In the polymerase chain reaction (PCR) technique, DNA is amplified in vitro by a series of polymerization cycles consisting of three temperature-dependent steps: DNA denaturation, primer-template annealing, and DNA synthesis by a thermostable DNA polymerase. The purity and yield of the reaction products depend on several parameters, one of which is the annealing temperature (Ta). At both sub- and super-optimal Ta values, non-specific products may be formed, and the yield of products is reduced. Optimizing the Ta is especially critical when long products are synthesized or when total genomic DNA is the substrate for PCR. In this article we experimentally determine the optimal annealing temperature (TaOPT) values for several primer-template pairs and develop a method for its calculation. The TaOPT is found to be a function of the melting temperatures of the less stable primer-template pair and of the product. The fact that experimental and calculated TaOPT values agree to within 0.7 degree C eliminates the need for determining TaOPT experimentally. Synthesis of DNA fragments shorter than 1 kb is more efficient if a variable Ta is used, such that the Ta is higher in each consecutive cycle.

Anisotropic shock sensitivity and detonation temperature of pentaerythritol tetranitrate single crystal Electrical resistivity of singlecrystal Al2O3 shockcompressed in the pressure range 91-220 GPa (0.91-2.20 Mbar) Experimental investigation of shear stress effects on shockinduced phase transition in InSb single crystal AIP Conf.The shock response of single crystals of the explosive pentaerythritol tetranitrate was studied from 3.4 to 19 GPa. Four orientations, (llO), (OOl), (lOl), and (100) were studied. The shock Hugoniot and shock initiation sensitivities were measured in wedge tests. Intermediate velocity transitions were noted. A sensitivity anomaly near 4 GPa was discovered for (110) orientations. Chemiluminescent phenomena associated with this anomaly were studied using framing photography and uv/visible spectroscopy. Both plane-wave explosive and gas-gun impact experiments were done. Analysis of the steric hindrance occurring during edge dislocation motion in the shear flow associated with shockinduced uniaxial strain was done. The results corroborate the hypothesis that the orientation dependence of shock initiation sensitivity is due to the anisotropy of plastic flow, that is, the relative ease of slip in different orientations. 3572

The IB␣ protein is a key molecular target involved in the control of NF-B/Rel transcription factors during viral infection or inflammatory reactions. This NF-B-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets IB␣ for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the IB␣ inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of IB␣ as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length IB␣ protein but not with N-terminal-deletion-containing versions of IB␣. IB␣ interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of IB␣. Importantly, in HeLa and 293 cells, endogenous and transfected IB␣ coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and IB␣ colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and IB␣ were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, IB␣ colocalized with ␣-tubulin filaments. Taken together, these results highlight an intriguing interaction between the IB␣ protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.The cytoskeleton can be considered a complex biological network that integrates signal transduction pathways and responds by altering the pattern of gene expression and by changing cell morphology. Reorganization of structural components within the cytoskeleton can achieve these changes by exposing or masking internal molecular binding sites (32). For example, changes in the arrangement of cytoskeletal filaments may expose sequestered RNA molecules to cytoplasmic enzymes to which they were otherwise resistant (50). Recently, the Rho family of G proteins has become a paradigm of the link between actin cytoskeleton modifications and gene expression in response to growth factors (for a review, see reference 18). For example, phosphatidylinositol 3-kinase-mediated activation of Rac proteins by platelet-derived growth factor or insulin receptors (52) leads to membrane ruffling on the one hand and stimulation of the p21-associated kinases that control JNK/SAPK (18) and the mitogen-activated protein kinases (MAPKs) (25) on the other hand, leading to gene expression.The microtubule cytoskeleton also is involved in modulation of gene expression. Microtubule-disrupting agents such as colchicine have been reported to induce interleukin-1 expression in monocytes (45) or AP1-regulat...

CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrAϳP exhibits high affinity (dissociation constant [K d ], <10 nM) for consensus TTAA-N7-TTAA binding sites with "typical" (N ‫؍‬ 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (K d , >500 nM) CtrA binding sites with "atypical" (N 7) spacing. Footprints demonstrated that phosphorylated CtrAϳP does not exhibit increased affinity for "atypical" sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 M) accumulate, which can drive CtrA binding to "atypical" sites. In vivo cross-linking showed that when the stable CtrA⌬3 protein persists during the cell cycle, the "atypical" sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrA⌬3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrAϳP phosphorylation that provides the dominant "activation" signal. Protein dimerization is one potential means of "activation." The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the "atypical" sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how "atypical" sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.