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.
Article: Structure of the gene for mouse thymidylate synthase. Locations of introns and multiple transcriptional start sites.[show abstract] [hide abstract]
ABSTRACT: We have isolated and analyzed the structure of the gene for thymidylate synthase from a 5-fluoro-2'-deoxyuridine-resistant 3T6 cell line that overproduces thymidylate synthase 50-fold by virtue of gene amplification. Three overlapping DNA segments containing the entire thymidylate synthase gene were identified in Charon 35 genomic libraries. Sequence analysis revealed that all of the coding regions were contained in a 12-kilobase segment of DNA. The gene has 6 introns ranging from 0.6 to 4.1 kilobases in length. The sequences at the 5' and 3' ends of each intron conformed to the consensus sequences for mammalian introns. S1 nuclease and primer extension assays showed that transcription of the thymidylate synthase gene initiates at several sites within a 66-nucleotide region. There are no TATAA or CCAAT sequences in the vicinity of the initiation sites. However, the region does contain DNA sequences that are known to stimulate binding of the transcription factors Sp1 and USF. These binding sites are adjacent to each other, suggesting that the two proteins may bind to the upstream region of the thymidylate synthase gene in a cooperative or competitive manner.Journal of Biological Chemistry 01/1987; 261(34):16000-5. · 4.77 Impact Factor
Article: Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns.[show abstract] [hide abstract]
ABSTRACT: Restriction endonuclease DNA fragment patterns have been used to examine the relationships among 28 isolates of Leishmania as well as Crithidia, Endotrypanum, and Trypanosoma cruzi. Fragments of nuclear DNA were generated with six restriction enzymes, and blots were hybridized with probes from three loci. Among the major lineages the fragment patterns are essentially completely different, while within the major lineages various degrees of divergence are found. Molecular evolutionary trees were constructed using the method of Nei and Li to estimate the percent nucleotide sequence divergence among strains from the fraction of fragments shared. Defined groups, such as species or subspecies within the major lineages, are also grouped by nuclear DNA comparisons. Within the donovani complex, we find Leishmania donovani chagasi and Leishmania donovani infantum to be as similar as strains within Leishmania donovani donovani, consistent with the proposal by other workers that New World visceral leishmaniasis originated quite recently.Proceedings of the National Academy of Sciences 02/1987; 84(2):484-8. · 9.68 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Thymidylate synthetase and dihydrofolate reductase exist as a bifunctional protein in a number of species of protozoa which span diverse groups of the subkingdom. The enzymes copurify upon gel filtration and on affinity chromatography columns specific for dihydrofolate reductase. The bifunctional protein has been found in species of Crithidia, Leishmania, Trypanosoma, Plasmodium, Eimeria, Tetrahymena and Euglena. For reasons unknown, neither enzyme could be detected in Entamoeba histolytica or E. invadens. Since neither enzyme has yet been found as a separate protein in protozoa, it is likely that the bifunctional protein is widespread among these primitive eukaryotes. In most cases, the apparent size of the native protein is approximately twice that of the subunit possessing thymidylate synthetase. Further, with one exception, the subunit sizes are close to the sum of the subunit sizes of the separate enzymes found in other sources.Molecular and Biochemical Parasitology 05/1984; 11:257-65. · 2.55 Impact Factor
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
Received October 28, 1986; Revised and Accepted March 12, 1987
Accession no. Y00124
reductase-thymidylate synthase (DHFR-TS) gene of the protozoan
parasite Leishmania major has
by growth phase or developmental
5' region of the DHFR-TS gene does not reveal
with other trypanosomatid genes, eukaryotic consensus genetic
elements, or the mammalian DHFR promoter element.
is especially significant as we show that the 5' region
coli DHFR gene exhibits homology
despite their greater evolutionary distance.
5' structure of mRNA
transcribed from the dihydrofolate
been characterized. S1 nuclease
stage. The DNA sequence
to the mammalian DHFR
number of novel molecular phenomena (1,2).
unusual structure of cellular mRNAs, which exhibit a bipartite
35 nucleotide extension
5' end (3,4). This sequence, the "mini-exon" (5) or "spliced
(6), is encoded by
a separate genetic locus from the body
absence of "consensus" genetic elements implicated
aspects of gene expression
the questions associated with gene
genes which have been studied
provide useful comparative
because they catalyze critical
of the family Trypanosomatidae exhibit
in other eukaryotes (reviewed
in other species would
in the de novo synthesis
©I RL Press Limited, Oxford, England.
Nucleic Acids Research
genes encoding these enzymes
transfection methodology, allowing localization of cis-acting
ends of DHFR and TS mRNAs (15,18,20,21,22),
the different mouse DHFR mRNAs being invariant thoughout the cell
DHFR and TS thus represent paradigms for the study of
structure and expression.
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-
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
at both the
with the profile of
a single gene (24-26).
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
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
Intracellular amastigotes were obtained from lesions initiated
Amastigotes were purified from the infected tissue by Percoll
in vitro in medium M199 as
MTX for the R1000
Nucleic Acids Research
RNA filter hybridization.
1% agarose gels (35).
with 50 mM NaOH, 10 mM NaCl for 45 minutes; 100 mM Tris-
for 45 minutes; and 20x SSPE for
10 mM NaPO4
nitrocellulose filters. Filters were subsequently baked under
3 hours at 800C and stained with methylene blue prior
for 12 hours
at 450C in
M Tris (pH 7.0), 0.1% SDS, 0.1% sodium pyrophosphate,
10 ug/ml poly-U,
10 ug/ml poly-C,
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).
with 50% formamide,
20 minute washes
at 550C with
to X-ray film.
was performed according
of Berk and Sharp (37).
Two probes were utilized:
labeled at the
5' Sau 3A site
and gamma 32P-ATP (ICN); 2) the 458 bp Pvu
on 6% formaldehyde,
Following electrophoresis the gels were
20 mM MOPS (pH
1 hour (lx SSPE=
M NaCl) and blotted overnight onto
100 ug/ml denatured
for 40 hours
of denatured radiolabeled
for 10 min
at 450C, followed by four
5 x 109 cpm/ug. Filters were washed
to the method
1) the 575 base
II fragment (positions
Nucleic Acids Research
+13 to -445),
by primed synthesis (M13 17 bp sequencing primer; New England
in the presence
of 32P-dCTP (Amersham),
stranded DNA from
an M13 recombinant containing this fragment
The fragment sizes reported are the
least four determinations,
technique (40) with alpha 35S-dATP (41) and Leishmania-derived
DNAs cloned into M13 vectors (38). The sequence from -760 to +1
on both strands,
in the mRNA-coding direction.
on the mRNA-complementary
rounded to the nearest
while that from -952 to -761
1; see Fig.
the DHFR-TS gene
S1 nuclease analysis using the
1D for location
line revealed five protected
of the DHFR-TS mRNA.
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
data not shown).
R1000 line reveals
RNA species of
its apparent intensity reflects
phase promastigotes or
575 bp Sau 3A-
Nucleic Acids Research
sizes 330, 380, 430, 510 and 570 nucleotides (nt; Fig. 1B).
two major Si-resistant
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.
nuclease protection profiles
with the -245 product being approximately two-fold more abundant
the -375 product.
We next examined mRNAs isolated from the wild-type LT252 line,
isolated from chronic mouse
Protected fragments of approximate sizes 390,
in the R1000 line (Fig. 1C, lane
fragments are evident
in the promastigotes
to the -195 S1 site and fully protected probe were not detected
detected in the LT252 amastigote preparation (Fig. 1C, lane 3),
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
the levels of the Si products are all elevated in
DNA seqence of the
of the DHFR-TS jene.
Blot hybridization analysis of RNA revealed that the
RI fragment immediately
similarly abundant polyadenylated RNA (unpublished data). This
suggested that the Sal
transcribed DNA separating the two mRNAs, which
of this fragment,
to positions -375and -245
a product the size of the
I fragment was observed which was not
1B, lane 1).
S1 mapping of
,lanes2 and 3)
1C shows the results
310 and 260 nt are
1) and similarly sized
from the parental
-445. sense+13 to
ntand260 ntfragments were
and wild type amastigotes.
0.7 kb Sal
5' of the Eco RI site (Fig.
- Eco RI fragment must
contain any non-
5' of the