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Aviv H & Leder P. Purification of Biologically Active Globin

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60) CCINUI~EB42 This Week’s Citation Classic !~OB~ 21,1991 Aviv H & Leder P. Purification of biologically activeglobin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Nat~Aco4 Sci. USA 69:1408-12, 1972. [Laboratory of Molecular Genetics. National Ina*nute ofChild Health and Human Development, National Institutes ofHealth. Bethesda. MDI ______________________ A convenient technique for the partial punfication of tairnng a thoit ~etth of thymkines (ollgo dl). It large quantities of functional, poly(adenyluc acid)-rich was not obvious,, howevee, that suds a ihoit A:T mRNA is described. Biologically active rabbit glabin hybrid would be stable. To syu1hcaLa~c ollgo dT mRNA was punfied by this procedureand assayed for diemleally hooked to cellulose, I fowida pnscedssie its ability to direct the syiitheais of rabbit globin in a used dut invoked aindemation using cell-free extract of asciws tunsy. 111w Sd ® indicates DCC, idsidiIs veryunwitive to water. knowkigve,y that this paper has been cited in more than 6,150 publications.l Hate orgamc d~.try, this was not encouraging *5*- for me. I sought advice from bead Sdiedd~of the Wesvnasn Institute who apent a _____ hi the I a whale How to Win Friends and Advance Science lab. set up the synthesis whids itsioked week of shaking. I did this with great h~sitatlo~ sure that it wouldni work. This was ..v.bJlly the Haim Aviv 5 Department of Plant Genetics only organicsynthesis that I ever did in my cases,, Weivnann institute of Science aid it worked. Relsovot 76100 Having lots of extracted reticulocyte RNA, I set Israel upthe procedure thatis described In this mod.citrd paper, and it worited rigid away. We got plenty of I came to Philip Leder’s laboratory at the National labeled amino acids incorporated into protein di- tnstitute of Child Health and Human Development rectedbymRNA. in 1970 after finishing my doctorate at the I immediately followed this ~ by run- Weumann ln~tituteof Science in IsraeL The main ning through this little column of oliga d1 using reieardl direction of the lab was to study imiTwno. RNA preparations extracted from niyelwna cells, globulin biosynthesis usmg molecular biology ~- which, ksc*Dy, I had not discarded.This opened up proaches. One of the firststeps In this endeavor was many studies on immunogiobin mRNA,,wisids were to isolate the immunogiobulin mRNA from my- ——4 eloma cells (MOPC-41). The isolation ofmRNA was Having lots of pure globin mINA., we joined thought to be essential in order to have a handle on forces with Jeff Rose and5 Ed Scoinids to synthesize the biosynthesis ofimmunoglobulins. coniplenientary DNA —ai 6.~xteta.Lmilestone hi After establishing a cell-free translation system the development of gene doning and moleadar usingan extract from a murme aacitescell line,’ we set up to establish an extraction procedure foe My procedure for mRNA purification made me tnRNA from the myeloma cells. However~for about lots of friends. Many colleagues have asked for urn- six months we could not get any counts incorpo- pies of this precious oiigo dT celhslose, width I was rated Irons labeled amino adds, whatever we tried. willing to sharewith then provided theymade their It was a very frustrating experience. Then I sug- owis batch under my guidance and we ~llt It. gested thatwe check out our extractionprocedures I also bad aimplaints that the column did not (parflcularIy the quality of freshly distilled phenol) work. It mostly turned out that the column was not using mRNA extracted from rabbit reticulocytes for — proped~ whids there were a number of published proce- In the nearly 20 years since the procethue was dures. This wasdone just asa control. It workedfine published, it ha been cited hi thousand,of psth&a- when mRNA was extracted from retics, but failed dons and is still the most airiness t.,aj~ij~j~of when extracted from myeloma tissue. mRNA purification. ~iy riall changes hi the pro- At that time, a number of papers were published cedure subsequendy have bees hdrodeced. Some showing that globin mRNA has a sfretd, of adeno2 - rave on thewashing step when purity Is not crucial, sines at the 3’ end whose ftmction was not clear. It some recycle the unbound material Ifa higher yield occurred to me that we might be able to separate Is desired. But to the best of my knowledge, most giobin mRNA from ribosonsal RNA and tRNA by researdier, have been happy with the procedureas hybridizing mRNA to a solid phase polymer con- originally described. I. Aviv H, Bourn. I & Led,r P. Protein-synthesis directed by encephalomyocarditis virua-RNA—propcrues oI* transfer RNA-drpcndenl system. Proc. Na:. Aca,L Sri. (iSA 68:2303-7. 1971 (Cited 135 times.) 2. Edmoods M & Cariniels M 6. Isolation and characterization of adenosinc monophosphate-rich polynucleoudes synthesized by Ehrlich arctics cells. J. Bk’!. Chem. 244:1314-24, l969. (Cited 230 timesj 3. GlIham PT. Synthesis of polynucleotide.ceUuloses and their use in fractionation ofpolynucleotiden. J.Amer. Chew. Soc. 86:4932-5, 1964. (Cited l9Otimen.) 4. Swan D, Aviv H& t.,eder 0. Purification and properties of biologically active messenger-RNA for a rnyelomalight ch~ia. Proc. Not. ,lcad. Sd. 1/5.4 69:1967-71, l972. (Cited 245 limes.) 5. Roan .1, Aviv H, Scoinlek E & Leder P. In-vitro synthesis of DNA complementary to purified rabbit globin messenger.RNA. Proc. Nat. ,jcad. Sc!. 03.4 69:264-8, 1972. (Cited 230 times.) Received February 8,1991 10 ©1991 by SI® CURRENT CONTENTS®.
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