Cystic Fibrosis One of the most common lethal mutations in humans autosomal recessive carried by 1/20 of Caucasians 1/400 couples will have children with the disease (1/4 of children will be afflicted) characterized by the production of abnormal secretions leading to a mucous build-up in pancreas this leads to poor digestion and malnutrition in lungs this leads to bacterial infections (usual cause of death) increased salt in sweat - not harmful, but useful for diagnosis probable cause of these different defects is a failure to properly transport salt through the cell membrane. if untreated 95% of affected children will die before age 5 with continuous and expensive treatment survival can be extended into 20's How to clone the Cystic Fibrosis gene? Problem - the mRNA is rare and and it is impossible to know which of the thousands of proteins in the affected cells is the product of the gene Solution - Positional cloning (also called "reverse genetics") find genes of unknown function by mapping them relative to a gene or marker that you can clone and then "walking" down the chromosome to the desired gene Works by using "Southern" blots to identify variable regions in the DNA genomic DNA from diferent individuals is digested with restriction enzymes the numerous restriction fragments created by the digestion are separated by size on agarose gels the DNA fragments are then transferred to a nitrocellulose or nylon membrane (blotting) radioactive probe DNA prepared from an already cloned DNA fragment is hybridized to the blot try answering this question about Southern blots from the Biology Project If there are different sized fragments these are called Restriction Fragment Length Polymorphisms (RFLPs) This can also be done using PCR -try answering this question about Huntington's disease and then this question 2 about Huntington's disease to see how this works the inheritance of of these RFLPs can be followed through a pedigree and mapped to the disease phenotype just like any other trait try to answer this question about clones that show linkage can then be used to isolate other overlapping clones in the same region of the chromosome (the "walk") Mapping the Cystic Fibrosis gene Tsui et. al. found a RFLP called DOCR-917 that was tightly linked to CF somatic cell hybrids showed that DOCR-917 is on Ch. 7 White et. al. linked CF to an RFLP from the met oncogene which was already known to be in the middle of the long arm of Ch. 7 Williamson et. al. mapped CF next to another RFLP in marker D7S8 in band q22 at the end of the long arm of Ch. 7 after much work by all parties the CF gene was mapped as being between met and D7S8, 1.3 map units from met and 0.9 map units from D7S8, a physical distance of approximately 1-2 million bp. Cloning the Cystic Fibrosis gene distance between met and D7S8 is too great for walking so chromosome jumping was also used after cloning 300 kb from the 500 kb region at the right distance from met and D7S8, how to find the gene? Zoo blots found four probes that cross hybridized with other mammalian species was near met, probably too far away had no open reading frame had no bands on Northerns had an open reading frame and hybridized to a 6.5 kb band on Northerns - finally a good candidate! clone 10-1 used 10-1 as a probe and cloned a cDNA from a normal sweat gland after much more work determined that the CF gene was 250 kb long and had 24 exons How do we know that they cloned the right gene? mRNA's found in all the right places (pancreas, sweat glands, lungs, etc.) mRNA would code for a transmembrane domain (as expected for a transporter molecule) most (70%) of CF patients have a 3 bp deletion that removes one phenylalanine residue expression of wild type CF in cells isolated from an affected individual cures the defect in chloride transport knocking the CF gene out in mice causes mice with similar problems to CF patients final cost - 170 million dollars! [ Biol 207 ] [ Bell ] [ CSU Chico ] [ Library ] This document is maintained by: Jeff Bell Last Update: Wednesday, April 12, 2000