Contractions and expansions of CAG/CTG trinucleotide repeats occur during ectopic gene conversion in yeast,by a MUS81-independent mechanism

CAG/CTG trinucleotide repeat tracts expand and contract at a high rate during gene conversion in Saccharomyces cerevisiae. In order to characterize the mechanism responsible for such rearrangements, we built an experimental system based on the use of the rare cutter endonuclease I-SceI, to study the fate of trinucleotide repeat tracts during meiotic or mitotic (allelic or ectopic) gene conversion. After double-strand break (DSB) induced meiotic recombination, (CAG)(98) and (CAG)(255) are rearranged in 5% and 52% of the gene conversions, respectively, with similar proportions of contractions and expansions. No evidence of a meiotic hot spot activity associated with trinucleotide repeats could be found. When gene conversion is induced by a DSB during mitotic growth of the cells, no rearrangement of the repeat tracts is detected when the donor sequence is allelic to the recipient site of the DSB. However, when the donor sequence is at an ectopic location, frequent contractions and expansions of the repeat tract are found. No crossing-over associated with gene conversion could be detected. Mutants for the MUS81 gene, involved in the resolution of recombination intermediates, show a frequency of rearrangements identical with that of the wild-type strain. We concluded that trinucleotide repeat rearrangements occur frequently during ectopic but not during allelic recombination, by a mechanism that does not require crossover formation.     

The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase

The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCF(SLY1)-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.     

Cationic phosphoramidate alpha-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation

A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural α-anomeric configuration. Cationic α-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic α-ON and their RNA targets were not substrates for RNase H. The penalty in T_m inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic α-ON in cell culture.     

Biological processes in organised media

Embedding a simple Michaelis-Menten enzyme in a gel slice may allow the catalysis of not only scalar processes but also vectorial ones, including uphill transport of a substrate between two compartments, and may make it seem as if two enzymes or transporters are present or as if an allosterically controlled enzyme/transporter is operating. The values of kinetic parameters of an enzyme in a partially hydrophobic environment are usually different from those actually measured in a homogeneous aqueous solution. This implies that fitting kinetic data (expressed in reciprocal co-ordinates) from in vivo studies of enzymes or transporters to two straight lines or a sigmoidal curve does not prove the existence of two different membrane mechanisms or allosteric control. In the artificial transport systems described here, a functional asymmetry was sufficient to induce uphill transport, therefore, although the active transport systems characterised so far correspond to proteins asymmetrically anchored in a membrane, the past or present existence of structurally symmetrical systems of transport in vivo cannot be excluded. The fact that oscillations can be induced in studies of the maintenance of the electrical potential of frog skin by addition of lithium allowed evaluation of several parameters fundamental to the functioning of the system in vivo (e.g., relative volumes of internal compartments, characteristic times of ionic exchanges between compartments). Hence, under conditions that approach real biological complexity, increasing the complexity of the behaviour of the system may provide information that cannot be obtained by a conventional, reductionist approach.     

Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle

Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.     

Characterization of a common deletion polymorphism of the UGT2B17 gene linked to UGT2B15

Members of the human UDP-glucuronosyltransferase 2B family are located in a cluster on chromosome 4q13 and code for enzymes whose gene products are responsible for the normal catabolism of steroid hormones. Two members of this family, UGT2B15 and UGT2B17, share over 95% sequence identity. However, UGT2B17 exhibits broader substrate specificity due to a single amino acid difference. Using gene-specific primers to explore the genomic organization of these two genes, it was determined that UGT2B17 is absent in some human DNA samples. The gene-specific primers demonstrated the presence or absence of a 150 kb genomic interval spanning the entire UGT2B17 gene, revealing that UGT2B17 is present in the human genome as a deletion polymorphism linked to UGT2B15. Furthermore, it is shown that the UGT2B17 deletion polymorphism shows Mendelian segregation and allele frequencies that differ between African Americans and Caucasians.     

Cloning, expression, and purification of the 27 kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis

A limited number of proteins of Mycobacterium tuberculosis have been characterized so far for their use as potential candidates for diagnosis and vaccine studies. This study was aimed at cloning, expression, and purification of a 27 kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR amplified using primers that contain specific restriction sites. The amplified product was inserted initially into pTOPO and then sub-cloned into pET15b and pET24d vectors, such that the recombinant protein is predicted to contain an N-terminal or a C-terminal histidine tag, respectively. The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) and the recombinant proteins were purified from the cytosolic fractions of the E. coli sonicates by nickel-NTA chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high performance liquid chromatography (HPLC), matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The purified proteins were found to be immunogenic and useful for immunodiagnostic studies of tuberculosis by enzyme linked immunosorbent assay (ELISA), with a sensitivity of 71% and specificity of 95%.     

Tick prevalence and species diversity on Aldabran giant tortoises (Dipsochelys dussumieri) in relation to host range and host size in a restored ecosystem, Kenya

Tick species density and diversity on Aldabran tortoises was investigated in relation to the habitat range and size of each tortoise. Identification of tick infestation patterns forms an important aspect of effective tick control. Ten Aldabran tortoises were de‐ticked and monitored over the course of 2 months. Tick species found were Amblyomma sparsum, Amblyomma nuttalli, Amblyomma hebraeum and Boophilus decoloratus, with the most prevalent species found being A. sparsum. Tick loads varied considerably from 20 to 214 ticks per tortoise, with most ticks collected from the head/neck region. Tortoises ranging outside Haller Park had higher tick loads (70–214) compared with tortoises ranging within Haller Park (20–99). Tick load was not correlated with tortoise size. Results indicate that tick loads are related to the habitat range of the tortoises and may indirectly also be related to food preference and host food availability. Implications of the findings and appropriate tick control measures are discussed.