CFTR – gene cloning and initial bioinformatic analysis Riordan et 12(*) et Tsui (1989) Science 245:1066 Carlow IT Bioinformatics November 2006 * Including Francis Collins, later leader of the Human Genome Sequencing Project

Cystic fibrosis Horrible inherited disease –Affecting lung, pancreas, sweat-glands Abnormally high trans-membrane electrical potential –Decreased Cl - ion membrane transport Often associated with failure to respond to ATP dependent kinase –no phosphorylation: no function

More symptoms etc. Difficult breathing Early death (1959 6mths, yrs) More prone to infections (thicker mucus) Can do pre-natal diagnosis or sweat test "Woe is the child who tastes salty from a kiss on the brow, for he is cursed, and soon must die“ German proverb 1700s We modify AMPs defensins: can make one effective in high salt environment??

Genetics & epidemiology Located on chr 7q Kb gene 1 in 25 europeans carries a CFTR mutation so 1:2500 live birth have the disease Males and female equally affected Life expect higher in males – nobody knows why Why so common? Cholera toxin requires normal CFTR Also possible connexion with typhus

Mapping Genetic association with markers pinpoints chromosome 7 Chromosome walking to zero in NO genome sequence in those days

Clone and sequence Why bother? –because we can! –? can predict features/functions –? Can compare CF v normal to identify mutation Working with cDNA not genomic Generate cDNA libraries from cells & cell-lines Screen for cDNAs that hybridise with known CFTR fragment Eventually (much hard work) got 19 overlapping cDNA clones

Fig 1 19 normal clones 2 CF clones

Fig3 - where expressed Patchy expression profile

Gene sequence Clones span 6.1kb of RNA ORF protein of 1480 amino acids –So bigger than 300AA average In 1989 << 1000 human genes sequenced Bioinformatic analysis possible then: –Start codon, consensus seq for transl start + AUG –2 nd structure prediction –Hydropathy plot –Homology searches (pre BLAST) –Glycosylation, Ser, Thr kinase sites

Start of ORF 5’- AGACCAUGCA-3’ in CFTR 5’-(CC)[A/G]CCAUGG(G) consensus –Convinced? –I’m not

The sequence 1 Exon splice Pred kinase sites 2 TM domains Trscr Start AA count RNA count

The sequence 2 First ATP Binding fold Is underlined Delta F 508 circled

Protein analysis Whole protein is two similar halves each with 6 membrane Spanning domains (hydropathic peaks) and two NBFs (hydrophilic regions) and a charged R region

Fig6 – homology/similarity  F508 Comparing two conserved regions in CFTR and other proteins: some with Two, some with one similar region, multidrug resistance, transporters etc. Conserved, hydrophobic Aromatic position at 508

Structure of the fold Two halves similar structure but low AA conservation (best is only 27/66 identities) Others in family have much tighter conservation No signal peptide says that orientation of first TM domain is (i – o) External loops very short …except between TM7 and TM8 where there is N glycosylation site

More… R domain is one exon 69/241 residues are polar alternating +ve and –ve charge regions Also most of the phosphorylation kinase sites All family members secrete something: –Chloride (CFTR) –Pigment (drosophila white gene) –lytic peptide (E. coli hemolysin) …so what about the “function unknown” mbpX gene in liverwort chloroplasts ?

More… Hypothesise that CFTR is the ion channel 10/12 of TM domains have >1 +ve AA –ie. amphipathic helix –cf. brain Na+ channel & GABA-R Cl- channel Contrast p-glycoprotein –Closely realted but no +ve TM AAs Big protein – maybe also other functions

Fig 7 a composite model Glycosylation

In colour from wikipedia

Conclude From very little data and very small DB N=basesN=seqs ,800,00020, ,762,58528, ,179,28539, ,101,066,28810,106,023 to compare with can make predictions about structure and function that have stood the test of time.

Postscript  F508 may be about delivery of protein to the membrane –Functions fine if you trick cells to deliver! By different mutations identified in the gene Last month 1531 different mutations at – With human genome, SNPs, ESTs much easier to interpret sequence information