Visualisation of human rad52 protein and its complexes with hRad51 and DNA.

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.

Native-like, long synthetic peptides as components of sub-unit vaccines: practical and theoretical considerations for their use in humans.

Vaccines have been used as a successful tool in medicine by way of controlling many major diseases. In spite of this, vaccines today represent only a handful of all infectious diseases. Therefore, there is a pressing demand for improvements of existing vaccines with particular reference to higher efficacy and undisputed safety profiles. To this effect, as an alternative to available vaccine technologies, there has been a drive to develop vaccine candidate polypeptides by chemical synthesis. In our laboratory, we have recently developed a technology to manufacture long synthetic peptides of up to 130 residues, which are correctly folded and biologically active. This paper discusses the advantages of the molecularly defined, long synthetic peptide approach in the context of vaccine design, development and use in human vaccination.

Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

The thermal stability of yellow fever vaccines

The assessment of yellow fever vaccine thermostability both in lyophilized form and after reconstitution were analyzed. Two commercial yellow fever vaccines were assayed for their thermal stability. Vaccines were exposed to test temperatures in the range of 8 (graus) C to 45 (graus) C. Residual infectivity was measured by a plaque assay using Vero cells. The titre values were used in an accelerated degradation test that follows the Arrhenius equation and the minimum immunizing dose was assumed to be 10 (ao cubo) particles forming unit (pfu)/dose. Some of the most relevant results include that (i) regular culture medium show the same degradation pattern of a reconstituted 17D-204 vaccine; (ii) reconstituted YF-17D-204 showed a predictable half life of more than six days if kept at 0 (graus) C; (iii) there are differences in thermostability between different products that are probably due to both presence of stabilizers in the preparation and the modernization in the vaccine production; (iv) it is important to establish a proper correlation between the mouse infectivity test and the plaque assay since the last appears to be more simple, economical, and practical for small laboratories to assess the potency of the vaccine, and (v) the accelerated degradation test appears to be the best procedure to quantify the thermostability of biological products.

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.

DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera: Psychodidae)

Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probles for positively separting individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. welcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, buth this is based on circumstantial evidence because of the difficulties of identifying female sandflies wothin the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligationg short fragments of Sau 3A-resistricted, genomic DNA into the plasmid vector PUC 18. Differential screening of 1316 library clones with total genomic DNA of Ps. Wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (=ca. 0.5 namograms) was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.

Structure of a multisubunit complex that promotes DNA branch migration.

The RuvA and RuvB proteins of Escherichia coli, which are induced in response to DNA damage, are important in the formation of heteroduplex DNA during genetic recombination and related recombinational repair processes. In vitro studies show that RuvA binds Holiday junctions and acts as a specificity factor that targets the RuvB ATPase, a hexameric ring protein, to the junction. Together, RuvA and RuvB promote branch migration, an ATP-dependent reaction that increases the length of the heteroduplex DNA. Electron microscopic visualization of RuvAB now provides a new insight into the mechanism of this process. We observe the formation of a tripartite protein complex in which RuvA binds the crossover and is sandwiched between two hexameric rings of RuvB. The Holliday junction within this complex adopts a square-planar structure. We propose a molecular model for branch migration, a unique feature of which is the role played by the two oppositely oriented RuvB ring motors.

Development of nuclear DNA probes for the typing of Trypanososma cruzi

We have developed and tested a new way of typing Trypanosoma cruzi, mamely the use of cloned nuclear DNA fragments as genetic markers. Restriction fragment length polymorphisms were verified on Soutern blots hybridized to random probes. Fragment patterns were analyzed and dendrograms constructed. Our results on well characterized laboratory strains correlate well to published isoenzyme studies. Some of the probes were also hybridized to chromosomes separated by pulse field gel electrophoresis a higher degree of heterogeneity was observed at this level.

Stability of isoenzyme and kinetoplast DNA (k-DNA) patterns in successively cloned Trypanosoma cruzi populations

Four Trypanosoma cruzi strains from zymodermes A, B, C and D were successively clonedon BHI-LIT-agar-blood BLAB). Twenty clones from the first generation (F1), 10 from The second (F2) and 4 from the third (F3) from the strains A138, B147 and C23 were isolated. The D150 strain provied 29 F1 and F2 clones. The strains and clones had their isoenzyme and K-DNA patterns determined. The clones from A138, Bl47 and C231 strains presented isoemzyme and K-DNA patterns identical between thewmselves and their respective parental strains. Therefore showing the homogenety and stability of isoenzyme and K-DNA patterns after successive cloning. The Dl50 strain from zymodeme D (ZD) showed heterogeneity. Twenty-eight out of 29 clones of the first generation were of zymodeme A and only one was of zymodeme C, confirming previous reports that ZD strains consisted of ZA and ZC parasite populations. The only D150 strain clone of zymodeme C showed a K-DNA pattern identical to its parental strain. The remining clones although similar among themselves were different from the parental strain. Thus the T. cruzi strains had either homonogeneus or heterogeneous populations. The clones produced by successive cloning provided genetically homonogeous populations. Their experimental use will make future results more reliable and reproducible.

A new repetitive DNA sequence from Trypanosoma cruzi

Tandemly repeated DNA sequences are found in the genome of higher eukaryotes, and have also been demonstrated in Trypanosoma cruzi. Repeated DNA sequences are potentially useful for the diagnostic detection of T. cruzi (A. Gonzales et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 3356-3360). We have isoleted two clones from a genomic library of T. cruzi (Y strain) that contain, in one clone a family of at least seven copies of a repetitive sequence of approximately 600 base pairs, and in the other an independent copy of the same sequence. One copy of the repetition (HSP) and the independent clone (HCR) were sequenced by the Sanger procedure (Fig.). This sequence hybridized to four strains of T. cruzi tested and did not hybridize to eleven species of trypanosotids from five different Genera, being a good candidate for diagnostic assays. GenBank accession numbers: HSP#m31919, HCR#31920.

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.