Bariloche protein symposium argentine society for biochemistry and molecular biology

45
BIOCELL, 27 (Suppl. I), 2003
MI-C4.
MOLECULAR CLONING, DNA SEQUENCING AND
EXPRESSION OF AN UBIQUITIN CONJUGATING
ENZYME GENE FROM TRYPANOSOMA CRUZI
Pravia, Carlos; Búa, Jaqueline; Bontempi, Esteban; Ruiz, Andrés.
INP “Dr. Fatala Chabén”, ANLIS/Malbrán, Buenos Aires.
E-mail: [e-mail]
The covalent conjugation of ubiquitin to other intracellular proteins
fullfils essential functions in the eukaryotic cells. The
ubiquitination requires the sequential actions of three enzymes.
The first one is an activating enzyme (E1) wich activates ubiquitin.
Following activation one of several ubiquitin conjugating enzymes
(E2 enzymes) transfers ubiquitin from E1 to a member of the
ubiquitin protein ligase family, E3, that catalyzes the covalent
attachment of ubiquitin to the substrate. We describe here the
identification and cloning of one Trypanosoma cruzi ubiquitin
conjugating enzyme, E2Tc. A cDNA clone 27o4 derived from CL
Brener cDNA library encodes a protein of 151 aminoacids with a
calculated MW of 17,5 kDa. This DNA sequence showed 81%
similarity and 67% identities Drosophila melanogaster ubiquitin
conjugating enzyme E2-17Kd, “bendless protein”. The 27o4 clone
hibridized with two chromosomal bands of approximately 1,350
and 760 Kbp. The RT-PCR from epimastigotes mRNA amplified
one main band of around 550 bp. In order to obtain the recombinant
E2Tc protein, the putative coding region was cloned into pMALp2
vector. A fusion protein, with a molecular mass close to the
calculated value was obtained. The enzymatic and immunological
characterization are currently being performed.
MI-C5.
EXPRESSION OF OAT ARGININE DECARBOXYLASE
(ADC) GENE IN Trypanosoma cruzi EPIMASTIGOTES
M.P. Serra, C. Carrillo, A. Huber, N.S. González and I.D.
Algranati.
Fundación Insttituto Leloir y Facultad de Ciencias exactas y
Naturales (UBA) .E-mail:[e-mail]
Trypanosoma cruzi epimastigotes cultivated in a semisynthetic
medium can normally proliferate only when putrescine or
spermidine is added to the medium. This auxotrophy for
polyamines is caused by parasite inability to synthesize putrescine.
Previous work from our laboratory has shown that wild-type strains
of T. cruzi do not contain ornithine decarboxilase (ODC) enzymatic
activity due to the absence of the corresponding gene in the parasite
genome. Recent experiments carried out to search for the putative
conversion of arginine into agmatine, which would allow putrescine
biosynthesis through the only other possible metabolic pathway
leading to polyamines, also gave negative results in all the strains
of T. cruzi assayed. We were able to demonstrate that the absence
of ADC enzymatic activity in wild-type T. cruzi is not caused by
inhibition of the enzyme expression inside the parasite. In fact
after transfections of T. cruzi with a recombinant plasmid bearing
the coding region of oat ADC inserted in an appropriate vector,
we have obtained transgenic T. cruzi showing an appreciable level
of transient expression of ADC enzymatic activity. The
heterologous ADC has been characterized by identification of its
products, agmatine and CO
2
, as well as by the stoichiometry of
the catalysed reaction and the specific inhibition by
difluoromethylarginine (DFMA).
MI-C6.
IDENTIFICATION AND ANALYSIS  OF
TRYPOMASTIGOTE STAGE-SPECIFIC GENES IN
Trypanosoma cruzi
Tekiel, V.; Agüero, F. and Sánchez, D.
Instit. de Investigaciones Biotecnológicas (IIB). UNSAM. Av Gral
Paz y Constituyentes, INTI, Edificio 24 (1650) San Martin, Buenos
Aires. E-mail: [e-mail]
T. cruzi, the aetiological agent of Chagas disease, affects 10% of
the Latin American population. The non-replicative trypomastigote
is one of the mammalian parasite-stages, and is involved in cell
invasion and tissue dissemination. We constructed a cDNA
epimastigote-subtracted library for trypomastigotes by suppressive
subtractive hibridization (library T-E). A total of 403 good-quality
sequences were obtained and a trypomastigote-specific database
constructed. More than 45% of the clones represent new genes as
no matches against any database available were found and 29
putative new T. cruzi genes were identified. Trypomastigote-
specific mRNA expression was confirmed by reverse Northern
blot, Northern-blot and semi-quantitative RT-PCR. Based on
sequence similarity, the T-E library was grouped into 23 clusters,
each containing several contigs. A conserved 
∼220 bp region,
present in about 25% of the clones, was identified and defined as
“T element“ (Te). Searches against T. cruzi genome database at
TIGR, revealed the presence of the Te in the 3’ UTR of a wide
range of putative proteins. Almost all ORFs flanking the T element
in the TIGR’s WGSs, were found in our T-E library. Differential
expression in trypomastigotes of some ORFs as well as the Te
were confirmed. The presence of the Te in the 3’ untranslated
region of mature transcripts was also determined. Results indicate
that the use of subtractive libraries is a useful tool for the
identification of novel and stage-specfic genes and stage-specific
regulatory elements.
MI-C7.
CLONING AND EXPRESSION OF A PLAUSIBLE
CYTOSOLIC TYPE MALATE DEHYDROGENASE (cMDH)
FROM LEISHMANIA MEXICANA
Aranda, A.; Leroux, A.; Cazzulo, J.J. and Nowicki, C.
Fac Farmacia y Bioquímica IQUIFIB (UBA-CONICET). Inst.
Invest. Biotecnológicas (IIB-INTECH, UNSAM-CONICET)
Buenos Aires, Argentina. E-mail: [e-mail]
L. mexicana, similarly to T. cruzi, possesses a broad specificity
aromatic aminotransferase but contrary to T. cruzi, this parasite
neither produces aromatic lactate derivatives as end products nor
possesses an enzymatic activity equivalent to the T. cruzi aromatic
L-
α-hydroxy acid dehydrogenase (AHADH). The latter, a cytosolic
enzyme, is a member of the MDH family, unable, however, to
reduce oxaloacetate. Preliminary purification protocols allowed
us to isolate from the crude extracts of L. mexicana three different
chromatographic fractions with MDH activity. In the present work
we report the cloning and functional expression in bacteria of two
of these three isoforms. Amino acid sequence alignments and the
protein targeting sequences suggest that one isoform may be located
in the mitochondria, and the other does not possess a typical
glycosome targeting sequence suggesting a possible cytosolic
localisation. Both recombinant MDHs were purified by Ni
2+
 affinity
chromatography with specific activities of 23 and 389 U/mg for
the putative mitochondrial and cytosolic type MDHs, respectively.
Glycosomal membranes are impermeable for molecules such as
NAD
+
/NADH; thus, trypanosomatids probably need a NADH
dependant dehydrogenase to regenerate the cytosolic NAD
+
. This
role might be played by AHADH in T. cruzi and by the cytosolic
type MDHs in T. brucei and L. mexicana.

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