle
both
post-transcriptional
using
DNA
as promoters,
etc.
(18,19).
5'and
at both the
with the profile of
3'
DHFRandTSexist
as
a
a single gene (24-26).
We and
circul ar
and
DNA
(29)
In
and
this
protein.
MATERIALS AND METHODS
Preparation of promastigotes and amastigotes.
The wild-type LT252 line of Leishmania major (30,31) and its
methotrexate (MTX) resistant derivative R1000-3 line (29) were
studied. Promastigotes were propagated
described
(25),
which contained
Stationary phase cells were harvested 48 hours after reaching
maximal cell density.
The viability of these cells was >95% as
determined by light microscopic examination of morphology and
motil ity.
Intracellular amastigotes were obtained from lesions initiated
by
intradermal
injection
of
promastigotes
into
the
dorsal
Amastigotes were purified from the infected tissue by Percoll
gradient
centrifugation
(32).
in vitro in medium M199 as
MTX for the R1000
1 mM
line.
2x
107
stationary
of
Balb/c
phase
mice.
hind
foot
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RNA
isolation.
Promastigote
parasites
precipitation
Polyadenylated
cellulose chromatography
RNA filter hybridization.
RNA was
fractionated
1% agarose gels (35).
treated
with 50 mM NaOH, 10 mM NaCl for 45 minutes; 100 mM Tris-
HCl
(pH 7.4)
for 45 minutes; and 20x SSPE for
10 mM NaPO4
[pH 7.0],
0.15
nitrocellulose filters. Filters were subsequently baked under
vacuum for
3 hours at 800C and stained with methylene blue prior
to
hybridization
(35),
allowing
identification
of
molecular
prehybridized
for 12 hours
at 450C in
NaCl, 0.15
M Tris (pH 7.0), 0.1% SDS, 0.1% sodium pyrophosphate,
0.2%bovine
serum
albumin,
0.2%
ficoll,
10 ug/ml poly-U,
10 ug/ml poly-C,
salmon sperm
DNA,
followed
by hybridization
above
solution
containing
107
cpm/ml
probe. The hybridization probe consisted of a 0.9 kilobase (kb)
Eco RI- Bgl
II fragment, corresponding to the genomic sequence
from -252 to +668 of the DHFR-TS gene (Fig. 1D).
was
radioactively
labeled
by
randomly primed
approximately
with 50% formamide,
5x SSPE,
0.1% SDS
20 minute washes
at 550C with
exposed
to X-ray film.
S1 nuclease
ma_pp
S1
nuclease
analysis
was performed according
of Berk and Sharp (37).
Two probes were utilized:
Sau
3A
-
Pst
restriction
fragment
relative
to
the
AUG
translation
labeled at the
5' Sau 3A site
using polynucleotide
and gamma 32P-ATP (ICN); 2) the 458 bp Pvu
and
M
of
RNA
amastigote
guanidinium
RNA
with
was
prepared
(34).
RNA
isothiocyanate
M
lithium
by
oligodeoxythymidylate
-was
isolated
by
lysing
in
5
followed
chloride
by
4
(33).
on 6% formaldehyde,
Following electrophoresis the gels were
20 mM MOPS (pH
7.0),
1 hour (lx SSPE=
M NaCl) and blotted overnight onto
quantitation
weight
markers.
of
RNA
and
were
0.75
Filters
50% formamide,
M
polyvinylpyrollidone,
100 ug/ml denatured
for 40 hours
of denatured radiolabeled
0.2%
in the
This fragment
synthesis
for 10 min
at 450C, followed by four
0.1x SSPE,
0.1% SDS,
(36)
to
5 x 109 cpm/ug. Filters were washed
dried
and
to the method
1) the 575 base
+135
ite;
Fig.
kinase
II fragment (positions
I
(positions
initiation
to
-440
1D),
(35)
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+13 to -445),
by primed synthesis (M13 17 bp sequencing primer; New England
Biolabs)
in the presence
of 32P-dCTP (Amersham),
stranded DNA from
an M13 recombinant containing this fragment
the
appropriate
orientation
(38).
annealed
with
the appropriate
digested
with
Si
nuclease
and analyzed
acrylamide/8M
urea gels
(39).
The fragment sizes reported are the
averages
of at
least four determinations,
10
nucleotides.
DNA
sequencin
DNA sequencing
was performed
technique (40) with alpha 35S-dATP (41) and Leishmania-derived
DNAs cloned into M13 vectors (38). The sequence from -760 to +1
was determined
on both strands,
obtained
solely
in the mRNA-coding direction.
uniformly
labeled
on the mRNA-complementary
strand
using single
in
Polyadenylated
DNA probe
for
by electrophoresis
RNA
at
was
3 hours
530C,
on
4%
rounded to the nearest
using
the dideoxynucleotide
while that from -952 to -761
was
RESULTS
Blot analysis
Total
3.2 kb
from the
lane
1; see Fig.
transcript
absent
in nonpolyadenylated
hybridizing
material
polyadenylated
contaminating
the
relative
abundance
small
Leishmania
the
only
RNA recognized
the DHFR-TS gene
amastigotes
(27;
S1
nuclease mappinj
amastigotes.
S1 nuclease analysis using the
(see
Fig.
1D for location
the R1000
line revealed five protected
of the DHFR-TS mRNA.
RNA from
the
when hybridized with the 0.9 kb Eco RI-
5' end of the Leishmania mai.or DHFR-TS
1D for the location of the probe). This
is present
in polyadenylated
RNA
evident
or
nonpolydenylated
the preparation;
of
the amplified
genome
(25). The
by hybridization
in
log
or stationary
data not shown).
ofDHFR-TS
R1000 line reveals
a single
RNA species of
Bgl
II fragment
gene (Fig.
1A,
3.2 kb
2) and
RNA
IA,
total
RNA
(Fig.
lane
1A,
3).
lane
Additional
but
not
to
(Fig.
the
in
RNA,
attributed
in
isDNA
its apparent intensity reflects
DHFR-TS
3.2
kb polyadenylated
probes
phase promastigotes or
gene
within
the
is
RNA
from throughout
mRNA
from
promastigqtes
and
575 bp Sau 3A-
and
polyadenylated
fragments
Pst
I fragment
RNA from
of approximate
of probes)
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sizes 330, 380, 430, 510 and 570 nucleotides (nt; Fig. 1B).
two major Si-resistant
products map
relative to the start of translation. Two minor prodcucts map to
positions -295 and -195. In addition,
fully protected Sau 3a- Pst
detected when RNA was omitted (Fig.
log
andstationaryphase
R1000
identical
nuclease protection profiles
with the -245 product being approximately two-fold more abundant
than
the -375 product.
We next examined mRNAs isolated from the wild-type LT252 line,
from
either
promastigotespropagated
isolated from chronic mouse
lesions.
of
an
Si
nuclease analysis
using
complementary
to theDHFR-TS
Protected fragments of approximate sizes 390,
evident
in the R1000 line (Fig. 1C, lane
fragments are evident
in the promastigotes
wild-type
LT252 line(Fig.
1C,
lane
to the -195 S1 site and fully protected probe were not detected
with
this
probe.Only
the390
detected in the LT252 amastigote preparation (Fig. 1C, lane 3),
however,
the signalintensity would be insufficient to reveal
less abundant species.
These data indicate that by Si analysis
thestructureof the 5'endof DHFR-TS
amplified and wild type promastigotes
In addition,
the levels of the Si products are all elevated in
the amplified
R1000
line.
DNA seqence of the
of the DHFR-TS jene.
Blot hybridization analysis of RNA revealed that the
I- Eco
RI fragment immediately
hybridized
to
both
the
DHFR-TS
similarly abundant polyadenylated RNA (unpublished data). This
suggested that the Sal
transcribed DNA separating the two mRNAs, which
cis-acting
transcriptional
elements.
nucleotide
sequence
of this fragment,
DHFR-TS translation
initiationcodon
The
to positions -375and -245
a product the size of the
I fragment was observed which was not
1B, lane 1).
promastigote
(Fig.
1B
S1 mapping of
mRNAs generated
,lanes2 and 3)
invitro,
1C shows the results
uniformly
labeled
strand from
310 and 260 nt are
1) and similarly sized
from the parental
Fragments
oramastigotes
Figure
a
probe
-445. sense+13 to
2).corresponding
ntand260 ntfragments were
mRNAs isthesame
in
and wild type amastigotes.
5' region
0.7 kb Sal
5' of the Eco RI site (Fig.
mRNA
as
well
1D)
as
to
another
I
- Eco RI fragment must
contain any non-
might encode
determined
952 bp
We
the
extending
(Fig.
5' of the
2).
3373