Bariloche protein symposium argentine society for biochemistry and molecular biology

48
BIOCELL, 27 (Suppl. I), 2003
MI-C16.
GENETIC VARIABILITY AND RECOMBINATION IN
ARENAVIRUSES
Goñi, S.
1
, Posik, D.
2
, Romanowski, V.
1,2
, Ghiringhelli, P.D.
1
 and
Lozano, M.E.
1
1
Laboratorio de Ingeniería Genética y Biología Celular y
Molecular. Universidad Nacional de Quilmes, Buenos Aires,
Argentina. 
2
IBBM, Universidad Nacional de La Plata, Argentina.
E-mail: [e-mail]
Arenaviruses possess a bipartite single stranded RNA genome.
Both molecules (RNAs S and L) present an ambisense coding
strategy, with two genes in each one, separated by an intergenic
region constituted by self-complementary sequences. Sources of
genetic variation can have diverse origins including the error rate
of the RNA replicases and, in multipartite viruses, the genomic
rearrangement during progeny generation. In addition, recent
evidences suggest that recombination among RNA molecules is a
quite frequent event, that can have an important role as genetic
variation source. In this work, we present alternative models of
RNA recombination based on the bioinformatic analysis of
complete S RNA sequences from arenaviruses. The main molecular
mechanisms that would enable the formation of recombinants
include copy-choice, primer-alignment-and-extension and trans-
esterification. Whitewater Arroyo virus, Bear Canyon virus (both
isolated in USA) and Rio Carcarañá virus (isolated in Argentina)
provide evidences of natural recombination among arenaviruses
detected by our theoretical analysis. All these possible genetic
sources of variation, added to ecological and human activity
changes, suggest that the number of arenaviruses would be
increased in the next years. Molecular-epidemiological surveillance
programs that contemplate these possibilities would be able to
help to minimize the risks of the emergency or re-emergency of
potential pathogens.
MI-C17.
GALACTOSIDES METABOLISM OF l. plantarum, GENES
AND THEIR FUNCTION: REGULATION IS THE KEY
Silvestroni, Aurelio
1
; Connes, C.
2
; LeBlanc, J-G.
1
; Piard, J-C.
2
;
Sesma, F.
1
 and Savoy de Giori, G.
1
1
CERELA, Tucumán, Argentina. 
2
INRA-CRJ, France. E-mail:
[e-mail]
In this study we present evidence that Lactobacillus plantarum
has sugar metabolism functional genes but fail to metabolize these
carbohydrates probably because they lack a crucial step of their
metabolic pathway. In L. plantarum ATCC8014, a 7.5 Kb gene
cluster involved in galactosides metabolism was identified and
sequenced. Their relevant genes, an á–galactosidase (melA) and a
galactosides permease (rafP) were cloned and expressed in
heterologous hosts showing enzymatic activity and lactose
transport, not present in the native strain. Using the cloned genes,
the á-Gal activity was evidenced in different E. coli strains and
Lactococcus lactis, while functional lactose transport was
demonstrated in E. coli. The metabolic capacity of two L.
plantarum  strains were compared as was the complete genome
region encoding the melA and rafP genes at the nucleotide and
amino-acid level. Only one of these strains was able to grow on
melibiose and lactose. However, sequence comparison showed no
relevant differences, suggesting that regulatory genes could be
responsible for these divergent results. This comparison provides
another good example that genes “are live and are functional”
only in a specific genetic environment.
MI-C18.
REGULATION OF EXPRESSION OF THE TWO-
COMPONENT SYSTEM CitST IN Bacillus subtilis
Sender, Pablo D.
1
; Blancato, Victor S.
1
; Lolkema, Juke
2
; and
Magni, Christian
1
.
1
Depto de Microbiología, FCByF, U.N.R., IBR-CONICET,
Argentina. 
2
Dept. of Microbiology, University of Groningen, The
Netherlands. E-mail: [e-mail]
The two-component signal transduction system is a ubiquitous
mechanism for sensing and responding to several environmental
stimuli in bacteria. In Bacillus subtilis, citS and citT encode a
two-component system involved at the regulation of the citrate
transporter CitM. Induction of citM is mediated by CitS (sensor
kinase), which recognizes external citrate, and CitT (response
regulator) that work as a transcriptional activator by binding to
the promoter region of citM. Due to the expression of citM depends
on the expression of the two-component citS and citT, in this period
we start the molecular characterization of the gene expression of
this two-component system to understand more about the exquisite
regulation in the citrate-Mg
+2
 transport in B. subtilis. Our
experiments shown that an expression of the two-component
system depends on the constitutive promoter located upstream
region of  citS. The control of the citS expression is under the
CcpA protein, which binds to the cre site present in the internal
region of this gene. The expression of citST is not repressed by
arginine in rich medium suggesting that this repression is operated
in the citM region.
BE-C1.
ENERGETIC MAPPING OF A PROTEIN-DNA INTERFACE
Ferreiro, Diego U.; Dellarole, Mariano; Centeno, Juan M.; Nadra,
Alejandro D. and Prat Gay, Gonzalo.
Fundación Instituto Leloir and FCEyN-UBA. Buenos Aires,
Argentina.  E-mail: [e-mail]
Protein-DNA interactions play a central role in cell physiology.
Within these, site-specific recognition is a primary topic that
underprints the expression profile of whole genomes. Although
many biological aspects of these non-covalent interactions are
becoming clearer, detailed biophysical understanding of site
specific recognition is lacking. Using the papillomavirus E2c
transcription factor as a model system we aim a physicochemical
dissection of its DNA binding properties. We set-up an
spectroscopic binding essay, readily done in solution, which
allowed us to quantify  DNA binding parameters in energetic terms,
detect conformational changes exerted on both macromolecules
upon binding and relate them to the high resolution structures
available. An extensive site-directed mutagenesis analysis over
the entire DNA binding region led us to assign the energetic
contributions of individual sidechains to the overall binding energy.
The small individual contribution of sidechains (< 1.0 kcal/mol)
correlates with a highly dynamic interface. This, together with
the additive free energy contributions by aminoacid-base
interactions, is indicative of absence of “hot-spots” at the interface.
Furthermore, the global energetics can be mapped into separate
patches that may be related to the interface solvent accessibility.
With the integration of structural, biophysical and computational
techniques, the molecular basis of this complex macromolecular
interactions are beginning to picture into thermodynamic models
that may help us grasp how these specific interactions are achieved.

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