|
|
|
|||||||
|
|||||||||
(Received for publication, April 21, 1995; and in revised form, July 12, 1995) From the
The genome of Trypanosoma cruzi contains tandemly
arrayed copies of the gene encoding amastin, an abundant protein on the
surface of the amastigote stage of the parasite. The transcription rate
of the amastin genes is the same in the different developmental stages,
but the steady state level of the 1.4-kilobase amastin mRNA is
50-85 times higher in amastigotes than in epimastigotes or
trypomastigotes(1) . Here we show that the amastin genes
alternate with genes encoding another protein, called tuzin, whose
1.7-kilobase mRNA is much less abundant in amastigotes. The
3`-untranslated region (UTR) of tuzin mRNA is only a few nucleotides in
length or even nonexistent, in contrast with the 630-nucleotide 3`-UTR
of amastin mRNA. No promoter elements were found upstream or within the
amastin/tuzin gene cluster. However, in amastigotes, the protein
synthesis inhibitor cycloheximide caused a 3-fold decrease in amastin
mRNA and a 7-fold increase in tuzin mRNA. Furthermore, when the amastin
3`-UTR plus its downstream intergenic region were fused behind the
luciferase coding region in a chimeric plasmid for transient
transfections, luciferase activity increased 7-fold in amastigotes and
decreased 5-fold in epimastigotes. Thus, developmental expression of
these alternating genes is regulated by different mechanisms.
Trypanosoma cruzi, the protozoan parasite that causes
Chagas' disease, constitutes a major public health problem
throughout much of Latin America. During its life cycle in the reduviid
bug vector and a mammalian host, the parasite goes through two
extracellular stages called the epimastigote and trypomastigote forms
and one intracellular stage called the amastigote form. In mammalian
hosts, the parasites exist mainly as intracellular amastigotes in a
variety of cell types. Relatively little is known about the biochemical
and immunological properties of this intracellular form compared with
the epimastigote form, primarily for the practical reason that
epimastigotes grow readily in cell-free culture, whereas amastigotes
can only be obtained from infected animals or cultures of mammalian
cell lines. Recently, we showed that the amastigote form of T.
cruzi has on its surface a family of closely related glycoproteins
collectively called amastin whose mRNA level is at least 50-fold higher
in amastigotes than in epimastigotes and trypomastigotes(1) .
The 174-amino acid sequence of nascent amastin has four distinct
hydrophobic domains of 20-30 amino acids each, suggesting that
the protein may span the outer membrane. The biological function of
amastin is not known, but its abundance and surface location suggest
that it plays an important role in the interaction between amastigotes
and their cytoplasmic environment. Amastin is encoded by eight or
more tandem genes of 1.2 kb ( Another unusual
feature of gene expression in trypanosomatids is the apparent lack of
promoters at the beginning of tandem gene clusters where transcription
by RNA polymerase II could be initiated. To date, the only identified
promoters for protein-encoding genes of trypanosomatids are in African
trypanosomes. In these organisms, the promoters for genes encoding two
different surface proteins, the variant surface glycoprotein (VSG) and
the procyclic acidic repetitive protein (PARP), were detected by their
ability to drive expression of a reporter gene in transfected
cells(11, 12) . However, these genes appear to
represent a special case because their transcription is resistant to
Two reports describing expression of foreign genes in T.
cruzi indicated that the presence of properly oriented 5`
sequences is essential for expression of a reporter gene. In one study
a segment of the T. cruzi SL gene inserted in front of
chloramphenicol acetyltransferase gene was shown to facilitate
expression of chloramphenicol acetyltransferase(15) , and in
another report, expression of the neomycin phosphotransferase and
chloramphenicol acetyltransferase genes was obtained following
transfection with a plasmid bearing these genes flanked by regions of
the glyceraldehyde 3-phosphate dehydrogenase gene(16) . Since
neither of these studies defined the sequences required for mRNA
expression in T. cruzi, we undertook the present study to
search for potential regulatory elements upstream of or within the
amastin gene family. We discovered that the 3-kb repeats contain a
previously unknown gene of 1.5 kb in addition to the 1.2-kb amastin
gene, leaving intergenic regions (IRs) of only 111 and 145 bp between
these two gene classes. The new gene potentially encodes a protein we
have named tuzin for T. cruzi expressed protein. Since the
steady-state levels of both amastin mRNA and tuzin mRNA vary
dramatically among the three developmental stages, the signals for
their respective post-transcriptional regulation likely occur within
their UTRs or short IRs. We show here that indeed the 3`-UTRs and IRs
contribute to the differential expression of amastin and tuzin mRNAs in
the different developmental stages.
To generate plasmid pL3T containing the 5`-flanking
region of the amastin cluster, a 2.9-kb HindIII-PstI
fragment located upstream the first amastin gene in recombinant phage
Figure 1:
Diagram of the 5` region of the
amastin/tuzin gene cluster and the sequence of an interior tuzin gene
plus its flanking regions. A,large and smallshadedrectangles indicate the amastin coding
regions and 3`-UTRs, respectively. Openrectangles represent the tuzin genes whose 3`-UTRs of 0-20 bp are too
small to show here. Restriction sites are for BamHI (B), BglII (Bl), EcoRI (E), PstI (P), and SphI (S). The openarrowhead indicates the
position at which the sequence preceding the entire gene cluster
diverges from the sequence preceding the interior tuzin genes, and
corresponds to the openarrowhead in panelB. The genomic DNA segment in bacteriophage clone
Figure 4:
Identification of sequences preceding or
within the amastin/tuzin gene cluster that facilitate expression of a
luciferase reporter gene in epimastigotes. Restriction fragments
indicated by the patternedboxes in Fig. 1A were cloned in either forward or reverse
orientation at the 5` end of the luciferase gene in the pGEM-luc vector. The smallblackboxes indicate
the locations of a 73-bp fragment containing the amastin SL addition
site and its upstream polypyrimidine tract. A 520-bp fragment
containing the 3`-UTR of the TCR27 gene and extending 135 bp beyond its
polyadenylation site was cloned downstream of the luciferase gene. T. cruzi epimastigotes were transfected with 100 µg of
each of the depicted plasmids and assayed for luciferase activity 48 h
later.
Figure 3:
Analysis of transcripts from the
5`-flanking region of the amastin/tuzin gene cluster. A,
photograph of an ethidium bromide-stained agarose gel containing the
cloned restriction fragments 1-5 shown in Fig. 1A (1-5), pBluescript (v), a fragment
encoding part of the 24 S
Figure 5:
Effect of different 3`-UTRs and downstream
IRs on luciferase expression in epimastigotes and amastigotes of T.
cruzi. Plasmid pLRT contains the promoter for a T. cruzi rRNA gene (24) inserted in the forward orientation at the HindIII site upstream of the amastin SL site in plasmid pLST.
In the pLRA and pLRC plasmids, the TCR27 3`-UTR of pLRT was replaced by
the amastin 3`-UTR plus downstream IR, and by the tuzin 3`-UTR plus
downstream IR, respectively. T. cruzi epimastigotes and
amastigotes were transfected with 20 and 100 µg, respectively, of
each plasmid and luciferase assays were performed 48 h later. In the Foldincrease columns, the numbers in brackets were obtained by normalizing the luciferase activity
units of pLRT to 1 and then expressing the luciferase activity units of
pLRA and pLRC relative to that normalized value. For example, in
amastigotes pLRA expresses 7.2-fold more luciferase than pLRT. The
ratio of these normalized values in amastigote transfectants versus epimastigote transfectants is shown in the last
column.
In addition to the above characterization, 3-kb fragments from BglII- or EcoRI-digested T. cruzi DNAs were
eluted from an agarose gel and ligated into pBluescript. After colony
hybridization with an amastin cDNA, two plasmids with 3-kb inserts,
named pAB3 and pAE3, were chosen for study. In both inserts, the
sequence between the stop codon of the upstream amastin gene and the
start codon of the downstream amastin gene was determined, and this
segment of the pAE3 insert is shown in Fig. 1B. This
intergenic sequence was compared with the sequence upstream of the
first amastin gene in the cluster. The two sequences are completely
different upstream of the openarrowhead shown in Fig. 1, A and B, and nearly identical
downstream of this position. In the intergenic sequence shown in Fig. 1B, distinctive polypyrimidine tracts occur just
upstream of the point of divergence. Polypyrimidine tracts have been
found to be essential for trans-splicing of the SL in Trypanosoma brucei and Leishmania
enrietti(29, 30) , and this motif provided the
first hint that the 3-kb repeats might contain another gene besides the
amastin gene.
To determine if the tuzin region is transcribed, we
prepared Northern blots of RNAs isolated from the epimastigote,
amastigote, and trypomastigote stages. The blot shown in Fig. 2was probed in succession with the coding regions for tuzin (panelB), amastin (panelA), and
24 S
Figure 2:
Northern blot analysis of transcripts from
the amastin/tuzin gene cluster. The blot containing total RNAs from
epimastigotes (E), amastigotes (A), and
trypomastigotes (T) was probed in succession with a
PCR-generated fragment from the tuzin coding region (panel B,
positions 1131-1648 in Fig. 1B), an amastin cDNA (panelA), and a fragment containing part of the 24
S
To map the 5` and 3` ends
of the tuzin mRNA, five tuzin cDNAs were isolated from about 10,000
clones of an amastigote cDNA library. The same number of library clones
yielded more than 100 amastin cDNA clones, consistent with the Northern
blot that suggested amastin mRNAs are about 20 times more abundant in
amastigotes than tuzin mRNAs. Partial sequencing of the five tuzin cDNA
clones demonstrated that in four cases the polyadenylation sites were
located either seven or 20 nucleotides beyond the stop codon, and in
one case polyadenylation occurred at the A residue within the UAG stop
codon itself, as indicated in Fig. 1B. The RNA molecule
that gave rise to this latter cDNA apparently did not have any 3`-UTR
nucleotides other than the poly(A), but its translation termination was
preserved because the UAG was converted to a UAA stop codon. Since
none of the five tuzin cDNAs contained a 5` SL, PCR amplification was
performed on an sample of the cDNA library using a SL primer and a
primer complementary to nucleotides 886-906 in the tuzin coding
region. The sequence of the resulting 140-bp amplification product
demonstrated that the AG dinucelotide at position 777 serves as the
main splice acceptor site for the SL (indicated in Fig. 1B). This dinucleotide is the first AG downstream
of the previously mentioned polypyrimidine tract. Five additional AG
dinucelotides occur between this site and the start codon, so some
tuzin mRNAs might have alternative SL addition sites as is the case
with amastin mRNAs(1) .
No luciferase activity above background was detected
when unmodified pGEM-luc or pLT, a derivative containing the
TCR27 3`-UTR, were introduced. However, when a 73-bp segment containing
the polypyrimidine tracts and SL addition site immediately preceding
the amastin coding region was placed in front of the luciferase gene of
pLT, a 400-fold increase in luciferase activity over the background
level was detected (pLST). This result provides strong evidence that a
SL addition site is necessary for the production of luciferase mRNA,
and this 73-bp segment was included in all subsequent plasmid
constructs. The three plasmids shown in the middle of Fig. 4(pL3T, pL2T, and pLIT) each contain a different region of
the amastin/tuzin gene cluster cloned in front of the luciferase gene
and the 73-bp segment. Thus, if any of these three regions contains
promoter activity, the amount of luciferase detected when its plasmid
is introduced into T. cruzi epimastigotes should increase
relative to that of pLST. As indicated in Fig. 1, the cloned
region in pL3T includes the first tuzin gene in the gene cluster plus
about 1.5 kb of unique sequence preceding this gene. The region in pL2T
is a complete internal 3-kb repeat including an amastin gene, a tuzin
gene, and their downstream IRs. The third region, cloned in pLIT,
contains only a tuzin gene and its 5`- and 3`-flanking intergenic
regions. For each of these constructs, the amount of luciferase
activity is about 700-fold above background (range =
692-747-fold) compared with 400-fold above background for pLST.
This 1.75-fold increase in luciferase activity might be due to a small
amount of promoter activity or due to the presence of additional
sequences upstream of the 73-bp segment that also influence trans-splicing. In either event, no region that clearly
contains substantial promoter activity was detected (see Fig. 5for an example of a T. cruzi DNA sequence that
does have promoter activity). In particular, note that in pL3T the
1.5-kb region immediately upstream of the gene cluster does not have
promoter activity. Regions still further upstream were not investigated
because the nuclear run on experiments (Fig. 3) indicated that
these regions, encompassing fragments 1 and 2 shown Fig. 1, were
not transcribed. When the orientation of these three fragments was
reversed, the luciferase activity was about the same as that for pLST
(not shown). The only trypanosomatid in which promoters for
protein-encoding genes have been identified is the African trypanosome, T. brucei, and in these cases the promoters have been shown to
be resistant to
Unfortunately, repeated attempts to transiently transfect amastigote
cells under a variety of electroporation conditions with plasmids pLIT
and pLST shown in Fig. 4, or with corresponding plasmids
containing the amastin 3`-UTR + IR, did not detect any luciferase
activity above background (not shown). These negative results suggest
that electroporation is much less efficient for transfection of
amastigotes than for epimastigotes. Therefore, we had to construct an
improved vector that directed much higher levels of luciferase
activity. The new construct was based on the observation that in
African trypanosomes high expression levels of a reporter gene are only
achieved using promoters for genes encoding rRNA, PARP, and the VSG,
each of which is a promoter for RNA polymerase I or a RNA polymerase
I-like enzyme. Since the promoter for a T. cruzi rRNA gene has
recently been reported(24, 25) , we inserted a 580-bp
fragment containing this promoter in front of the amastin SL addition
site of pLST and obtained the results shown in Fig. 5. The
presence of this T. cruzi rRNA promoter increased the amount
of luciferase activity in transfected epimastigotes so dramatically
that we reduced the amount of plasmid in a typical transfection from
100 to 20 µg and conducted the luciferase assays with a 1:50
dilution of the cell extracts. Thus, the results with pLST shown in Fig. 5are not directly comparable with the results shown in Fig. 4because of the differing transfection and assay
conditions. When transfected into epimastigotes, pLRT containing the T. cruzi rRNA promoter and the 3`-UTR of the TCR27 gene
stimulated about 2,050 times more luciferase activity (271,098- versus 132-fold) than did the corresponding plasmid without
the promoter (pLST) or with the promoter in the opposite orientation.
When transfected into amastigotes, pLRT containing the correctly
oriented promoter resulted in only a 94-fold increase in luciferase
activity rather than the 2,050-fold increase seen in epimastigotes,
consistent with the prediction that transfection efficiency is much
lower in amastigotes than in epimastigotes. However, this value in
amastigotes still represents a substantial number of luciferase units
above background (15,000 versus about 170), so plasmids in
which luciferase expression is driven by the rRNA gene promoter were
used to compare the effects of different 3`-UTRs + IRs. As shown
in Fig. 5, in epimastigotes the 3`-UTR of the constitutively
expressed TCR27 gene (pLRT) conferred the highest level of luciferase
expression, whereas the presence of the amastin 3`-UTR + IR (pLRA)
resulted in a 5-fold drop in luciferase expression. The opposite effect
was seen in amastigote cells. Luciferase activity was 7.2-fold higher
with the amastin 3`-UTR + IR than with the TCR27 3`-UTR. As
indicated in the right-hand column of Fig. 5, the resulting
normalized difference between amastigotes and epimastigotes is 36-fold
(5 Furthermore, in amastigotes, the
presence of the tuzin 3`-UTR + IR (pLRC) had the opposite effect
of the amastin 3`-UTR + IR on luciferase activity. When
transfected into amastigotes, the tuzin region caused the luciferase
activity to drop to 0.3 of that expressed with the TCR27 3`-UTR,
whereas the amastin 3`-UTR + IR increased it by 7.2-fold. In
contrast, in epimastigotes the tuzin and amastin 3`-UTRs + IRs had
similar affects. Compared with the TCR27 3`-UTR, the tuzin 3`-UTR
+ IR caused a 10-fold drop, and the corresponding amastin region
caused a 5-fold drop. Thus, the tuzin 3`-UTR + IR resulted in a
lower level of the chimeric luciferase mRNA in both epimastigotes and
amastigotes, whereas the amastin 3`-UTR + IR caused an increase in
amastigotes and a decrease in epimastigotes.
T. cruzi epimastigotes and amastigotes were incubated in the presence of
either 200 or 500 ng/ml cycloheximide for 4 h and total RNA isolated
for Northern blots that were probed in succession with the coding
regions of tuzin, amastin, and rRNA (Fig. 6). Again,
trypomastigotes were not used because of the difficulty in obtaining a
sufficient number for this experiment. Densitometric measurements of
the rRNA signals (panelC) were used to adjust for
differences in RNA loaded in each lane. After this normalization, the
relative signal intensities in panelsA and B were determined and several conclusions were drawn. First, the two
different cycloheximide concentrations had very similar effects in all
cases, so the RNA levels in the two concentrations could be averaged.
Second, the tuzin autoradiogram shown in Fig. 6was exposed
about 10 times longer than the amastin autoradiogram, again reflecting
the lower abundance of tuzin mRNA. Third, in amastigotes the effect of
cycloheximide on amastin RNA and tuzin RNA was quite different. At this
developmental stage, the drug caused a 3-fold drop in amastin RNA and a
7-fold increase tuzin RNA. The magnitude of this 7-fold increase is not
readily apparent in panelB because of the adjustment
that must be made for more RNA in the 0 drug lane of amastigotes as
shown in panelC. Fourth, in epimastigotes the
presence of cycloheximide did not affect substantively the steady-state
level of amastin mRNA (i.e. 1.3-fold more with cycloheximide),
whereas the tuzin mRNA level increased by 4.3. Finally, no differences
were observed between 4 h of incubation in cycloheximide (Fig. 6) and 2 or 8 h of incubation (not shown).
Figure 6:
Effect of cycloheximide on the
steady-state levels of amastin and tuzin mRNAs. For these Northern
blots, total RNAs were extracted from epimastigotes (E) and
amastigotes (A) that had been incubated for 4 h in the
presence of 0, 200, or 500 ng of cycloheximide/ml. Filters were probed
in succession with the coding regions for tuzin (panelB), amastin (panelA), and 24
The
interpretation of these cycloheximide results is tempered by several
factors (see ``Discussion''), but one clear observation is
that in amastigotes the inhibition of protein synthesis causes a
decrease in amastin mRNA and an increase in tuzin mRNA. In contrast, in
epimastigotes, this inhibition has no effect on amastin mRNA but causes
an increase in tuzin mRNA similar to that seen in amastigotes (4.3-fold
increase versus 7-fold increase). Thus, translation inhibition
affects the two mRNA species differently. The results described here suggest that different molecular
mechanisms regulate the RNA levels of the alternating amastin and tuzin
genes in the three developmental stages of T. cruzi. The
simplest model consistent with our results and with previous findings
in other trypanosomatids (8, 34, 39) is that
the two alternating genes are constitutively transcribed into large
polycistronic precursor RNAs, which are processed into amastin and
tuzin mRNAs whose levels are post-transcriptionally regulated by
different mechanisms. The first gene in the cluster, a tuzin gene, does
not contain the upstream polypyrimidine tract and AG acceptor site for
SL addition that precede internal tuzin genes (see Fig. 1B), suggesting that this first gene is not
processed into a functional mRNA. No evidence was obtained for the
presence of a strong promoter either preceding or within the
amastin/tuzin gene cluster. One of several possible explanations for
this lack of a promoter is that polycistronic transcription of the gene
cluster is initiated weakly at a number of sites within the cluster and
that the overall chromatin configuration surrounding the cluster
imparts the signals for the unidirectional transcription. Regions
that likely participate in the different post-transcriptional
regulatory mechanisms of these two coding regions are their flanking
5`- and 3`-UTRs and their flanking IRs. Consistent with this
possibility are reports from other experimental systems that 3`-UTRs
are involved in phenomena as diverse as self-regulation of mRNA
degradation(40, 41) , rates of
translation(42) , mRNA localization (43) , cellular
growth and differentiation(44) , and tumor suppression (45) . Recently, a stem-loop structure in the 3`-UTR of the
PARP gene of African trypanosomes was reported to participate in its
differential regulation(46) . In addition, sequences of both
the 3`- and 5`-UTR of hsp83 mRNA in Leishmania were found to
be involved in its temperature-dependent regulation(47) . The
IR sequences downstream of the 3`-UTRs also have been found to be
important. This region contributes to the differential expression of
the three gene classes encoding the surface protease gp63 of Leishmania chagasi, whereas the 3`-UTRs by themselves have
little effect (34) . Likewise, within the IR following the
dihydrofolate reductase-thymidylate synthase gene of Leishmania
major, and between the Amastin mRNA has a short 5`-UTR (17
nt) and a long 3`-UTR (630 nt), whereas tuzin mRNA has a long 5`-UTR
(137-nucleotide) and a short (<20-nucleotide) or nonexistent 3`-UTR.
None of these UTRs nor the intervening IRs of 145 and 111 bp display
any obvious sequence similarities, consistent with the possibility that
these sequences participate in the differential expression of amastin
and tuzin mRNAs. In the case of tuzin mRNA, its short or nonexistent
3`-UTR suggests that this sequence does not contribute much to the
differential regulation of tuzin mRNA. The complete absence of a 3`-UTR
in one tuzin cDNA and the creation of its UAA stop codon by 3`
polyadenylation is reminiscent of a similar phenomenon in mammalian
mitochondrial DNA in which the termination codons in the mRNAs of 9 of
the 13 protein-encoding genes are created during poly(A) addition (49) . Two general models have been proposed to account for
those cases in other biological systems where cycloheximide affects the
level of an mRNA in a post-transcriptional
manner(42, 50) . One model invokes the existence of a
labile regulatory protein that either stabilizes a specific mRNA or
targets it for degradation(50) . The other model, shown for
mammalian Transient
transfections of the luciferase reporter gene containing the different
3`-UTRs + IRs also indicated that different regulatory mechanisms
control the amastin and tuzin mRNA levels (Fig. 5). From these
experiments, we cannot determine whether it is the 3`-UTRs or the IRs,
or both, that are responsible for this difference. However, when
similar reporter gene transfections were conducted in Leishmania using the different 3`-UTRs and the downstream IRs of the three
gp63 gene classes of L. chagasi, it was found that the 3`-UTRs
and the IRs each contribute to the differential expression of the three
gene classes(34) . By analogy, it seems likely that a similar
scenario will hold for the differential expression of the amastin/tuzin
gene cluster. Finally, the deduced amino acid sequence of tuzin (Fig. 1C) does not have any substantive similarities
with the protein sequences currently in the data bases. In contrast to
amastin, which is an extremely hydrophobic protein, tuzin is a highly
charged molecule lacking a signal peptide or other sequence motifs that
might provide insight into its cellular location and biological
function. The relatively low abundance of its mRNA suggests that tuzin
is a rare protein that will be difficult to detect in Western blots of
subcellular fractions or in cellular immunolocalization assays. It also
is difficult to say whether the alternating arrangement of the amastin
and tuzin genes means that their gene products are associated
physiologically or structurally. Other known co-transcribed, multi-gene
clusters of trypanosomatids encode proteins such as ubiquitin and
calmodulin(6) , a phosphatase, and an RNA polymerase
subunit(7) , and the VSG, a transferrin receptor and adenylate
cyclase(54) . It is possible that these gene products are
associated, i.e. the phosphatase could act on the RNA
polymerase, or the transferrin and adenylate cyclase might be attached
to the same membrane as the VSG, but in no case has such a relationship
been demonstrated. Thus, the reason for the alternating nature of the
amastin and tuzin genes remains unclear.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank(TM)/EMBL Data Bank with accession
number(s) U25030[GenBank].
Volume 270,
Number 38,
Issue of September 22, pp. 22586-22594, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)each that are located within
3-kb DNA repeats. These genes are constitutively transcribed at the
same rate in all developmental stages, indicating that their elevated
expression in amastigotes must be regulated
post-transcriptionally(1) . Many tandem genes in T. cruzi and other trypanosomatids, such as Leishmania species and
African trypanosomes, are known to be transcribed into polycistronic
precursor
RNAs(2, 3, 4, 5, 6, 7) .
These precursor transcripts are processed into individual mRNAs by
intergenic cleavages followed by addition of a 39-nucleotide spliced
leader (SL) to the 5` ends in a trans-splicing reaction and
addition of a poly(A) to the 3` ends via
polyadenylation(8, 9, 10) . Thus, if the
amastin gene cluster is transcribed polycistronically, the
post-transcriptional control of amastin mRNA abundance is likely to be
regulated at these processing steps or by stability conferred on the
mRNA by the 5`- or 3`-untranslated regions (UTRs).
-amanitin, suggesting that similar to rRNA genes, they are
transcribed by RNA polymerase I or a modified form of RNA polymerase
II(13, 14) . In contrast, transcription of the amastin
genes of T. cruzi and the other protein-encoding genes of
trypanosomatids examined to date is inhibited by -amanitin,
indicating that they are transcribed by a conventional RNA polymerase
II.
Parasites
The Tulahuén
strain of T. cruzi(17) was used for all experiments.
Epimastigotes were maintained in logarithmic growth phase at 26 °C
in supplemented liver digest neutralized tryptose medium as described
previously(18) . Culture-derived trypomastigotes and
amastigotes were obtained by infecting monolayers of the renal
carcinoma cell line RA786 (19) grown in RPMI medium at 37
°C and purified by centrifugation in metrizamide gradients as
described previously(1) .Recombinant DNA Procedures
A T. cruzi genomic DNA library in bacteriophage FIX (Stratagene, La
Jolla, CA) was screened with an amastin cDNA labeled with
P using a random oligonucleotide primer kit (Amersham
Corp.). DNAs from several identified phage clones were purified using
procedures described by the supplier of an ion-exchange column (Qiagen,
Chatsworth, CA) and analyzed by restriction enzyme digestions.
Restriction fragments derived from phage clone
-A11 were purified
from agarose gels and subcloned into pBluescript (Stratagene). In
addition, total T. cruzi DNA was digested to completion with EcoRI or BglII, separated in 0.7% agarose gels, and
fragments of about 3 kb were excised and ligated into pBluescript. Escherichia coli DH5
strain was transformed with the
recombinant plasmids and the bacterial colonies screened with an
amastin cDNA probe. Plasmid DNAs were prepared using the Qiagen method
(Qiagen). Total RNA was isolated from parasite cultures using LiCl and
urea as described previously(20) . For Northern blots, 4 µg
of total RNA were separated in 1.2% agarose gels containing
formaldehyde, transferred to nitrocellulose membranes, and hybridized
to DNA probes(21) . Southern blots were performed as described
previously(22) . DNA sequencing was conducted with the dideoxy
chain termination method (Sequenase, Amersham Corp.).Plasmid Constructions
All constructs used in the
transfection experiments were derived from the pGEM-luc vector
(Promega Corp.) except for plasmid pHDT. To generate plasmid pLT and
most of the other constructs, a 520-bp HincII-StuI
fragment containing the 3`-UTR of the TCR27 gene (23) plus 135
bp downstream of its polyadenylation site was inserted at a SalI site just beyond the luciferase coding region of
pGEM-luc. This fragment was isolated from recombinant plasmid
p39, which contains part of the TCR27 gene cloned into pBluescript and
was kindly provided by K. Otsu (University of Iowa). To construct
plasmid pLIT, a 1.8-kb fragment containing the region between two
amastin genes was generated by PCR amplification using primers P1 and
P2 from the 5` and 3` ends of the amastin genes
(5`-GCTGGGCCCTTCCTACGTGAGAGCT and 5`-CGATGAATTTTGATTGTG, respectively).
The 5` primer has an ApaI site to facilitate cloning. The
template for this PCR was plasmid pAB3, which has a 3.0-kb genomic DNA
fragment containing the amastin and tuzin genes. The ApaI-digested PCR product was ligated to plasmid pLT
(pGEM-luc vector containing the TCR27 3`-UTR), which had been
linearized with NotI and ApaI. A 5` deletion of the
fragment containing the region between two amastin genes was obtained
by a complete digestion of plasmid pLIT with NsiI followed by
a partial digestion with AatII. Treatment with T4 DNA
polymerase and T4 DNA ligase resulted in a plasmid, named pLST,
containing the 73-bp sequence immediately upstream of the amastin start
codon. This 73-bp sequence includes the amastin 5`-UTR, a SL addition
site and a polypyrimidine-rich region, and it precedes the luciferase
gene in pLST.-A11 was used to replace a HindIII fragment containing
the intergenic region in plasmid pLIT. Plasmid pL2T was prepared by
replacing the same segment in pLIT with a 3-kb HindIII
fragment in
-A11 containing both the amastin and tuzin genes.
Plasmids containing the T. cruzi rRNA gene promoter (pLRT)
were generated from plasmid pLST by inserting a 580-bp HincII T. cruzi rRNA gene spacer fragment into the unique HindIII site. This fragment was isolated from plasmid
pTc18S(24, 25) . Plasmids pLRA and pLRC are
derivatives of pLRT in which the TCR27 3`-UTR was replaced by fragments
corresponding to the 3`-UTR and polyadenylation sites of an amastin
gene and a tuzin gene, respectively. The amastin 3`-UTR fragment was
obtained by digesting plasmid pAB3 with SphI and MscI, generating an 860-bp fragment containing the complete
3`-UTR plus a 200-bp segment spanning the polyadenylation site and SL
addition site of the downstream tuzin gene. The tuzin 3`-UTR was
obtained by PCR amplification of plasmid pAB3 using primers
corresponding to the 5` end of the amastin coding region (P1) and the
3` end of the tuzin gene (P3). Primers P1 (see above) and P3
(5`-TGCTGTCGACAGTAGCCAGCCATG) contain ApaI and SalI
sites, respectively, to facilitate cloning. Finally, plasmid pHDT was
constructed by deleting the 360-bp BamHI-PstI
fragment containing the PARP 3`-UTR in the pHD1 plasmid (26) and inserting the 520-bp HincII-StuI
fragment containing the 3`-UTR of the TCR27 gene.
Transcription in Isolated Nuclei
Nuclei were
isolated from epimastigotes, and nuclear run on transcripts were
labeled with [-P]UTP as described
previously(1) . Hybridization to subcloned fragments 1-5
in Southern blots of Fig. 1was carried out for 3 days at 65
°C using 2-5
10
cpm of labeled RNA that
was purified using Sephadex G-50 QuickSpin columns (Boehringer
Mannheim) in 5 ml of hybridization solution followed by filter washings
as described previously(27) .
-A11 is denoted by the hatchedline. Three
fragments derived from either the 5` end or interior regions of the
gene cluster that were used in the transfection experiments shown in Fig. 4are denoted by the patternedboxes. Lines labeled 1-5 indicate fragments
that were probed in the nuclear run on experiment shown in Fig. 3. B, the sequence of a 2411-bp region between the
coding regions of adjacent amastin genes with the amastin stop and
start codons at the 5` and 3` ends, respectively. The start and stop
codons of the intervening tuzin gene are denoted by the underlyingblackbox and blackoval,
respectively. BoxedAG dinucleotides indicate SL
addition sites. Polypyrimidine sites upstream of these SL sites are
also boxed. Blackarrowheads indicate
poly(A) addition sites. The openarrowhead between
the SL addition site and start codon of the tuzin gene indicates the
location at which the unique sequence upstream of the gene cluster
diverges from the intergenic sequence. The indicated restriction sites
were used to construct the plasmids shown in Fig. 4and Fig. 5. C, the deduced amino acid sequence of
tuzin.
rRNA (r) and two fragments of
the T. cruzi TCR27 gene (T). B,
autoradiogram of labeled run on transcripts hybridized to the DNA
fragments shown in panelA. Migrations of molecular
size markers in kb are shown on the left.
DNA Transfections and Luciferase Assays
Plasmid
DNAs to be used for transfections were prepared by alkaline lysis of
host bacteria followed by column purification (Qiagen). Transfections
of epimastigotes and amastigotes were performed by electroporation with
20 or 100 µg of circular plasmid DNA, and each plasmid was
transfected in triplicate experiments. Briefly, 10
parasites were washed in cold phosphate-buffered saline and
suspended in 0.4 ml of phosphate-buffered saline containing 0.5
mM MgCl
and 0.1 mM CaCl. The
cell suspensions were mixed with 50 µl of plasmid DNA, and after
incubation for 15 min on ice, they were subjected to one pulse at 300 V
and 960 microfarads for epimastigotes or 500 V and 450 microfarads for
amastigotes using a Gene Pulser (Bio-Rad). 10 min after
electroporation, epimastigotes were transferred to 5 ml of liver digest
neutralized tryptose medium and incubated at 26 °C for 48 h.
Amastigotes were transferred to 25-cm tissue culture flasks
containing 50% confluent monolayers of RA786 cells and 5 ml of RPMI
medium and incubated at 37 °C for 48 h. For luciferase assays,
cells were harvested and washed in phosphate-buffered saline,
transferred to Eppendorf tubes, and centrifuged for 10 s at 10,000
g. The pellets were suspended in 100 µl of lysis
buffer (100 mM potassium phosphate, pH 7.8, 0.4% Triton X-100,
and 2 µg/ml leupeptin). After centrifuging for 1 min at 10,000
g, 50 µl of undiluted supernatant or 5 µl of a
1:5 dilution were assayed as described previously(28) .
Identification of the 5` End of the Amastin Gene
Cluster
The amastin genes are located within tandem repeats of 3
kb in the genomes of several T. cruzi strains(1) . To
isolate the unique region immediately upstream of the amastin gene
cluster, a genomic library of T. cruzi DNA was screened with a
cloned amastin cDNA. Several independent recombinant FIX phage
clones were identified and examined. All of them have 3-kb repeats, and
one phage,
-A11, has an additional nonrepetitive region.
Restriction digests, Southern blot analyses, and PCR amplifications
were used to generate the restriction map shown in Fig. 1A. The 14-kb insert of phage
-A11 has two
amastin genes and, as described below, 2.5 copies of the previously
unknown tuzin gene. It also has 6.5 kb of unique sequence upstream of
the first gene in the cluster. The sequence extending from the first EcoRI site indicated in Fig. 1A through most
of the first amastin gene was determined. This sequence has a point
mutation in the first amastin gene that abolishes an EcoRI
site located 172 bp downstream of the start codon in all but one of the
amastin cDNAs previously sequenced(1) . Moreover, the presence
of a 5.5-kb EcoRI fragment at the 5` end of the cluster is
consistent with the results of Southern blots of T. cruzi genomic DNA probed with amastin cDNA, in which weakly hybridizing EcoRI fragments of 5.5 and 1.0 kb were observed (not shown).
Another Gene Is Located Between Adjacent Amastin
Genes
The distance between the 3` poly(A) addition site of one
amastin gene, and the 5` SL addition site of the adjacent downstream
amastin gene is about 1,743 bp (these addition sites vary slightly in
different cDNAs). Analysis of this sequence in pAE3 revealed the
presence of a 447-codon open reading frame potentially encoding a
45-kDa protein that we have named tuzin. The deduced amino acid
sequence, shown in Fig. 1C, had no substantive
similarity to protein sequences in the data bases in April, 1995.
Southern blots of partially restricted genomic DNAs probed with this
coding region confirmed that the tuzin and amastin genes alternate
head-to-tail in the same 3-kb repeats (not shown). The corresponding
sequence of the 3-kb repeat in pAB3 as well as partial sequences of
several tuzin cDNAs (see below) indicate that, similar to amastin
genes, the tuzin genes are heterogeneous in sequence with frequent base
substitutions. rRNA (31) (panelC). The amastin
and tuzin RNAs are 1.4 and 1.7 kb, respectively, indicating from a
comparison with their respective gene lengths that each has a 3`
poly(A) tail of about 200 nucleotides. A densitometer tracing of a
short exposure of the autoradiogram shown in panelA indicated that the ratio of amastin RNA in epimastigotes,
amastigotes, and trypomastigotes is 1:85:4.8, respectively. The
corresponding ratio for the tuzin RNA was 1:3.5:0.1, whereas that for
rRNA was 1:1:1, demonstrating that equal amounts of total RNA were
added to each lane. In addition, the tuzin autoradiogram shown in Fig. 2was exposed about 10 times longer than the amastin
autoradiogram, indicating that in the amastigote and trypomastigote
stages amastin RNA is much more abundant than tuzin RNA. Our earlier
measurements suggested that amastin RNA is about 50-fold more abundant
in amastigotes than in epimastigotes(1) , but the difference
between that value and the 85-fold found here could reflect differences
in the growth phases of the different developmental stages when the RNA
was collected. Thus, it is likely that the two alternating gene classes
are transcribed into large polycistronic precursor RNAs, which are
processed into amastin and tuzin mRNAs whose steady state levels are
quite different in the amastigote and trypomastigote stages but much
more similar in the epimastigote stage. Since the specific activities
of the radioactive amastin and tuzin gene probes used in panelsA and B may be different, we cannot use the
Northern blots to quantitate the relative abundances of amastin and
tuzin mRNAs in epimastigotes. However, about the same number of amastin
and tuzin cDNA clones were detected in a T. cruzi epimastigote
cDNA library, suggesting that the levels of their corresponding mRNAs
are similar at this developmental stage.
rRNA gene (panelC). Exposure times of the
autoradiograms were 4.5 h (A), 48 h (B), and 15 min (C). In panelB, the blackspot just above the band in the Tlane is an imperfection in the x-ray film and not due to probe
hybridization.
Search for a Transcription Initiation Site at the 5` End
of the Amastin/Tuzin Gene Cluster
Since the amastin and tuzin
genes are transcribed to an equal extent in all developmental
stages(1) , we examined the region at the 5` end of the
amastin/tuzin gene cluster for a site where this transcription might
begin. Nuclear run on assays were conducted using nuclei prepared from
epimastigotes, and P-labeled nascent RNAs were used to
probe Southern blots containing DNA fragments 1-5 indicated in Fig. 1A. Fig. 3shows that no detectable
transcription occurs from fragments 1 and 2, which contain
approximately 3 kb of unique sequence located upstream of the first
tuzin gene in the cluster. As expected, strong hybridization to
fragments 3-5, which contain the tuzin and amastin genes,
occurred. Strong hybridization also occurred to control DNA fragments
containing a rRNA gene and the TCR27 gene, which we have previously
shown to be a single copy gene encoding a T. cruzi cytoskeletal protein(22, 23, 32) . Thus,
if a unique transcription start site exists for this gene cluster, it
is located within the approximately 400-bp sequence between the
upstream EcoRI site indicated in Fig. 1A and
the start of the first tuzin gene. Further efforts to demonstrate
transcription within this region were not successful. Therefore, it is
not possible to distinguish from these experiments whether
transcription begins a short distance upstream of the first gene and
extends through the cluster or whether transcription initiation occurs
at one or more sites within the cluster. Hybridization of nuclear run
on RNA to the individual strands of the amastin gene cloned into
bacteriophage M13 demonstrated that only the coding strand of these
genes is transcribed (1) , so at the minimum there must be a
signal that imparts a directionality to the transcription of this gene
cluster.
Identification of 5` Sequences Important for Expression
Using Transient Transfection Assays
Since the approximate
location of a promoter for the amastin/tuzin gene cluster was not
identified in the nuclear run on experiments, we conducted transient
transfections to look for 5` sequences important for amastin and tuzin
expression using a series of constructs derived from pGEM-luc (Promega). This plasmid contains a luciferase reporter gene
flanked by restriction sites into which different fragments were
inserted. In the plasmids tested, a 520-bp fragment that includes the
entire 3`-UTR and 135 bp beyond the polyadenylation site of the TCR27
gene, which is constitutively expressed during the epimastigote and
amastigote stages of T. cruzi, ()was inserted
downstream of the luciferase gene. Epimastigotes were transfected with
each construct, and whole cell extracts were prepared 48 h later for
luciferase assays. At least three independent transfections and assays
were conducted with each recombinant plasmid, and the means and
standard deviations of the luciferase activities are shown in Fig. 4.-amanitin, which is a characteristic of RNA
polymerase I promoters (11, 12, 13, 14) . Since our results
suggest that the signals for SL addition alone are sufficient for
luciferase activity in transfected epimastigote cells, we tested
whether the promoter for one such -amanitin resistant gene of
African trypanosomes, the PARP gene(12, 26) , would
stimulate luciferase expression in T. cruzi. As shown at the
bottom of Fig. 4, plasmid pHDT has a 290-bp fragment containing
the PARP promoter and SL addition site (26) inserted upstream
of the luciferase gene, and the 3`-UTR of the TCR27 gene inserted
downstream. This plasmid directs about 900-fold more luciferase
activity than background, or only 1.3-fold (900- versus 700-fold) more than pLIT, pL2T, or pL3T, which contain regions
derived from the amastin/tuzin gene cluster. Although it is not
possible to interpret unambiguously the result of this heterologous
PARP transfection, it does suggest that the 290-bp fragment of T.
brucei contributes functional SL addition signals for the
luciferase gene but little, if any, promoter activity in T.
cruzi. Thus, a promoter for a protein-encoding gene of African
trypanosomes is not recognized as a promoter by epimastigotes of T.
cruzi.The Role of the Amastin and Tuzin 3`-UTRs Plus IRs in the
Developmental Expression of Their RNAs
The 3`-UTR of amastin
mRNA (630 nucleotides) is larger than its coding region (525
nucleotides). The presence of this large 3`-UTR in the abundant,
amastigote-specific amastin mRNA and a small or nonexistent 3`-UTR in
the less abundant tuzin mRNA, suggests that the amastin 3`-UTR might be
important in the control of the stage-specific expression of amastin.
This prediction is consistent with the fact that all developmentally
regulated genes studied so far in trypanosomatids appear to be
controlled at least in part by post-transcriptional events (26, 33, 34) . Thus, we examined whether the
amastin and tuzin 3`-UTRs plus their downstream IRs affect expression
of the luciferase gene when transfected into T. cruzi amastigotes and epimastigotes. Transfections into trypomastigotes
were not attempted because of difficulties in obtaining sufficient
numbers of trypomastigotes for this type of experiment. 7.2), which is not much less than the 50-85-fold
difference in the steady-state level of amastin mRNA observed on
Northern blots (see Fig. 2). Likewise, for the tuzin 3`-UTR
+ IR, the normalized ratio in luciferase activity between
amastigotes and epimastigotes is 3, consistent with the Northern blot
in Fig. 2, indicating that there is 3.5 times more tuzin mRNA in
amastigotes than epimastigotes.The Effect of Inhibition of Protein Synthesis on the
Steady State Levels of Amastin and Tuzin mRNAs
Inhibitors of
protein synthesis have been shown to affect the steady-state levels of
mRNAs encoding several mammalian proteins(35) , gp63 in Leishmania(36) , and PARP in African
trypanosomes(37) . For the gp63 and PARP genes, the 10-fold or
more increase in their mRNAs in the presence of cycloheximide suggests
that a labile negative regulator might target these transcripts for
degradation(36, 37) . Since gp63 and PARP are both
abundant stage-specific surface proteins, as is amastin(1) , we
decided to investigate the effect of cycloheximide on the accumulation
of amastin and tuzin transcripts in T. cruzi. A cycloheximide
concentration of 250 ng/ml has been shown previously to inhibit protein
synthesis in T. cruzi by 97%(38) .
rRNA (panelC). The signal intensities on short
exposures of the autoradiograms were determined by densitometry. The
rRNA signals in each lane of panelC were normalized
to each other, and the relative signals of the amastin and tuzin mRNAs
were adjusted to this normalized value. Exposure time for the tuzin
autoradiogram (panelB) was about 10 times longer
than for the amastin autoradiogram (panelA).
- and -tubulin genes of T.
brucei, the SL addition site of the downstream gene directs the
location of the upstream gene's poly(A) addition
site(8, 48) .-tubulin mRNA, involves the co-translational degradation
of mRNAs by a ribosome-associated RNase(51, 52) . In T. cruzi amastigotes, cycloheximide caused a decrease in the
abundant amastin mRNA and an increase in the less abundant tuzin mRNA,
consistent with the presence of both the positive and negative
regulatory aspects of the first model. In epimastigotes, the
cycloheximide had no effect on amastin mRNA and caused an increase in
tuzin mRNA similar to that observed in amastigotes. This result
suggests that the cycloheximide-affected mechanism regulating tuzin
mRNA levels may be the same in amastigotes and epimastigotes, whereas
the cycloheximide-affected mechanism contributing to the elevated
amastin mRNA level in amastigotes does not operate in epimastigotes.
More experiments will be required to elucidate the details of these
cycloheximide-affected mechanisms, including measurements of the mRNA
decay rates and the use of other translation inhibitors such as
pactamycin or puromycin that act on different steps in protein
synthesis than does cycloheximide(53) .
))
We thank J. A. N. Batista for advice and technical
help, S. Schenkman and K. S. Kim for providing plasmids containing
promoters for the T. cruzi rRNA gene and the T. brucei PARP gene, respectively, and K. Otsu for providing the T.
cruzi genomic library.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  D. C. Bartholomeu, G. C. Cerqueira, A. C. A. Leao, W. D. daRocha, F. S. Pais, C. Macedo, A. Djikeng, S. M. R. Teixeira, and N. M. El-Sayed Genomic organization and expression profile of the mucin-associated surface protein (masp) family of the human pathogen Trypanosoma cruzi Nucleic Acids Res., June 1, 2009; 37(10): 3407 - 3417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Nardelli, A. R. Avila, A. Freund, M. C. Motta, L. Manhaes, T. C. L. de Jesus, S. Schenkman, S. P. Fragoso, M. A. Krieger, S. Goldenberg, et al. Small-Subunit rRNA Processome Proteins Are Translationally Regulated during Differentiation of Trypanosoma cruzi Eukaryot. Cell, February 1, 2007; 6(2): 337 - 345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. McNicoll, M. Muller, S. Cloutier, N. Boilard, A. Rochette, M. Dube, and B. Papadopoulou Distinct 3'-Untranslated Region Elements Regulate Stage-specific mRNA Accumulation and Translation in Leishmania J. Biol. Chem., October 21, 2005; 280(42): 35238 - 35246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Myung, J. K. Beetham, M. E. Wilson, and J. E. Donelson Comparison of the Post-transcriptional Regulation of the mRNAs for the Surface Proteins PSA (GP46) and MSP (GP63) of Leishmania chagasi J. Biol. Chem., May 3, 2002; 277(19): 16489 - 16497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Irmer and C. Clayton Degradation of the unstable EP1 mRNA in Trypanosoma brucei involves initial destruction of the 3'-untranslated region Nucleic Acids Res., November 15, 2001; 29(22): 4707 - 4715. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. C. Coughlin, S. M. R. Teixeira, L. V. Kirchhoff, and J. E. Donelson Amastin mRNA Abundance in Trypanosoma cruzi Is Controlled by a 3'-Untranslated Region Position-dependent cis-Element and an Untranslated Region-binding Protein J. Biol. Chem., April 14, 2000; 275(16): 12051 - 12060. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Di Noia, I. D'Orso, D. O. Sanchez, and A. C. C. Frasch AU-rich Elements in the 3'-Untranslated Region of a New Mucin-type Gene Family of Trypanosoma cruzi Confers mRNA Instability and Modulates Translation Efficiency J. Biol. Chem., March 31, 2000; 275(14): 10218 - 10227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Mahmood, J. C. Hines, and D. S. Ray Identification of cis and trans Elements Involved in the Cell Cycle Regulation of Multiple Genes in Crithidia fasciculata Mol. Cell. Biol., September 1, 1999; 19(9): 6174 - 6182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Abuin, L. H. G. Freitas-Junior, W. Colli, M. J. M. Alves, and S. Schenkman Expression of trans-Sialidase and 85-kDa Glycoprotein Genes in Trypanosoma cruzi Is Differentially Regulated at the Post-transcriptional Level by Labile Protein Factors J. Biol. Chem., May 7, 1999; 274(19): 13041 - 13047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Quijada, M. Soto, C. Alonso, and J. M. Requena Analysis of Post-transcriptional Regulation Operating on Transcription Products of the Tandemly Linked Leishmania infantum hsp70 Genes J. Biol. Chem., February 14, 1997; 272(7): 4493 - 4499. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Charest, W.-W. Zhang, and G. Matlashewski The Developmental Expression of Leishmania donovani A2 Amastigote-specific Genes Is Post-transcriptionally Mediated and Involves Elements Located in the 3'-Untranslated Region J. Biol. Chem., July 19, 1996; 271(29): 17081 - 17090. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. D'Orso and A. C. C. Frasch TcUBP-1, a Developmentally Regulated U-rich RNA-binding Protein Involved in Selective mRNA Destabilization in Trypanosomes J. Biol. Chem., September 7, 2001; 276(37): 34801 - 34809. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |