Resumen: We have cloned and expressed in Escherichia coli a 702-base pair gene coding for the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypanosoma cruzi. The DHFR domain was purified to homogeneity by methotrexate-Sepharose chromatography followed by an anion-exchange
chromatography step in a mono Q column, and displayed a single 27-kDa band on SDS-PAGE. Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the wild-type bifunctional enzyme with Km values of 0.75 microM for dihydrofolate and 16 microM for NADPH and a kcat value of 16.5 s-1. T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the inhibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors that form an initial complex which isomerizes slowly to a tighter complex and are referred to as 'slow, tight-binding' inhibitors. While the slow-binding step of inhibition was apparently unaffected in the individually expressed DHFR domain, the overall inhibition constant was two-fold higher as a consequence of the superior inhibition constant value obtained for the initial inhibitory complex.
Resumen: Recent studies have proposed the use of molecular coancestry coefficients as a measure of genetic variability and as a useful tool for conservation
purposes. Using simulated data, molecular coancestry
has been shown to become constant very quickly after
separation of populations, leading to population diversity remaining constant.
However, the use of molecular coancestry information to study the genetic relationships between breeds has not yet been widely explored. Here we analyze the polymorphism of 14 microsatellites in 222 unrelated individuals belonging to seven native Spanish breeds to ascertain the usefulness of molecular coancestry-based methodologies in providing information on their genetic relationships. Average kinship distance
(Dk) and average molecular coancestry coefficients
(fij) were compared with well-known genetic distances, such as between-breed Reynolds’ distance (DR), Nei’s standard distance (Ds), and shared allele distance (DAS). Kinship distance and fij have moderate to low correlations with the other genetic distances, showing that they provide different information: both Dk and fij account for the allele frequencies in the founder population, whereas DR, Ds, and DAS characterize the shortterm evolution of the populations. Furthermore, Dk and fij were only moderately correlated (−0.500). The present study used field data to confirm previous research pointing out the ability of molecular coancestry coefficients
to assess genetic differentiation of an ancestral
origin. In this respect, molecular coancestry-based parameters may be used with classical genetic parameters to obtain information on population dynamics in livestock breeds. This study additionally presents reliable evidence on the history of these sheep breeds.
Resumen: The mechanisms by which pore-forming toxins are able to insert into lipid
membranes are a subject of the highest interest in the field of lipid–protein
interaction. Eight mutants affecting different regions of sticholysin II, a
member of the pore-forming actinoporin family, have been produced, and
their hemolytic
and lipid-binding properties were compared to those of the
wild-type protein. A thermodynamic approach to the mechanism of pore
formation is also presented. Isothermal titration calorimetry experiments
show that pore formation by sticholysin II is an enthalpy-driven process
that occurs with a high affinity constant (1.7×108 M−1). Results suggest that
conformational flexibility at the N-terminus of the protein does not provide
higher affinity for the membrane, although it is necessary for correct pore
formation. Membrane binding is achieved through two separate mechanisms,
that is, recognition of the lipid–water interface by a cluster of aromatic
residues and additional specific interactions that include a phosphocholinebinding
site. Thermodynamic parameters derived from titration experiments
are discussed in terms of a putative model for pore formation.
