Analysis of the factor V Leiden mutation using the READIT Assay.

BACKGROUND: A variety of methods exist for the detection of single-nucleotide polymorphisms (SNPs) present in amplified segments of genomic DNA. We show the application of a novel SNP scoring tool for analysis of the factor V Leiden mutation. METHODS AND RESULTS: We have developed a novel method for analyzing SNPs. The luciferase-based technique, known as the READIT Technology (Promega Corp, Madison, WI), was used to analyze 510 residual human samples sent for factor V Leiden testing from three independent testing laboratories. A blinded retrospective analysis of the factor V Leiden mutation was used to determine the accuracy and throughput capabilities of the technology. One hundred percent concordance was observed between the READIT Assay and genotype assignments made in the testing laboratories. In addition, greater than 6 SDs of separation were observed between the means of wild-type and heterozygote sample populations. Repetitive sample measurements with representative wild-type, heterozygote, and mutant samples showed that greater than 9 SDs separated the means of heterozygote and homozygote sample populations. Confidence intervals based on the means of wild-type, heterozygote, and mutant sample populations were determined. CONCLUSION: Perfect concordance using the READIT Assay showed its effectiveness as a SNP scoring tool. The design of the factor V READIT Assay was straightforward, requiring the design of two unmodified oligonucleotides that differ at the 3' penultimate position to form perfect hybrids with the wild-type or Leiden form of the factor V sequence. The use of previously published amplification primers and conditions minimized the time needed to optimize and validate the assay. The READIT Calculator supplied with the assay allowed automated genotype assignments and statistical analysis from the READIT Assay data. Confidence-interval analysis validated the ability to distinguish between wild-type, heterozygote, and mutant samples using the READIT Assay.

Retinoid X receptor isotype identity directs human vitamin D receptor heterodimer transactivation from the 24-hydroxylase vitamin D response elements in yeast.

Unlike estrogen and progesterone receptors that operate as homodimers on response elements, retinoid X receptors (RXRs) and vitamin D receptors (VDRs) can function as heterodimers. Studies concerning the significance of heterodimeric partnerships are usually performed utilizing mammalian or insect cells. These cells express endogenous nuclear receptors, making it impossible to assign a role for one receptor subtype over another while studying the function of transfected receptor(s). Yeast lacks endogenous VDRs and RXRs and their ligands and provides a unique cellular context to study nuclear receptor function. We examined the interaction between human VDR and human RXR alpha, mouse RXR beta 2, and mouse RXR gamma to identify physiologically important receptor interactions. DNA binding studies on consensus, osteocalcin, or the rat 24-hydroxylase vitamin D response elements (VDREs) indicated that although RXR complexes can form on the consensus DNA elements, RXR:VDR heterodimers preferentially interact with the natural VDREs. The interaction is RXR isotype-specific and affected by ligands. Transactivation studies using the rat 24-hydroxylase VDREs indicated that VDR preferentially associated with RXR alpha or RXR gamma to stimulate transcription, and the activity was potentiated by ligand. Although RXR beta 2:VDR bound tightly to DNA, the resulting heterodimer transactivated poorly. The regulation of the 24-hydroxylase promoter observed in yeast is similar with respect to transactivation potential of specific VDRE and fold activation observed in osteosarcoma cells. Ligand binding to both receptors in a RXR:VDR complex is required for maximal transcriptional activity, indicating that the isotype-specific RXR partner significantly contributes to the ability of RXR:VDR heterodimers to transactivate from target response elements in yeast.

Functional analysis of Drosophila factor 5 (TFIIF), a general transcription factor.

Factor 5 is a Drosophila RNA polymerase II initiation factor that also affects the elongation phase of transcription. We have used a cDNA encoding the large subunit of factor 5 (F5a) to produce recombinant F5a (rF5a). Antibodies directed against peptides deduced from the sequence of the F5a cDNA recognized rF5a and the large subunit of factor 5 purified from Kc cells. A chimeric human/fly factor composed of the small subunit of human TFIIF (RAP30) and rF5a stimulated elongation by Drosophila RNA polymerase II when assayed using a dC-tailed template. In addition, the chimeric human/fly factor functioned during initiation in either the Drosophila or human system. Therefore, the structure of the large subunit of TFIIF is sufficiently conserved from human to fly to allow functional interaction with both the small subunit of TFIIF and RNA polymerase II from either species. Analysis of deletion mutants of F5a indicated that almost all of the protein was required for initiation while only the NH2-terminal region was required for stimulating transcriptional elongation. A comparison of our results with those obtained with RAP74 suggest that the carboxyl terminal region of the protein may be involved in interactions with RNA polymerase II or other factors during initiation.

Stability of Drosophila RNA polymerase II elongation complexes in vitro.

We show that nuclear extract from Drosophila Kc cells supports efficient elongation by RNA polymerase II initiated from the actin 5C promoter. The addition of 0.3% Sarkosyl, 1 mg of heparin per ml, or 250 mM KCl immediately after initiation has two effects. First, the elongation rate is reduced 80 to 90% as a result of the inhibition of elongation factors. Second, there is an increase in the amount of long runoff RNA, suggesting that there is an early block to elongation that is relieved by the disruptive reagents. Consistent with the first effect, we find that the ability of factor 5 (TFIIF) to stimulate the elongation rate is inhibited by the disruptive agents when assayed in a defined system containing pure RNA polymerase II and a dC-tailed template. The disruptive agents also inhibit the ability of DmS-II to suppress transcriptional pausing but only slightly reduce the ability of DmS-II to increase the elongation rate twofold. The pause sites encountered by RNA polymerase II after initiation at a promoter and subsequent treatment with the disruptive reagents are also recognized by pure polymerase transcribing a dC-tailed template. It has been suggested that 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits RNA polymerase II during elongation, but we find that the purine nucleoside analog has no effect on elongation complexes containing RNA over 500 nucleotides in length or on the action of factor 5 or DmS-II in the defined system.

Molecular cloning of a putative tetrodotoxin-resistant rat heart Na+ channel isoform.

Voltage-gated Na+ channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na+ channels are blocked by 1-10 microM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity [3H]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire alpha subunit of a cardiac-specific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles.