Gary F. Gerard

Mutation detection using Surveyor™ nuclease

We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3 side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA; (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.

RNA-Directed DNA Polymerase—Properties and Functions in Oncogenic RNA Viruses and Cells1

Publisher Summary This chapter provides description of the properties and classification of the oncornaviruses and related virus particles that possess RNA→DNA polymerase molecules. The chapter describes the properties of the DNA polymerase activities of oncornaviruses and related particles including the endogenous reaction, the reaction products, and the utilization of external templates. The chapter also deals with the studies on the inhibitors of the viral DNA polymerase, their mechanism of action and their effect on cell transformation and tumor induction. The properties of purified RNA→DNA polymerase and the mechanism of DNA synthesis are also explored. The chapter provides evidence for the in vivo function of the viral RNA→DNA polymerase and a concise description of the present understanding of the molecular events of oncornavirus replication and cell transformation. The chapter also discusses the analysis of viral related base sequences in normal and cancer cells by molecular hybridization with the viral DNA product of the RNA→DNA polymerase, and studies on RNA→DNA polymerase in normal and cancer cells.

Poly(2′-O-methylcytidylate)·oligodeoxyguanylate, a template-primer specific for reverse transcriptase, is not utilized by hela cell γ DNA polymerases

Summary The ability of y DNA polymerase activities purified from the cytoplasm and nucleus of HeLa cells (Spadari, S. and Weissbach, A. (1974) J. Biol. Chem. 249 , 5809) to copy poly(Cm)·oligo(dG) was tested, and compared with the template activity of poly(C)·oligo(dG). The major HeLa cell nuclear γ DNA polymerase effectively copied poly(C)·oligo(dG), while none of the γ DNA polymerase activities purified from HeLa cells were capable of utilizing poly(Cm)·oligo(dG) as template. These results support the contention that poly(Cm)·oligo(dG), but not poly(C)·oligo(dG), is a specific template-primer for RNA tumor virus RNA-directed DNA polymerases.

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases.

Abstract Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). AF ABDMP (2,5-dimethyl-4-N-benzyl demethyl rifampicin), AF 013 (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.

A method for clone sequence confirmation using a mismatch-specific DNA endonuclease.

Site-directed mutagenesis and polymerase chain reaction (PCR)-based cloning are well-established methods carried out routinely in most modern molecular biology laboratories. Application of these methods requires confirmation of the DNA sequence of the target gene by sequencing of DNA purified from multiple colonies, a laborious process. We have developed an alternative approach to screen DNA amplified directly from colony DNA for both desired and undesired mutations. This approach is based on the use of a plant mismatch DNA endonuclease, Surveyor Nuclease, to directly screen clones derived by site-directed mutagenesis. We have also used this approach to identify error-free clones of three genes from celery cDNA produced by PCR and TOPO® cloning. Sequence confirmation using Surveyor Nuclease provides a fast and simple approach to obtain desired clones from site-directed mutagenesis and PCR-based cloning methods without the necessity of sequencing DNAs purified from multiple clones.

Studies on the DNA polymerase activity contained in particles released from human embryo cell monolayers.

Abstract Cell monolayers derived from a whole human embryo released particles when grown at 39°C, but not at 36°C, that had RNAase and actinomycin D-sensitive endogenous DNA polymerase activity which banded in sucrose density gradients at 1.17 g/cm 3 . Monolayers from embryonic skin and muscle, kidney and lung also released such particles. The release phenomenon appears to be spespecific to human embryonic cells, since similar endogenous DNA polymerase activity could not be detected in culture fluid from several established human cell lines of different origin grown at 39°C. DNA polymerase activity purified from these human embryo particles by salt and neutral detergent extraction, followed by DEAE-cellulose and phosphocellulose chromatography (i) had a molecular weight of 105000 as determined by Sephadex G-200 gel filtration; (ii) copied activated calf thymus DNA most efficiently among the template-primers tested and poly(dC) · (dG) 12–18 much less effectively, and did not respond to poly(A) · (dT) 12–18 or (dT) 12–18 alone; (iii) was not inhibited by N -ethylmaleimide at a concentration of 1 · 10 −3 M; and (iv) was stimulated by the presence of KCl in reaction mixtures up to a concentration of 70 mM. The DNA polymerase from the human embryo particles does not therefore possess the properties of viral RNA-directed DNA polymerase, even though the particles themselves exhibit several of the properties of an RNA tumor virus. Moreover, all properties determined for the human embryo enzyme were similar or identical to those of human mitochondrial DNA polymerase (Fry, M. and Weissbach, A. (1973) Biochemistry 12, 3602–3608).

Biochemical suppression of tumor virus activity.

DNA and RNA polymerases and their templates play a central role in the control of gene expression. As an approach to a scientific chemotherapy to control nucleic acid‐directed disease, we have used the highly purified RNA‐directed DNA polymerase of the avian myeloblastosis virus (AMV) as a test enzyme to search for inhibitors of DNA polymerase. Three kinds of inhibitors with different mechanisms of action were studied: 1) rifamycin SV derivatives; 2) polycyclic amines; and 3) homopolymers composed of 2′O‐methyl and 2′‐O‐ethyl substituted poly(A), poly(U), poly(I), and poly(C). The biological activity in vitro and in vivo of these inhibitors and their mechanisms of action are discussed.

Acquisition of mitochondrial dysregulation and resistance to mitochondrial-mediated apoptosis after genotoxic insult in normal human fibroblasts: a possible model for early stage carcinogenesis

Acquisition of death-resistance is critical in the evolution of neoplasia. Our aim was to model the early stages of carcinogenesis by examining intracellular alterations in cells that have acquired apoptosis-resistance after exposure to a complex genotoxin. We previously generated sub-populations of BJ-hTERT human diploid fibroblasts, which have acquired death-resistance following exposure to hexavalent chromium [Cr(VI)], a broad-spectrum genotoxicant. Long-term exposure to certain forms of Cr(VI) is associated with respiratory carcinogenesis. Here, we report on the death-sensitivity of subclonal populations derived from clonogenic survivors of BJ-hTERT cells treated with 5 μM Cr(VI) (DR1, DR2), or selected by dilution-based cloning without treatment (CC1). Following Cr(VI) treatment, CC1 cells downregulated expression of the anti-apoptotic protein Bcl-2 and exhibited extensive expression of cleaved caspase 3. In contrast, the DR cells exhibited no cleaved caspase 3 expression and maintained expression of Bcl-2 following recovery from 24 h Cr(VI) exposure. The DR cells also exhibited attenuated mitochondrial-membrane depolarization and mitochondrial retention of cytochrome c and SMAC/DIABLO following Cr(VI) exposure. The DR cells exhibited less basal mtDNA damage, as compared to CC1 cells, which correlates with intrinsic (non-induced) death-resistance. Notably, there was no difference in p53 protein expression before or after treatment among all cell lines. Taken together, our data suggest the presence of more resilient mitochondria in death-resistant cells, and that death-resistance can be acquired in normal human cells early after genotoxin exposure. We postulate that resistance to mitochondrial-mediated cell death and mitochondrial dysregulation may be an initial phenotypic alteration observed in early stage carcinogenesis.