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Garry R Cutting

Publications and source records attributed to Garry R Cutting.

23 records · Page 2Linked to original sources
PubMed2002

Optimized regulation of gene expression using artificial transcription factors.

A major focus in the basic science of gene therapy is the study of factors involved in target-specific regulation of gene expression. Optimization of artificial or "designer" transcription factors capable of specific regulation of target genes is a prerequisite to developing practical applications in human subjects. In this paper, we present a systematic and combinatorial approach to optimize engineered transcription factors using designed zinc-finger proteins fused to transcriptional effector domains derived from the naturally occurring activators (VP16 or P65) or repressor (KRAB) proteins. We also demonstrate effective targeting of artificial transcription factors to regulate gene expression from three different constitutive viral promoters (SV40, CMV, RSV). Achieving a desired level of gene expression from a targeted region depended on several variables, including target site affinities for various DNA-binding domains, the nature of the activator domain, the particular cell type used, and the position of the target site with respect to the core promoter. Hence, several aspects of the artificial transcription factors should be simultaneously evaluated to ensure the optimum level of gene expression from a given target site in a given cell type. Our observations and our optimization approach have substantial implications for designing safe and effective artificial transcription factors for cell-based and therapeutic uses.

Cytomegalovirus↗
PubMed2002

Five percent of normal cystic fibrosis transmembrane conductance regulator mRNA ameliorates the severity of pulmonary disease in cystic fibrosis.

Estimates of the level of transcripts from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene required to develop a CF phenotype range from 4-20% of normal. Due to the importance of obtaining reliable data on this issue for therapeutic strategies, we developed a novel polymerase chain reaction-based method to quantify CFTR transcripts and applied it to the analysis of nasal epithelium RNA of five patients with CF and the 3272-26A>G/F508del genotype. We calculated that 8.2 +/- 0.84% of the total CFTR RNA present in these five patients is normal full-length CFTR mRNA. We then demonstrated (in nasal samples from F508del carriers, n = 30) that the abundance of full-length F508del CFTR transcripts is reduced compared with wild-type transcripts, and estimated that the average ratio of F508del/wild-type transcripts is 0.87 +/- 0.06. To determine the amount of full-length transcripts relative to levels found in normal individuals, we corrected for the lower abundance of the F508del transcripts and calculated that the five patients with CF have, on average, 4.7 +/- 0.45% of the normal level of wild-type CFTR mRNA. Because these patients have mild CF compared with F508del homozygotes, this CFTR mRNA level appears to be sufficient to avoid the severe complications of the disease.

Alternative Splicing↗
PubMed2001

A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.

We identified a novel cystic fibrosis transmembrane conductance regulator (CFTR)-associating, PDZ domain-containing protein, CAL (CFTR associated ligand) containing two predicted coiled-coiled domains and one PDZ domain. The PDZ domain of CAL binds to the C terminus of CFTR. Although CAL does not have any predicted transmembrane domains, CAL is associated with membranes mediated by a region containing the coiled-coil domains. CAL is located primarily at the Golgi apparatus, co-localizing with trans-Golgi markers and is sensitive to Brefeldin A treatment. Immunoprecipitation experiments suggest that CAL exists as a multimer. Overexpression of CAL reduces CFTR chloride currents in mammalian cells and decreases expression, rate of insertion and half-life of CFTR in the plasma membrane. The Na(+)/H(+) exchanger regulatory factor, NHE-RF, a subplasma membrane PDZ domain protein, restores cell surface expression of CFTR and chloride currents. In addition, NHE-RF inhibits the binding of CAL to CFTR. CAL modulates the surface expression of CFTR. CAL favors retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR. Thus, the regulation of CFTR in the plasma membrane involves the dynamic interaction between at least two PDZ domain proteins.

Adaptor Proteins, Signal Transducing↗