Mitchell L. Drumm

Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer

We have used retrovirus-mediated gene transfer to demonstrate complementation of the cystic fibrosis (CF) defect in vitro. Amphotropic retroviruses were used to transduce a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into CFPAC-1, a pancreatic adenocarcinoma cell line derived from a patient with CF that stably expresses the chloride transport abnormalities characteristic of CF. CFPAC-1 cells were exposed to control virus (PLJ) and CFTR-expressing virus (PLJ-CFTR); viral-transduced clones were isolated and subjected to molecular and physiologic analysis. RNA analysis detected a viral-derived CFTR transcript in all of the PLJ-CFTR clones that contained unrearranged proviral sequences. Agents that increase intracellular cAMP stimulated 125I efflux in PLJ-CFTR clones but not PLJ clones. Whole-cell patch-clamp performed on three responding clones showed that the anion efflux responses were due to cAMP stimulation of Cl conductance. Our findings indicate that expression of the normal CFTR gene confers cAMP-dependent Cl channel regulation on CF epithelial cells.

Cystic fibrosis gene

The cystic fibrosis gene, located at 7q31, spans about 230 kb of genomic DNA and contains 27 exons. The cDNA of 6.2kb would predict an 1480 amino acid protein, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR has a high degree of homology with members of the ABC-transporter super family. The predicted protein structure consists of two membrane-spanning domains, each of 6 sub-units, anchoring CFTR in the apical membrane of specialized epithelial cells, 2 nucleotide binding folds (NBF) and a regulatory (R) domain. Disease-associated mutations in the CF gene are mainly clustered in the nucleotide-binding folds. The most common mutation, occurring in 70% of CF genes in Northern Europe and North America, is the deletion of amino acid phenylalanine at position 508 in the first NBF (ie delta F508).

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Anion Binding as a Probe of the Pore

We compared the effects of mutations in transmembrane segments (TMs) TM1, TM5, and TM6 on the conduction and activation properties of the cystic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby, to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution studies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conduction property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord with the notion that anion-water interactions are important determinants of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mutations in TM5 and TM6 also dramatically reduced the sensitivity of CFTR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected if these TMs are intimately involved in the physical process that opens and closes the channel.

Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

We complemented the Cl‐ conductance defect in cystic fibrosis lymphocytes by transfection with wild‐type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non‐CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector‐specific sequences. Both fluorescence and patch‐clamp assays showed that transfectants expressing wild‐type CFTR acquired properties previously associated with Cl‐ conductance (GCl) regulation in non‐CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist‐induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild‐type CFTR corrects the defects in Cl‐ conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR‐mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.

Physical mapping of the cystic fibrosis region by pulsed-field gel electrophoresis

The gene for cystic fibrosis (CF) is known to be flanked by the closely linked DNA markers met and J3.11 on chromosome 7. Using the technique of pulsed-field gel electrophoresis, we have constructed a complete overlapping restriction map of approximately 3000 kb of DNA in this region. The met and J3.11 probes are found to be between 1300 and 1800 kb apart, which compares well with their genetic distance of 1-2 cM. The CF gene must be located within this interval, and the availability of this physical map should be of considerable utility in mapping additional clones as the search for the gene proceeds.

2 – Molecular Biology of Cystic Fibrosis

The past decade of research in cystic fibrosis has produced a wealth of information about the underlying defect responsible for the disease. The initial finding that the physiological disturbance in CF is one of abnormal electrolyte transport across epithelial tissues led to the elucidation of a pathway in which epithelial chloride transport is normally elicited in response to beta-adrenergic stimuli and involves the second messenger cAMP to activate protein kinase A. While that pathway was being described, work on the genetic front was concurrently providing information about the genomic location of the gene causing CF, which ultimately led to the identification and cloning of the gene encoding the cystic fibrosis transmembrane conductance regulator. The cloned CFTR gene provided a powerful reagent to use in the next generation of cell physiology experiments, in which it was determined that CFTR is not only the substrate of PKA phosphorylation, a step previously determined to be in the activation pathway of the chloride channel, but is in fact a cAMP-dependent chloride conducting channel itself. Further analysis of the gene has shown that although there is a single mutation that accounts for most of CF, there are well over 200 other lesions within the gene that can cause disease as well. Identification of these mutations has provided information into the normal function of CFTR by studying these variants in heterologous expression systems. As a result, the molecular mechanism of CFTR function is beginning to unfold, as well as the mechanism by which particular mutations impair that function. From a clinical perspective, the research brings optimism from two directions. First, understanding how disease-causing mutations impair function may culminate in pharmacologic approaches that can restore function to some of these mutants. Second, treating the disease at the level of the gene appears to be a realistic goal: Gene transfer experiments in cultured CF cells have shown that the procedure will restore cAMP-dependent chloride conductance to the cells, laying the groundwork for somatic cell gene therapy as a feasible treatment for CF. Currently, work is rapidly progressing in developing delivery systems for this purpose. Finally, animal models that should not only aid in understanding the physiology of electrolyte transport in epithelia but should serve as indicators for tests of therapeutic approaches to treating CF are being developed, either by pharmacological means or by gene delivery protocols.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic Markers for Oncogenes, Growth Factors, and Cystic Fibrosis

The techniques of molecular biology have had a dramatic effect on the advancement of human genetics. In particular, the development of restriction fragment length polymorphisms (RFLPs) has allowed researchers to generate genetic markers for virtually any region of the human genome. Most RFLPs occur when a mutation creates or deletes a recognition site for a restriction enzyme, generating a DNA fragment of altered size. In the simplest case this will create two alleles. A DNA probe which hybridizes to this fragment will detect the presence of these alleles in the DNA from different individuals. Probes used to detected RFLPs have been derived from both cloned genes and randomly isolated DNA segments. Thus, each RFLP is a genetically inherited marker for a precise location on a chromosome.