A StrategyforIsolationof cDNAs Encoding Proteins AffectingHuman IntestinalEpithelialCell Growth and Differentiation:Characterization of a Novel Gut-specificN-myristoylated Annexin

The human intestinal epitheliumisrapidly and perpetuallyrenewed asthedescendantsofmulti- potentstem cellslocatedincryptsundergoprolifera- tion,differentiation, and eventualexfoliation duringa verywellorganizedmigrationalongthecrypttovillus axis.The mechanisms thatestablishand maintainthis balancebetween proliferation and differentiation are largelyunknown .We haveutilizedHT 29 cells,derived from a human colonadenocarcinoma,as a model sys- tem foridentifyinggene productsthatmay regulate theseprocesses.Proliferating HT-29 cellsculturedin theabsenceofglucose(e.g.,usinginosineas thecar- bon source)have some ofthecharacteristics ofundif- ferentiated butcommitted cryptepithelial cellswhile postconfluentcellsculturedintheabsenceofglucose resembleterminallydifferentiated enterocytesor goblet cells.A cDNA library,constructedfrom exponentially growingHT-29 cellsmaintainedininosine-containing media,was sequentially screenedwithaseriesofprobes depletedofsequencesencodinghousekeepingfunc- tionsand enrichedforintestine-specific sequencesthat areexpressedinproliferating committed,but notdif- ferentiated, epithelial cells.Of 100,000recombinant phage surveyed,one was found whose cDNA was de- rivedfrom an apparentlygut-specific mRNA .Iten- codesa 316 residue,35,463-Dproteinthatisa new member oftheannexin/lipocortin family.Other family members havebeen implicatedinregulationofcellular growth and insignaltransductionpathways.RNA blot