Biomedical subjects

Penny Zhu

Publications and source records attributed to Penny Zhu.

5 recordsLinked to original sources

PubMed2006

Long-range cooperative binding effects in a T cell receptor variable domain.

Although cellular processes depend on protein-protein interactions, our understanding of molecular recognition between proteins remains far from comprehensive. Protein-protein interfaces are structural and energetic mosaics in which a subset of interfacial residues, called hot spots, contributes disproportionately to the affinity of the complex. These hot-spot residues can be further clustered into hot regions. It has been proposed that binding energetics between residues within a hot region are cooperative, whereas those between hot regions are strictly additive. If this idea held true for all protein-protein interactions, then energetically significant long-range conformational effects would be unlikely to occur. In the present study, we show cooperative binding energetics between distinct hot regions that are separated by >20 A. Using combinatorial mutagenesis and surface plasmon resonance binding analysis to dissect additivity and cooperativity in a complex formed between a variable domain of a T cell receptor and a bacterial superantigen, we find that combinations of mutations from each of two hot regions exhibited significant cooperative energetics. Their connecting sequence is composed primarily of a single beta-strand of the T cell receptor variable Ig domain, which has been observed to undergo a strand-switching event and does not form an integral part of the stabilizing core of this Ig domain. We propose that these cooperative effects are propagated through a dynamic structural network. Cooperativity between hot regions has significant implications for the prediction and inhibition of protein-protein interactions.

Bacterial Toxins↗

PubMed2006

Whole genome amplification of plasma-circulating DNA enables expanded screening for allelic imbalance in plasma.

Apoptotic and necrotic tumor cells release DNA into plasma, providing an accessible tumor biomarker. Tumor-released plasma-circulating DNA can be screened for tumor-specific genetic changes, including mutation, methylation, or allelic imbalance. However, technical problems relating to the quantity and quality of DNA collected from plasma hinder downstream genetic screening and reduce biomarker detection sensitivity. Here, we present a new methodology, blunt-end ligation-mediated whole genome amplification (BL-WGA), that efficiently amplifies small apoptotic fragments (<200 bp) as well as intermediate and large necrotic fragments (>5 kb) and enables reliable high-throughput analysis of plasma-circulating DNA. In a single-tube reaction, purified double-stranded DNA was blunted with T4 DNA polymerase, self-ligated or cross-ligated with T4 DNA ligase and amplified via random primer-initiated multiple displacement amplification. Using plasma DNA from breast cancer patients and normal controls, we demonstrate that BL-WGA amplified the plasma-circulating genome by approximately 1000-fold. Of 25 informative polymorphic sites screened via polymerase chain reaction-denaturating high-performance liquid chromatography, 24 (95%) were correctly determined by BL-WGA to be allelic retention or imbalance compared to 44% by multiple displacement amplification. By enabling target magnification and application of high-throughput genome analysis, BL-WGA improves sensitivity for detection of circulating tumor-specific biomarkers from bodily fluids or for recovery of nucleic acids from suboptimally stored specimens.

Allelic Imbalance↗

PubMed2005

High-throughput scanning of breast tumor surgical specimens for low-level mutations.

Large numbers of mutations are postulated to occur as early events in carcinogenesis. For certain types of human tumors (mutator phenotypes) these mutations can be a driving force in generating clonogenic, causative genetic changes leading to multistage carcinogenesis. These low-level mutational events are highly significant due to their potential use as molecular markers for early identification of genomic instability that can lead to cancer and to their potential influence on the ability of tumors to resist drug treatment and/or metastasize. Detecting the presence and diversity of such genetic changes in human tumors is desirable due to their potential prognostic value. However, identification of these low-frequency genetic changes is difficult, since most mutations exist at mutant/wild-type ratios of <10(-3). We recently developed inverse PCR-based amplified restriction fragment-length polymorphism (iFLP), a new technology that combines inverse PCR, RFLP, and denaturing HPLC to allow scanning of the genome at several thousand positions per experiment for low-level point mutations. Using iFLP we previously demonstrated low-level mutations (mutation frequency <10(-3)) in human colon cancer cells that harbor mismatch repair deficiency and in sporadic colon cancer surgical specimens. In the present work we investigated whether low-level mutations are also present in sporadic breast cancer surgical specimens. Using iFLP we identified widespread low-level mutations in two out of ten surgical specimens examined (20%). Examination of the microsatellite instability status of these samples demonstrated that the samples are stable (MSI-S). We conclude that low-level mutations are less frequent in breast cancer than in colon cancer; however, single nucleotide instability that generates such mutations may still be present in a fraction of breast cancers.

Base Sequence↗

PubMed2004

Inverse PCR-based RFLP scanning identifies low-level mutation signatures in colon cells and tumors.

Detecting the presence and diversity of low-level mutations in human tumors undergoing genomic instability is desirable due to their potential prognostic value and their putative influence on the ability of tumors to resist drug treatment and/or metastasize. However, direct measurement of these genetic alterations in surgical samples has been elusive, because technical hurdles make mutation discovery impractical at low-mutation frequency levels (<10(-2)). Here, we describe inverse PCR-based amplified restriction fragment length polymorphism (iFLP), a new technology that combines inverse PCR, RFLP, and denaturing high-performance liquid chromatography to allow scanning of the genome at several thousand positions per experiment for low-level point mutations. Using iFLP, widespread, low-level mutations at mutation frequency 10(-2)-10(-4) were discovered in genes located on different chromosomes, e.g., OGG1, MSH2, PTEN, beta-catenin, Bcl-2, P21, ATK3, and Braf, in human colon cancer cells that harbor mismatch repair deficiency whereas mismatch repair-proficient cells were mutation free. Application of iFLP to the screening of sporadic colon cancer surgical specimens demonstrated widespread low-level mutations in seven out of 10 samples, but not in their normal tissue counterparts, and predicted the presence of millions of diverse, low-incidence mutations in tumors. Unique low-level mutational signatures were identified for each colon cancer cell line and tumor specimen. iFLP allows the high-throughput discovery and tracing of mutational signatures in human cells, precancerous lesions, and primary or metastatic tumors and the assessment of the number and heterogeneity of low-level mutations in surgical samples.

Base Pair Mismatch↗

PubMed2004

DNA amplification method tolerant to sample degradation.

Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in an unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA-RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA-RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification. When intact genomic DNA is used, RCA-RCA retains gene-amplification differences (twofold or higher) between complex genomes on a genome-wide scale providing highly improved concordance with unamplified material as compared with other amplification methodologies including multiple displacement amplification. Using RCA-RCA, formalin-fixed samples of modest or substantial DNA degradation were successfully amplified and screened via array-CGH or Taqman PCR that displayed retention of the principal gene amplification features of the original material. Microsatellite analysis revealed that RCA-RCA amplified genomic DNA is representative of the original material at the nucleotide level. Amplification of cDNA is successfully performed via RCA-RCA and results to unbiased gene expression analysis (R(2) = 0.99). The simplicity and universal applicability of RCA-RCA make it a powerful new tool for genome analysis with unique advantages over previous amplification technologies.

Cell Line, Tumor↗