Trypanosoma cruzi: Mutations in the 3' untranslated region of calmodulin gene are specific for lineages T. cruzi I, T. cruzi II, and the Zymodeme III isolates

Populations of Trypanosoma cruzi can be clustered in two main phylogenetic lineages, T. cruzi I and T. cruzi II and a third group denominated Zymodeme III (ZIII) has been described. Using 23 isolates representing the two major T. cruzi groups and the Zymodeme III, the 3' untranslated region (3'UTR) of the calmodulin gene was analyzed. Several mutations located on a 330 bp segment of this 3'UTR were observed, among which three important insertion/deletion events, namely (1) a dinucleotide AG present only in ZIII isolates; (2) a 13 bases purine block missing only in ZIII; and (3) a five base GT block in T. cruzi II. Minimum free energy dot plots show that T. cruzi I and T. cruzi ZIII exhibit similar patterns of optimal and sub-optimal folding of this segment. These mutations in 3'UTR of calmodulin raise the possibility that T. cruzi I and ZIII group are sharing common functional routes.     

Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium

Owing to the cell membrane barriers, most macromolecules and hydrophilic molecules could not freely enter into living cells. However, cell-penetrating peptides (CPPs) have been discovered that can translocate themselves and associate cargoes into the cytoplasm. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) can form stable complexes with plasmid DNA at the optimized nitrogen/phosphate ratio of 3 and deliver plasmid DNA into Paramecium caudatum in a noncovalent manner. Accordingly, the transported plasmid encoding the green fluorescent protein (GFP) gene could be expressed in cells functionally assayed at both the protein and DNA levels. The efficiency of gene delivery varied among these CPPs in the order of HR9 > PR9 > SR9. In addition, these CPPs and CPP/DNA complexes were not cytotoxic in Paramecium detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diohenyltetrazolium bromide (MTT) assay. Thus, these results suggest that the functionality of arginine-rich CPPs offers an efficient and safe tool for transgenesis in eukaryotic protozoans.     

Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant

Arabidopsis thaliana APETALA3 (AP3) and Antirrhinum majus DEFICIENS (DEF) MADS box genes are required to specify petal and stamen identity. AP3 and DEF are members of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. In order to investigate the molecular mechanisms underlying organ development in early diverging clades of core eudicots, we isolated and identified an AP3 homolog, FaesAP3, from Fagopyrum esculentum (buckwheat, Polygonaceae), a multi-food-use pseudocereal with healing benefits. Protein sequence alignment and phylogenetic analyses revealed that FaesAP3 grouped into the euAP3 lineage. Expression analysis showed that FaesAP3 was transcribed only in developing stamens, and differed from AP3 and DEF, which expressed in developing petals and stamens. Moreover, ectopic expression of FaesAP3 rescued stamen development without complementation of petal development in an Arabidopsis ap3 mutant. Our results suggest that FaesAP3 is involved in the development of stamens in buckwheat. These results also suggest that FaesAP3 holds some potential for biotechnical engineering to create a male sterile line of F. esculentum.     

The nucleoporin Nup358 associates with and regulates interphase microtubules

The nucleoporin Nup358 resides on the cytoplasmic face of the interphase nuclear pore complex (NPC). During metaphase, its recruitment to kinetochores is important for correct microtubule-kinetochore attachment. Here, we report that a fraction of endogenous Nup358 interacts with interphase microtubules through its N-terminal region (BPN). Cells overexpressing the microtubule targeting domain of Nup358 displayed dramatic alteration in the microtubule organization including increased microtubule bundling and stability. Ectopic expression of BPN and full-length Nup358 exhibited significantly higher levels of acetylated microtubules that were resistant to nocodazole, a microtubule depolymerizing agent. Furthermore, RNAi mediated depletion of Nup358 affected polarized stabilization of microtubules during directed cell migration, confirming the in vivo role of Nup358 in regulating interphase microtubules.     

pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.     

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other’s inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield.     

Leishmania mexicana amazonensis: development of a peptide tag useful for labeling and purifying biotinylated recombinant proteins

A number of peptide tags are available to facilitate the characterization of recombinant proteins. We have tested the bacterial oxaloacetate decarboxylase biotinylation domain for its efficacy in tagging recombinant proteins in vivo in Leishmania. To achieve efficient biotinylation, Leishmania also had to be co-transformed with the gene for bacterial biotin protein ligase (birA gene product). The recombinant chimeric protein could be detected on blots probed with avidin-horseradish peroxidase and purified on immobilized monomeric avidin resins.     

Genetic variability of Xrcc3 and Rad51 modulates the risk of head and neck cancer

A case-control study was conducted to analyze the possible associations between the head and neck cancer (HNC) risk and fourteen single nucleotide polymorphisms (SNPs) and haplotypes in Xrcc3 and Rad51 genes. This study involved 81 HNC cases and 111 healthy control subjects. A significant risk-increasing effect of rs3212057 (p.Arg94His) SNP in Xrcc3 (OR=6.6; p<0.01) was observed. On the other hand, risk-decreasing effect was found for rs5030789 (g.3997A>G) and rs1801321 (c.-60G>T) in 5' near gene and 5'UTR regions of Rad51, respectively (OR=0.3 and OR=0.2, p<0.05, respectively). Moreover, these effects were shown to be modulated by tobacco-smoking status and gene-gene interactions. Concluding, the genetic variability of Xrcc3 and/or Rad51 genes might be of relevance with respect to HNC risk.     

Arabidopsis thaliana MTP1 is a Zn transporter in the vacuolar membrane which mediates Zn detoxification and drives leaf Zn accumulation

The Arabidopsis thaliana metal tolerance protein 1 (MTP1) of the cation diffusion facilitator family of membrane transport proteins can mediate the detoxification of Zn in Arabidopsis and yeast. Xenopus laevis oocytes expressing AtMTP1 accumulate more Zn than oocytes expressing the AtMTP1(D94A) mutant or water-injected oocytes. An AtMTP1-GFP fusion protein localizes to the vacuolar membrane in root and leaf cells. The analysis of Arabidopsis transformed with a promoter-GUS construct suggests that AtMTP1 is not produced throughout the plant, but primarily in the subpopulation of dividing, differentiating and expanding cells. RNA interference-mediated silencing of AtMTP1 causes Zn hypersensitivity and a reduction in Zn concentrations in vegetative plant tissues.     

Plant degreening: evolution and expression of tomato (Solanum lycopersicum) dephytylation enzymes

Chlorophyll is the most abundant pigment on earth and even though it is known that its high photo-excitability necessitates a tight regulation of its degradation pathway, to date there are still several steps in chlorophyll breakdown that remain obscure. In order to better understand the ‘degreening’ processes that accompany leaf senescence and fruit ripening, we characterized the enzyme-encoding genes involved in dephytylation from tomato (Solanum lycopersicum). A single pheophytinase (PPH) gene and four chlorophyllase (CLH) genes were identified in the tomato genome. A phenetic analysis revealed two groups of CLHs in eudicot species and further evolutionary analysis indicated that these enzymes are under diverse selection pressures. A comprehensive expression profile analysis also suggested functional specificity for these dephytylating enzymes. The integrated analysis allows us to propose three general roles for chlorophyll dephytylation: i) PPH, which is under high selective constraint, is responsible for chlorophyll degradation during developmentally programed physiological processes; ii) Group I CLHs, which are under relaxed selection constraint, respond to environmental and hormonal stimuli and play a role in plant adaptation plasticity; and iii) Group II CLHs, which are also under high selective constraint, are mostly involved in chlorophyll recycling.