Showing papers by "Stratagene published in 1998"

Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal.

Abstract: DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps. Despite extensive biochemical, genetic and structural studies, the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragments with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

Collections of uniquely tagged molecules

Abstract: A method for functionally labeling large numbers of molecular species in a mixture of different species is provided. Each molecular species is labeled with a species-unique tag which allows for the rapid identification of each labeled species. The species-unique tag is identifiable by a uniquely identifiable property or characteristic.

Identification of thyroid hormone response elements in the human fatty acid synthase promoter

Abstract: To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5′-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides −870 to −650) and TRE2 (nucleotides −272 to −40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter−luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides −716 to −731) represents TRE1 and that the sequence GGGTCC (nucleotides −117 to −112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.

Polymerase enhancing factor (pef) extracts, pef protein complexes, isolated pef protein, and methods for purifying and identifying

Abstract: The invention provides novel extracts, proteins, and complexes that improve the polymerization activity of nucleic acid polymerases. Included within the aspects of the invention are methods for identifying compositions with a polymerase enhancing activity, methods for purifying and using these compositions, and specific extracts, proteins, and complexes that function to enhance polymerase activity. As an example, specifically described is nucleotide and amino acid sequence information for a Pyrococcus furiousus PEF (P45), which was used to produce a recombinant PEF.

Hepatocarcinogenesis (Z#2)/mutagenesis during initiation stage

Abstract: Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human α-1-antitrypsin (α1AT) Z gene transgenic mouse (Z#2). We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies. We crossed the Z#2 line and mice transgenic for lambda/lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis. Five month old double transgenic mice (Z#2+/lacI+) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10−5–10−4) useful to mutagenesis studies. In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (

Highly transformable bacterial cells and methods for producing the same

Abstract: The invention provided herein includes novel gram negative bacteria cells containing the Hte mutation. Other aspects of the invention include methods for rendering gram negative bacterial cells bearing the Hte region, such as E. coli cells competent for DNA transformation using any of a variety of competency inducing procedures. The competent cells of the subject invention may be frozen so as to provide for prolonged storage.

Versatile Epitope Tagging Vector for Gene Expression in Mammalian Cells

Abstract: We have constructed an epitope-tagging vector, pCMV-Tag 1, for gene expression in mammalian cells. This vector, which allows for N-terminal, C-terminal and internal tagging of the gene product of interest with the FLAG® and/or c-myc epitopes, enables researchers to rapidly and efficiently characterize gene products in vivo.