Bariloche protein symposium argentine society for biochemistry and molecular biology

62
BIOCELL, 27 (Suppl. I), 2003
MI-P10.
EXOPROTEASE ACTIVITY  OF  PSYCHROTROPHIC
MARINE PSEUDOALTEROMONAS SP. STRAIN 41, UNDER
DIFFERENT CONDITIONS
Estevao Belchior, Silvia and Alvarez, Héctor M.
Departamento de Bioquímica. Facultad de Ciencias Naturales.
Universidad Nacional de la Patagonia San Juan Bosco. E-mail:
[e-mail]
Extracellular protease activity has been detected in the marine
bacterium  Pseudoalteromonas sp, during cultivation under
different conditions. The strain was isolated from intestinal tract
of hake, collected from the San Jorge Gulf (Patagonia, Argentina).
To determine whether de novo protein biosynthesis was required
for exoprotease activity, 5h starved cells were washed and
subdivided into three samples. The samples were suspended (i)
in mineral base medium (MBM) containing chloramphenicol
(250
µg/ml), (ii) without addition of antibiotic and (iii) the other
sample was cultivated with casein as sole protein source, at 20°C
during 24 h. In the absence of substrate, extracellular activity was
detected at low basal levels, which increased significantly after
addition of casein suggesting the occurrence of an inducible
process. Low basal rates of exoprotease activity were found in the
chloramphenicol-treated cells during starvation. These
observations indicated that de novo protein synthesis during
starvation was not required and the presence of protease activity
in the supernatant fluid, from antibiotic–treated cells, could be a
result of secretion of protease preformed and accumulated
intracellularly. In gelatin zymograms, extracellular activity showed
two proteolityc bands with apparent molecular masses of
approximately 31,6 and 62 kDa.
MI-P11.
VARIABILITY OF NUCLEORPROTEIN AND
HEMAGGLUTININ GENES OF MEASLES VIRUS
A. Belizán, M.H. Argüelles, G. Glikmann.
Laboratorio de Inmunología y Virología, Universidad Nacional
de Quilmes. E-mail: [e-mail]
Measles virus (MV), which is a member of the Morbillivirus genus
of the Paramixoviridae family, is an enveloped virus containing a
negative sense RNA genome. Although MV is considered to be
monotypic, genetic variability has been observed and numerous
genotypes have been defined. Molecular epidemiology is based
on analysis of nucleoprotein (N) and hemagglutinin (H) genes,
which contain the most variable regions of the genome. The aim
of this study was to characterize the genetic variability of wild
type strains collected during the 1998 Argentinean outbreak.
The 3’ end of N gene (605 bp) was amplified by RT-PCR. As this
region is known to have the highest variability within the genome,
the restriction digested 605-bp fragments were analyzed by Single
Strand Conformation Polymorphism (SSCP) to characterize these
strains. The nucleotide sequence analysis was only applied to those
samples with different SSCP electrophoresis profiles. Besides, the
hemagglutinin gene was amplified by RT-PCR in two fragments
of 1100 and 1500 bp, with an overlapping region of 400 bp. The
amplified fragments were sequence analyzed.
The sequences obtained were compared to previously published
sequences belonging to different genotypes. Although some
nucleotide differences were found among different isolates, both
in N and H genes, all samples showed to belong to the genotype
D6.
MI-P12.
CITRATE METABOLISM BY ENTEROCOCCUS FAECALIS
ATCC29212
Blancato, Víctor S. and Magni, Christian.
Dpto. de Microbiología, FCBYF, U.N.R., IBR-CONICET. Suipacha
531, Rosario, Argentina. E-mail: [e-mail]
Enterococci compose the microbial association of a variety of
fermented foods such as cheese, fermented sausages and fermented
vegetables. However members of the genus Enterococcus have
distinguished themselves from other lactic acid bacteria by their
role in human infection, harboring a number of identified virulence
factors, and for their acquired resistance to antibiotics.  The purpose
of the present report was to study the Citrate metabolism in E.
faecalis. Our results shown that it could grow in MRS glucose,
MRS citrate but it grew better when both compounds are present
(MRSGC), indicating that citrate was co metabolized. PCR and
Southern blot experiments indicate that the cit cluster present in
the strain ATCC29212 is similar to that found in the sequenced
genome of E. faecalis V583. The cluster include the structural
genes coding for the citrate lyase subunits (citD, citE, citF) and
the accessory genes required for the synthesis of an active citrate
lyase complex (citC, citX and citG), the citM, a putative
oxaloacetate decarboxylase, and two genes encoding the
oxaloacetate decarboxylase complex biotin dependent (aodA,
oadB),  citN the transporter and a putative regulator protein. Dot
Blot and RT-PCR experiments suggest that the expression of the
citrate lyase complex in E. faecalis is activated by citrate.
MI-P13.
IDENTIFICATION OF TzCYC2 A CYCLIN- LIKE PROTEIN
OF  Trypanosoma cruzi
Etchegoren JI, Santori MI, Rojas F, Muñoz MJ, Téllez-Iñón MT.
INGEBI-CONICET and FCEyN-University of Buenos Aires,
Buenos Aires. E-mail [e-mail]
Several genes from the family of cdc2-related protein kinases,
CRKs, have been cloned in trypanosomatids. The participation of
these proteins in the control of the parasites’ cell division cycle is
under study. Two CRK genes, CRK1 and CRK3, had been cloned
in  Trypanosoma cruzi. Using the yeast two-hybrid system we
identified three novel cyclins-like proteins able to associate to
TzCRK1, named TzCYC2, 4 and 5. TzCYC2 belongs to the PHO
family of proteins which phosphorylate transcription factors
controlling phosphate metabolism. Its deduced amino acid
sequence (230 aa.) indicates that the identity is restricted to their
cyclin box domains (Gómez et al., 2001). Southern blot analysis
reveals that TzCYC2 is codified by a single copy gene. Northern
blot assays show that the mRNA is expressed in all life cycle stages
of the parasite, with higher levels of expression in the amastigote
form. The TzCYC2 was cloned in the bacteria expression vector
pET22(b)+, with a Histidine tag at the C-terminal. The expressed
cyclin was purified with a Ni-agarose matrix. The recombinant
protein was used to immunize rabbits to obtain a specific antibody.
In a functional complementation assay the full-length of TzCYC2
was able to rescue Saccharomyces cerevisiae deficient for G1
cyclins. These results suggest that TzCYC2 could be involved in
cell cycle progression of the parasite.
Supported by WHO, CONICET and UBA.

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