Shanchuan Zhao

Bio: Shanchuan Zhao is an academic researcher from Harvard University. The author has contributed to research in topics: Complementary DNA & Differential display. The author has an hindex of 6, co-authored 6 publications receiving 445 citations.

New primer strategy improves precision of differential display

Abstract: To increase the reproducibility and to reduce the false positives in the initial mRNA differential display, modified long composite primers were developed based on both mRNA differential display and RNA arbitrarily primed PCR fingerprinting methods. Ten-base nucleotides were added at the 5' ends of the primers used in the initial mRNA differential display. These included a restriction site to aid cloning. PCR began with one low-stringency cycle (40 degrees C for annealing) followed by 35 high-stringency cycles (60 degrees C for annealing). The modified method significantly improved the reproducibility and sensitivity of the mRNA differential display while still keeping the characteristics of the original method.

Cyclin E and cyclin A as candidates for the restriction point protein.

Abstract: Progression of cells into S phase is proposed to be determined by accumulation of a labile protein (the restriction point protein R; A. B. Pardee, Proc. Natl. Acad. Sci. USA, 71: 1286–1290, 1974). We report here that cyclin E and cyclin A proteins as well as their dependent histone H1 kinases satisfy all of the criteria for the R protein, which includes late G 1 phase increase, an excess delay of appearance after inhibition of protein synthesis in nontransformed cells, and a faster recovery in transformed cells. We suggest that the molecular basis of the R protein could be cyclin production and inactivation.

Better gel resolution and longer cDNAs increase the precision of differential display.

Abstract: become a widely used method for the rapid identification of differentially expressed genes in a variety of eukaryotic systems (4). In the past three years, technical modifications have reduced the redundancy of anchored primers, decreased the number of reverse transcription reactions needed, increased reproducibility, reduced the incidence of false positives and increased the overall efficiency of the method (5). The original method separates cDNAs on a denaturing polyacrylamide gel. However, conventional DNA sequencers sometimes do not provide adequate gel resolution, resulting in the inadvertent isolation of several cDNAs from what appears to be a single band. These cDNAs may represent independent cDNA fragments of the same or very similar molecular weight (2), separated strands of a unique doublestranded cDNA molecule with or without Taq DNA polymerase-mediated addition of 3′-terminal adenosine nucleotide (1), truncated polymerase chain reaction (PCR) products of a unique cDNA (unpublished data) and fragments representing a unique cDNA with multiple polyadenylation sites (3). Here we report the use of a new programmable DNA sequencer, called the GenomyxLR (Genomyx, Foster City,

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

Abstract: We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system.

Abstract: Conditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor. After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition. We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts. We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity. Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence. However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition. Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E. These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence.

Induction of apoptosis in uninfected lymphocytes by HIV-1 tat protein

Abstract: Infection by human immunodeficiency virus-type 1 (HIV-1) is typified by the progressive depletion of CD4 T lymphocytes and deterioration of immune function in most patients. A central unresolved issue in acquired immunodeficiency syndrome (AIDS) pathogenesis is the mechanism underlying this T cell depletion. HIV-1 Tat protein was shown to induce cell death by apoptosis in a T cell line and in cultured peripheral blood mononuclear cells from uninfected donors. This Tat-induced apoptosis was inhibitable by growth factors and was associated with enhanced activation of cyclin-dependent kinases.

Cyclin E and Survival in Patients with Breast Cancer

Abstract: Background Cyclin E, a regulator of the cell cycle, affects the behavior of breast-cancer cells. We investigated whether levels of cyclin E in the tumor correlated with survival among patients with breast cancer. Methods Tumor tissue from 395 patients with breast cancer was assayed for cyclin E, cyclin D1, cyclin D3, and the HER-2/neu oncogene with the use of Western blot analysis. Full-length, low-molecular-weight, and total cyclin E were measured. Immunohistochemical assessments of cyclin E were also made of 256 tumors. We sought correlations between levels of these molecular markers and disease-specific and overall survival. Results The median follow-up was 6.4 years. A high level of the low-molecular-weight isoforms of cyclin E, as detected by Western blotting, correlated strongly with disease-specific survival whether axillary lymph nodes were negative or positive for metastases (P<0.001). Among 114 patients with stage I breast cancer, none of the 102 patients with low levels of cyclin E in the tumor...