Biomedical subjects

Haiwei H Guo

Publications and source records attributed to Haiwei H Guo.

4 recordsLinked to original sources

PubMed2005

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting.

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50,000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.

Cell Separation↗

PubMed2004

Protein tolerance to random amino acid change.

Mutagenesis of protein-encoding sequences occurs ubiquitously; it enables evolution, accumulates during aging, and is associated with disease. Many biotechnological methods exploit random mutations to evolve novel proteins. To quantitate protein tolerance to random change, it is vital to understand the probability that a random amino acid replacement will lead to a protein's functional inactivation. We define this probability as the "x factor." Here, we develop a broadly applicable approach to calculate x factors and demonstrate this method using the human DNA repair enzyme 3-methyladenine DNA glycosylase (AAG). Three gene-wide mutagenesis libraries were created, each with 10(5) diversity and averaging 2.2, 4.6, and 6.2 random amino acid changes per mutant. After determining the percentage of functional mutants in each library using high-stringency selection (>19,000-fold), the x factor was found to be 34% +/- 6%. Remarkably, reanalysis of data from studies of diverse proteins reveals similar inactivation probabilities. To delineate the nature of tolerated amino acid substitutions, we sequenced 244 surviving AAG mutants. The 920 tolerated substitutions were characterized by substitutability index and mapped onto the AAG primary, secondary, and known tertiary structures. Evolutionarily conserved residues show low substitutability indices. In AAG, beta strands are on average less substitutable than alpha helices; and surface loops that are not involved in DNA binding are the most substitutable. Our results are relevant to such diverse topics as applied molecular evolution, the rate of introduction of deleterious alleles into genomes in evolutionary history, and organisms' tolerance of mutational burden.

Amino Acid Substitution↗

PubMed2003

Tumbling down a different pathway to genetic instability.

Ulcerative colitis (UC), a chronic inflammatory condition associated with a predisposition to colon cancer, is frequently characterized by DNA damage in the form of microsatellite instability (MSI). A new report links inflammation in UC with increases in the DNA repair enzymes 3-methyladenine DNA glycosylase and apurinic/apyrimidinic endonuclease, and, paradoxically, with increased MSI. These findings may represent a novel mechanism contributing to MSI in chronic inflammation.

Base Sequence↗

PubMed2002

Endogenous mutagenesis and cancer.

Mutations in DNA accrue relentlessly, largely via stochastic processes. Random changes accumulate, eventually disabling genetic components which result in the formation of the cancer phenotype. Given the infrequency of measured nucleotide changes and the requirement for several mutations to occur in the same cell, it has been postulated that the rate of mutation must become elevated early in the course of evolution of the cancer. Recently, large scale sequencing of tumor DNA has sought to directly measure random mutations. We discuss the implications of these findings and the factors that must be considered in order for fruitful determination of whether a mutator phenotype is a necessary precursor for cancer.

Base Sequence↗