Hanlin Gao

Polymerase with 3' to 5'exonulcease plays an important role in the maintenance of in vivo DNA replication fidelity. In order to develop more reliable SNP assays, we revisit the underlying molecular mechanisms by which DNA polymerases with 3' exonucleases maintain high fidelity of DNA replication. In addition to mismatch removal by proofreading, we recently discovered a premature termination of polymerization by a new mechanism of OFF-switch. This novel ON/OFF switch turns off DNA polymerization from mismatched primers and turns on DNA polymerization from matched primers. Two SNP assays were developed based on the proofreading and the newly identified OFF-switch respectively: terminal labeled primer extension and the ON/OFF switch operated SNP assay. These two new methods are well adapted to conventional techniques such as electrophoresis, real time PCR, microplates, and microarray. Application of these reliable SNP assays will greatly facilitate genetic and biomedical studies in the post-genome era.

XK469 (NSC 697887) is a synthetic quinoxaline phenoxypropionic acid derivative that possesses unusual solid tumor selectivity and activity against multidrug-resistant cancer cells. We report here that XK469 and its S(-) and R(+)-isomers induce reversible protein-DNA crosslinks in mammalian cells. Under protein denaturing conditions, the protein-DNA crosslinks are rendered irreversible and stable to DNA banding by CsCl gradient ultracentrifugation. Several lines of evidence indicate that the primary target of XK469 is topoisomerase IIβ . Preferential targeting of topoisomerase IIβ may explain the solid tumor selectivity of XK469 and its analogs because solid tumors, unlike leukemias, often have large populations of cells in the G1/G0 phases of the cell cycle in which topoisomerase IIβ is high whereas topoisomerase IIα , the primary target of many leukemia selective drugs, is low.

Piwi-interacting RNAs (piRNAs) are a distinct group of small non-coding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Due to their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that at least had two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found 3 piRNAs, piR-32051, piR-39894 and piR-43607, to be derived from the same piRNA cluster at chromosome 17. We confirmed the 3 selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We fu...

CHAPTER 8 Mutation Discovery Using High-Throughput Mutation Screening Technology KAI LI, HANLIN GAO, HONG-GUANG XIE, WANPING SUN, and JIA ZHANG ... Accumulating evidencehas well documented that genetic diseases and cancers are caused by gene mutations ...

Strand asymmetries in DNA evolution, including indel and single nucleotide substitutions, were reported in prokaryotes. Recently, an excess of G>A over C>T substitutions in hemophilia B patients was recognized in our molecular diagnostic practices. Further analysis demonstrated biased point mutations between sense and antisense strands when unique changes in factor IX were counted. Similar mutation spectra of factor IX and the HGMD prompted us to speculate that the excess of G>A over C>T may be present in genes other than factor IX. Data from nine genes (each has ≥ 100 missense mutations) retrieved from HGMD, international factor IX database, and Dr. Sommer's lab database in the City of Hope National Medical Center, Duarte, CA, USA were analyzed for their point mutation spectra. Similar to factor IX, all genes selected in this study have biased G>A over C>T unique mutations when nonsense mutations were excluded. The biased missense point mutations were recently convincingly documented by the statistic data of categorized missense mutation in HGMD. The consistence of the genetic observation and the genomic data from HGMD strongly indicate that biased point mutations, possibly a phenotypic selection, are more widespread than previously thought. The biased mutations have immediate clinical impact in molecular diagnostics.

Abstract: The changes in physical and chemical characteristics derived from genetic alteration are the basis for the development of mutation assays. Among the ever-increasing number of mutation assays available, most are variants of allele-specific primer extension ...

Cancer-secreted microRNAs (miRNAs) are emerging mediators of cancer-host crosstalk. Here we show that miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is a potent regulator of migration through targeting the tight junction protein ZO-1. In endothelial monolayers, exosome-mediated transfer of cancer-secreted miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-105 in nonmetastatic cancer cells induces metastasis and vascular permeability in distant organs, whereas inhibition of miR-105 in highly metastatic tumors alleviates these effects. miR-105 can be detected in the circulation at the premetastatic stage, and its levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer.

Very virulent (vv) infectious bursal disease virus (IBDV) Gx strain with high pathogenicity was attenuated through replication in specific-pathogen-free (SPF) chicken embryos and in chicken embryo fibroblast (CEF) cell cultures. The changes in VP2 nucleotide and the deduced amino acid sequences were obtained during attenuation of vvIBDV in CEF culture. Sequence analysis of selected passages from numbers 0 to 20 in CEFs (designated here Gx to CEF-20) showed that no changes were detectable in the VP2 gene before CEF-7. There were a few changes in the nucleotide sequence of the VP2 gene but no amino acid substitutions at CEF-8. The virus of CEF-9 was an intermediate with some amino acid changes that possibly were related to virulence. CEF-10 virus had become similar to CU-1 strain. The VP2 gene sequence remained the same from CEF-10 to CEF-20. The results of pathogenicity tests showed that the mortalities of Gx, CEF-5, CEF-8, and CEF-9 in 4-wk-old SPF chickens were 64%, 60%, 60%, and 32%, respectively; whereas CEF-10, CEF-15, and CEF-20 were nonpathogenic. Virus neutralization tests with Gx strain showed that the antigenicities are similar from Gx to CEF-20.