A microarray system for genotyping 150 single nucleotide polymorphisms in the coding region of human mitochondrial DNA

We established a genotyping system for a panel of 150 SNPs in the coding regions of mitochondrial DNA based on multiplex tag-array minisequencing. We show the feasibility of this system for simultaneous identification of individuals and prediction of the geographical origin of the mitochondrial DNA population lineage of the sample donors by genotyping the panel of SNPs in 265 samples representing nine different populations from Africa, Europe, and Asia. Nearly 40,000 genotypes were produced in the study, with an overall genotyping success rate of 95% and accuracy close to 100%. The gene diversity value of the panel of 150 SNPs was 0.991, compared to 0.995 for sequencing 500 nucleotides of the hypervariable regions I and II of mtDNA. For 17 individuals with identical sequences in the hypervariable regions of mtDNA, our panel of SNPs increased the power of discrimination. We observed 144 haplotypes that correspond to previously determined mitochondrial "haplogroups," and they allowed prediction of the origin of the maternal population lineage of 97% of the analyzed samples.     

A direct role of Mad1 in the spindle assembly checkpoint beyond Mad2 kinetochore recruitment

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying entry into anaphase until all sister chromatids have become bi‐oriented. A key component of the SAC is the Mad2 protein, which can adopt either an inactive open (O‐Mad2) or active closed (C‐Mad2) conformation. The conversion of O‐Mad2 into C‐Mad2 at unattached kinetochores is thought to be a key step in activating the SAC. The “template model” proposes that this is achieved by the recruitment of soluble O‐Mad2 to C‐Mad2 bound at kinetochores through its interaction with Mad1. Whether Mad1 has additional roles in the SAC beyond recruitment of C‐Mad2 to kinetochores has not yet been addressed. Here, we show that Mad1 is required for mitotic arrest even when C‐Mad2 is artificially recruited to kinetochores, indicating that it has indeed an additional function in promoting the checkpoint. The C‐terminal globular domain of Mad1 and conserved residues in this region are required for this unexpected function of Mad1.     

Antiproliferative effect of Angelica archangelica fruits

The aim of this work was to study the antiproliferative effect of a tincture from fruits of Angelica archangelica and the active components using the human pancreas cancer cell line PANC-1 as a model. Significant dose-dependent antiproliferative activity was observed in the tincture with an EC50 value of 28.6 microg/ml. Strong antiproliferative activity resulted from the two most abundant furanocoumarins in the tincture, imperatorin and xanthotoxin. The contribution of terpenes to this activity was insignificant. Imperatorin and xanthotoxin proved to be highly antiproliferative, with EC50 values of 2.7 microg/ml and 3.7 microg/ml, respectively, equivalent to 10 and 17 microM. The results indicate that furanocoumarins account for most of the antiproliferative activity of the tincture.     

Interactions of the antibiotics neomycin B and chlortetracycline with the hammerhead ribozyme as studied by Zn2+-dependent RNA cleavage

We have investigated the interactions of two antibiotics, neomycin B and chlortetracycline (CTC), with the hammerhead ribozyme using two Zn(2+) cleavage sites at U4 and A9 in its catalytic core. CTC-dependent inhibition of Zn(2+) cleavage was observed in all cases. In contrast, we unexpectedly observed acceleration of A9 cleavage by neomycin under low ionic strength conditions similar to those used to study inhibition of hammerhead substrate cleavage by this antibiotic. This result provides evidence that the inhibitory mechanism of neomycin does not include competition with the metal ion bound to the A9/G10.1 metal-ion binding site, as previously proposed. Under high ionic strength conditions, optimized for Zn(2+)-dependent cleavage, we observed neomycin-dependent inhibition of cleavage at both A9 and U4. The ability of neomycin to both inhibit and accelerate Zn(2+) cleavage suggests that there is either more than one neomycin binding site or multiple binding modes at a single site in the hammerhead ribozyme. Furthermore, the accessibilities and/or affinities of disparate neomycin binding sites or binding modes are dependent on the ionic strength and the pH of the medium.     

EPR spectroscopic analysis of TAR RNA-metal ion interactions

Metal ion-induced changes in HIV-1 TAR RNA internal dynamics were determined by the changes in EPR spectral width for TAR RNAs containing spin-labeled nucleotides (U23, U25, U38, and U40). This gave a dynamic signature for each of 10 metal ions studied, which fell into one of three distinct groups. While Li(+) and K(+) had little effect on TAR RNA internal dynamics, Na(+) unexpectedly had a dynamic signature that was similar to Ca(2+) and Sr(2+), with a decrease in mobility at U23 and U38, little or no change at U25, and an increase in mobility at U40. Mg(2+), Co(2+), Ni(2+), Zn(2+), and Ba(2+) had similar effects on U23, U38, and U40, but the mobility of U25 was markedly increased. Our results show that RNA dynamics change upon metal binding to the TAR RNA bulge, indicating that RNA structure adapts to accommodate metal ions of different size and coordination properties.     

Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation

Saccharomyces cerevisiae Rad51, Rad54, and replication protein A (RPA) proteins work in concert to make heteroduplex DNA joints during homologous recombination. With plasmid length DNA substrates, maximal DNA joint formation is observed with amounts of Rad51 substantially below what is needed to saturate the initiating single-stranded DNA template, and, relative to Rad51, Rad54 is needed in only catalytic quantities. RPA is still indispensable for optimal reaction efficiency, but its role in this instance is to sequester free single-stranded DNA, which otherwise inhibits Rad51 and Rad54 functions. We also demonstrate that Rad54 helps overcome various reaction constraints in DNA joint formation. These results thus shed light on the function of Rad54 in the Rad51-mediated homologous DNA pairing reaction and also reveal a novel role of RPA in the presynaptic stage of this reaction.     

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.