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Function and evolution of secondary metabolites — no easy answers
biotopics
Function and evohtion of secondary rnetabolites - no easy answers riptv-t Exactly why crlls tnakc scconda~~ mewbolitcs, and how their biofunctions baw S~dlC~?i ard evolved, hw puzzled scientiatu for around half a cri1tury. Rcccndy. the Ciba foundation* gathered togcrhrr a group of scirutisa (chemists, physiologists. evolutionary rnd niolecular biologists) under the chairmanship of J. Davies (Ul;iv. British Columbia. Cnnada), ro debate these questions. With scientists present iiom such diverse backgrounds, thcrc was a lot of new and unt;lmiliar infonnatiou fix participants to digest. To start with, how might antibiotics have arisen? Primordial antibiotics - primitive catalysts: We know that many of toda>+ antibiotics act ou the ribosomc. and that resistance to them is oftcu due to moditication of ribosomal RNA at specitic sites. Perhaps this is telling us something of the role of ancestral antibiotic structures-did they act on primordial RNA? One schoo1 of thought on the origi!ia of protein synthesis assumc4 that tlic first pcptide bonds were formed through the action of ‘caulytic RNA’ in tbc primordial soup. Nowadays, we see catalytic RNP. at work in the splkiug ofintrons but RNA splicing may have prc-dated translation of mRNA as WC know it today. Using self-Tplicing of Group I illtrons as a model. J. Davies and co-workers haw Irsvcstigated the cftkt cf antibiotics on the splicing
l!BlECHMAY 1992 (!/OL10)
waction. Smprisinglv. streptomycin inhibited splicing. but csccss GTI’ could rcvcrsc r!lc in!libi:ion. The antiblotic (which has some structural similari~ to GTP) w35 sirnpIy intcrtking wth r!~c sddidx: ofguanosinc r&dues to the intron -an obligatory step in the splicing nwchanisn~. Howcvcr, spurr~cd by this glimmer of hope, tlwy tcstcd other antibiotics. Many of the arninoglycosides (which xt acl antibiodcs by bi&ing to ribosotnal RNA, thus caukg mistraodation) arc indeed inhibitors ofsclf-splicing, and their &initics of binding to introns arc‘ as high as their atfinities ofhinding to the ribo:om.~l RNA. Was their ancestral function to bind LO introns? Addition or‘viomycin tt> the srlfsplicing reaction gave an uncxpcctcd result, promoting ol;gomrrization of the introns which had been cut out by the splicing mechanism. The antibiotic thus promoted a primitive ‘RNA-&se’-litc reaction - taking small RNA tnoleculcs and polymcrizing thctn. This is couccivably how longer RNAs first arose and perhaps how primitive ribosornal RNA was buiit up. Dkcs and collsagues thrrcforc believe that mccstrai antibiotics may have pl.;yctd an important role as ‘scaffolds’ to promote RNA assembly and hcncc translation. This primi&r iunction lids cvolvcd into au antagonistic role in today’s antibiotic structures wvhich attack and prcvcnt the cell&r functions that, originally, rlrcy wcrr r:.sponsiblc for. How did ancestral antibiotic s;ructures first arise? Versions of them have been detected as products when the prrccived chctnical constituents of the primordial soup are wbjected to t!x reaction conditions thought to bc rxtant at that time.
Secondary metabolites - agents of cellular communication? Many cornpomlds which wcrc isolated initiallv as antibiotics, also have other biological propertics. Vancom~;citi, for csalnplc, is also a hypotcnsivr agent. c’ry:!:ro:I;:;cil: is 3 murili~~ .tgonist, and the antifungal antibiotic cyclosporin A intlucnccs production of intrrlcukin (IL-?). In addition, some microbes have now been shown to Ixvc vcrtcbr.xl~-lilw receptors. c.s_ for humdn chorionic gonadotrophm (WCC) (Ps~wfo~~~o~~,7.c thyroid ctimulatin~ rrwltophiiin) ami horrnooc (TSH) (E. i!l/i). Taken togcthcr. this could tncan that the ccl!ular connnunication ;ictxvorks wl~ich arc so csscntial in mct.lbolicnlly cotnplcs. multiorgan spccics dcvclopcd earlier in much simpler orgamsms, and that the relics ofthcsc procwcs arc stili to bc found i17 microbes. It may simply bc fortuitous hd\.C that sow of :hcw mctabolitcs antibiotic .~ctivir~. It is now the norm for ph3rmaccutiial compmics to use receptorb.lscd scrc’cns (comtesy of ci>NA cloning and heterologous cspwrsinn in tractdblr hosts) to starch for natural products with now1 pharn~acologic~l activities. I_ Nisbct (Xcnova, UK) has founded a company based on such screcniug operations, and rcportcd that thcri- SCCIIICto be no shortage of ‘receptor-accivc compounds produced by microbes. Nnturc 11‘1s had cstcnstvc opportuuity to csperiincnt with molccillar structures, and optimize how whey might tit and react lvith their targets. There is, therefore, much to be gaiwd wit11 rrgxd to the construction of functional molecules with cnhanccd potency from carrful observatiou of how ‘cftktor-receptor’ iutcrxxiom of the natural products arc tailored exquisitely, for the task that they perform. The mtcraction of
Q 1992. Elsevier Sctence Publishers Ltd IUKI
vancorny& and dcrivativcs with cell walls. studied and modcllcd by D. Williams (Cambridge Univ., UK), provided an cxcclicnt case in point. Most secondary mctabolitcs arc made only for a &ort time in the devciopmcntal cynic ofthc organism. K. Chatcr (John lnncs Inst., Norwich, UK) described how, as more information bccomcs Ivaiiablc on the regulatory cascadck which control the csprcssion of antibiotic production, it wcms that the antibiotics arc made in rcsponsc to cellular regulatory effcctors, and not (as was once thought) bccausc the antibiotics thcmsclvcs arc the etk:ors. Thus it may bc dangerous to gcncraiizc and to assume that all such mctnbolitcs arose to satis@ a sin& global function. It is a giant evolutionary icap from primitive cells using thcsc structures as scaffolds for assembly ofprimitivc RNA, to some of tk exotic cffccts that sccondnry mctabolitcs arc now shown to have. Evolution - a thorny issue 130~5 connidcring the cvoiution of secondary metabolitcs ht-lp us to focus on their timction? T. CavaiicrSmith (Univ. British Columbia. Canada) dcmonstratcd that inspcccion of phyiogcnctic trcrs scc’ms uninformative in pointing to the gcncsis ofsccondary metabolism. and how it has subscqucntly been dispcrscd. Taking a physioiogict’s approach. it is cicar (L. Vining. D~lhousic Univ., Canada) th.u production of secondary mctaboiitcs scc‘nis to correlate with specif:c x0iogical n&es. For cxampic, rhc fiinmcl:tous bacteria arc very distant in cvuiutionary time Co111 rhr tiiamrntour fungi, but both occupy the salnc ecological niche and both arc prodigiously active in the biosynthesis of secondary mrtabolitcs. Species which arc more closely r&cc.: XJ cithcr the bacteria or tb~ tu:,kj, but occupy dificrcnt n’--ks, do not make thcsc products. kkbitat. it seems, Is much more in-,pcrtant than phyiogcny. This obserx+atioia is vital to the starch for new mcr;;boiites bccatL
it rcprccrntr one of the m$or goats of the pixrmaccutical industv. WC should not restrict our search for candidatc producer\: to Inicroor~~cliinnls: many hi&r lit? forms produce 1_condar?, mctabolitcs of potential intcrest. K. Rinrhart (Univ. Illinois, IL, indicated USA) that mark mxroorganismc product a wide range ofsccondary mctabolitrs. sonic of which arc not tijund in tcrrcstriai (though compound5 isoiatcd 5omc marine species (c.g. from sponges] may actually bc prodnccd by symbiotic 1nicroar~a:;lnisllls). I<. Lchrcr (UCLA School of Mcdicinc, CA, USA) dcscribcd the cndog:cnous antimicrobial pcptidcs, dcfcnrins, which arc pmduccd by the majority of higher organisms, including man. The molecular structure, drrivation and mode of action of defcnsins has been worked out in detail: they arc of particular interest bccausc of their broad-spectrum antimicrobial actn+y against bacteria. fim;j, mycobactrria, spirochaetcs ;,!ld $OIIlC viruses. ikcccnt work on plnnts, whtxc rcscarch into i,atural products began, was dcscribcd bv I’. Waterman (Univ. Strathciydc, UK) who discussed the possibk roles oipinnt SLYondary mctaboiitcs in plant dcfcncc, pollination or srcd dirpersaj. or in adaptation to abiotic factors. From what wc now know (via ciolling and DNA scqucncin$ ofthc biochcmist5; and pcptide seyucnccs of cnzymcq mvoiwd in bio~ynrhcric of wcondary mctaboiiccs. thcrc unusual is nothing charactcri;tiialiy or special about them: the components of secondav metabolism cannot bc sharply distinguished from their comerparts in primac metabolism. They probably cvol14 (by gcnc duplication, and subscqucnt mutation and diwrgcnce) from a:xcstral genes inrolwd in primxy mctaboiism to function solely as !encs ofscconda~ rnctabolism. This mdicatcs th.tt primary metnbolism was csscntiallv already i!l csistcncc and ilmction~n~ etkctivcly before scrondary metabo!+m was added ta tbc rqxrtoire of tnctabolic xtkity. Howe\-cr, it must bc rcmcmbcrcd that gene transfer between specks is much more common than xx-c prrviou ;Iy kclirr-cd possible. Evolution of” a accolld~~-tnu~~bolit~ pathway may not hnvc t;lkcn pixc in tbc dnccstors of the spscics -.vhcre it is now found - it (or parts of it) cou!d just as ensiiy have been trans
past. ‘This compiicatcs enormously the analysis ofcvolutionary trees. but rhc data arc conristcnt with gene duplication followed by scparatc cvoiution of primary and secondary pathway-s. Rcmcmbcr too that what WC pcrcciw as the end point of a pathwa) may not bc the name compound that is driving the evolutionary sclcction ofthat pathway. D. Hopmood (Jc!!n Inncs Inst., Norwich, UK) pointed out that aincc we know so lit& about the natural roles ofthcse compounds iI? the producer organisms. it is possible that :n intcrmcdiatc ofthc pathwry could bc more import;rnt th,jn an end-product tu the bioio~~ of the orgmism. Wc should not bc too focused on the Sinai product of a pathway. bcc~msc who k~xxvs what intcrestmg phnrmacoiogicdl xciviticc some of the intcrrncdiatrc might have? IJcrxusc they 3rc 1n3de in minute ciuantitics. or r!lcir isolation has not bet-n punucd, they- have yet to bc rccognizcd as active in bioloical x-rccnk is it time to rc-csnminc this issue, in p3nllci \vitil the more usual cfiort of scrxniiir: ior novel mctrbolites from fresh .isotntcG At the moment, due to i_gnorancc. \vc could be panning tor goid and isolating the gold dust white throwing away the more valudbic nu@!ts. The
search for definition Sccondnn_ mctabolitcs arc nirr.rdv cst~hiishcd as ctktivc nnrimicrobials, irnmunosupprcss~nts. antihclrninthics. herbicide; and choicstcruiacmic drug. The x-cry nature of the subject means that a debate o: I c-x-oiutimnl-,d its consequences can, at times. bc drivcu more by prcjudicc than by scicntiiic fact. Howc*:x. thcrr was Sood agccmcnt among tbc sytnposium participants th.It. although they wcrc not es&y sure hnxv secondary mctnbrrlitcs had cv<>lvcd, they \\-crc very pieascd tbar it had bappcncd and thx mankind is the bcnekiary in terms ofbettcr hcuithcare. Throughout the mcctin$. 3 definition of-sccondat)rnct.lbol& 1~3s bring pursued. i’c4~rps the beut suggestion \cts: ~biocbcmirri pathIY’;I)‘swhich arc not ncccssary fix the vowth or reproduction of.m organtsm. but which cdn bc dcmonstrxcd gcnctically, pl”~iolo~icaiiy or biochcrnic~alh-‘.
146
the chemical stfuctufcs
havr
been
dcttxmincd and the biochemist? of rhr biosynthetic pathways studred. Compared with primaT metabolism, the c~~rymolo~y and regulation ofthe patlLways is stili ieis-well understood. However, now that molecular biology has opened a IWV door
on cl:“: problems,
the hope
is
tlm it will provide a unitkd view CC how expression of the gcncs which
make the tnetabolites is controlled and, fro111an understanding ufthcsc signals, the role ofthe mctnbolitcs for chc organisw m:?y bc made nmrc clcx. For the blotcchnolog indusof try, the cloning and rspfrasion genes CnGdiirg whole ix parrial
MessageAmplification Pkenotyping(MAPPing)*principles,practicean potential James W. Larrick Message amplification nhenotyaing (MAPPing)* is
a sensitive PoWerase
chain
reaction (PCRtbased technique for the rapid determination of the mRNA phenotype of small numbers of ceils. Isolated mRNA is reverse-transcribed (RT) into DNA, and then the DNA fragments corresponding to proteins or genes of interest are specifically amplified by PCR. The technique, also called RT-PCR, has wide application in biology and medicine, and comparism with bioassays demonstrates that MAPPing saves significant time and material. The teckique
shoirld be applicable
to analyse mRNAs of virtually any tissue or cell type. Wound healing, inflammation and cancer, a- well ns the normal processes of cellular growth and diffcrcntiation, arc co-ordinatcd by a complcs array of protein mcdiaton; cytokines, growth &ctors, cnzymcs and their rcccpton’-4. No single mediator is responsible for any particular biologjcal response; rather, a wide spectrum of cytokines and growth factors interact to detcrminc the fate of various ccl1 subpopulations. These mediators and the cells involved in these processes xc often obtainable for analysis only in small quantities. The dcvclopmcnt of scnsitivc tcchniyucs for the dctcction and quantification of mRNA is thcrcfore important to the cell biologist. The trcmcndous amplification obt;rinablc through PCR technology’ has contributed to the dcvclopmcnt of a method called message amplification phenotyp?ng (MAPPin@-+ (xc Fig. 1). Thr iechniquc incorporates a micro-procedure for isolating RNA, rcversc rran-
scnption oftotal cellular RNA to product cDNA, and enzymatic ampiitication of target DNA fragmcnr:, using PCR. The overall pattern of mcssengcr RNAs prcscnt in rmnll numbers of cells can therefore bc dcccrmincd siruply , without the complexity and time required for individual assays (c-g. bioassays, cnzymc immunoassay [HA] or radibimmunoassay [RIA]) for the transcribed proteins. The MAPPing technique has been used to detect and cotnparc the rclativc amounr~ of spccitic mRNAs from man:; ccllulrr sources (FCC
Table l), alld rcccnt advances permit quantification of individual mRNA spccics. This tcchniyuc should be appiicablc to cells from virtually any source. Esprcssion of cckaryotic mRNAs has traditionally been studied using the northcm b!ot technique’. Eowevcr, this method is inscnsitivc, requiring microgram quantities of purified polyadenylated RNA
[ poly(A)+ IINA], and cumbcrsornc, requiring the use ofspccihc probes for dctccting ml
1992.Elwer
Soence PubishersLtd(UK1
Function and evohtion of secondary rnetabolites - no easy answers riptv-t Exactly why crlls tnakc scconda~~ mewbolitcs, and how their biofunctions baw S~dlC~?i ard evolved, hw puzzled scientiatu for around half a cri1tury. Rcccndy. the Ciba foundation* gathered togcrhrr a group of scirutisa (chemists, physiologists. evolutionary rnd niolecular biologists) under the chairmanship of J. Davies (Ul;iv. British Columbia. Cnnada), ro debate these questions. With scientists present iiom such diverse backgrounds, thcrc was a lot of new and unt;lmiliar infonnatiou fix participants to digest. To start with, how might antibiotics have arisen? Primordial antibiotics - primitive catalysts: We know that many of toda>+ antibiotics act ou the ribosomc. and that resistance to them is oftcu due to moditication of ribosomal RNA at specitic sites. Perhaps this is telling us something of the role of ancestral antibiotic structures-did they act on primordial RNA? One schoo1 of thought on the origi!ia of protein synthesis assumc4 that tlic first pcptide bonds were formed through the action of ‘caulytic RNA’ in tbc primordial soup. Nowadays, we see catalytic RNP. at work in the splkiug ofintrons but RNA splicing may have prc-dated translation of mRNA as WC know it today. Using self-Tplicing of Group I illtrons as a model. J. Davies and co-workers haw Irsvcstigated the cftkt cf antibiotics on the splicing
l!BlECHMAY 1992 (!/OL10)
waction. Smprisinglv. streptomycin inhibited splicing. but csccss GTI’ could rcvcrsc r!lc in!libi:ion. The antiblotic (which has some structural similari~ to GTP) w35 sirnpIy intcrtking wth r!~c sddidx: ofguanosinc r&dues to the intron -an obligatory step in the splicing nwchanisn~. Howcvcr, spurr~cd by this glimmer of hope, tlwy tcstcd other antibiotics. Many of the arninoglycosides (which xt acl antibiodcs by bi&ing to ribosotnal RNA, thus caukg mistraodation) arc indeed inhibitors ofsclf-splicing, and their &initics of binding to introns arc‘ as high as their atfinities ofhinding to the ribo:om.~l RNA. Was their ancestral function to bind LO introns? Addition or‘viomycin tt> the srlfsplicing reaction gave an uncxpcctcd result, promoting ol;gomrrization of the introns which had been cut out by the splicing mechanism. The antibiotic thus promoted a primitive ‘RNA-&se’-litc reaction - taking small RNA tnoleculcs and polymcrizing thctn. This is couccivably how longer RNAs first arose and perhaps how primitive ribosornal RNA was buiit up. Dkcs and collsagues thrrcforc believe that mccstrai antibiotics may have pl.;yctd an important role as ‘scaffolds’ to promote RNA assembly and hcncc translation. This primi&r iunction lids cvolvcd into au antagonistic role in today’s antibiotic structures wvhich attack and prcvcnt the cell&r functions that, originally, rlrcy wcrr r:.sponsiblc for. How did ancestral antibiotic s;ructures first arise? Versions of them have been detected as products when the prrccived chctnical constituents of the primordial soup are wbjected to t!x reaction conditions thought to bc rxtant at that time.
Secondary metabolites - agents of cellular communication? Many cornpomlds which wcrc isolated initiallv as antibiotics, also have other biological propertics. Vancom~;citi, for csalnplc, is also a hypotcnsivr agent. c’ry:!:ro:I;:;cil: is 3 murili~~ .tgonist, and the antifungal antibiotic cyclosporin A intlucnccs production of intrrlcukin (IL-?). In addition, some microbes have now been shown to Ixvc vcrtcbr.xl~-lilw receptors. c.s_ for humdn chorionic gonadotrophm (WCC) (Ps~wfo~~~o~~,7.c thyroid ctimulatin~ rrwltophiiin) ami horrnooc (TSH) (E. i!l/i). Taken togcthcr. this could tncan that the ccl!ular connnunication ;ictxvorks wl~ich arc so csscntial in mct.lbolicnlly cotnplcs. multiorgan spccics dcvclopcd earlier in much simpler orgamsms, and that the relics ofthcsc procwcs arc stili to bc found i17 microbes. It may simply bc fortuitous hd\.C that sow of :hcw mctabolitcs antibiotic .~ctivir~. It is now the norm for ph3rmaccutiial compmics to use receptorb.lscd scrc’cns (comtesy of ci>NA cloning and heterologous cspwrsinn in tractdblr hosts) to starch for natural products with now1 pharn~acologic~l activities. I_ Nisbct (Xcnova, UK) has founded a company based on such screcniug operations, and rcportcd that thcri- SCCIIICto be no shortage of ‘receptor-accivc compounds produced by microbes. Nnturc 11‘1s had cstcnstvc opportuuity to csperiincnt with molccillar structures, and optimize how whey might tit and react lvith their targets. There is, therefore, much to be gaiwd wit11 rrgxd to the construction of functional molecules with cnhanccd potency from carrful observatiou of how ‘cftktor-receptor’ iutcrxxiom of the natural products arc tailored exquisitely, for the task that they perform. The mtcraction of
Q 1992. Elsevier Sctence Publishers Ltd IUKI
vancorny& and dcrivativcs with cell walls. studied and modcllcd by D. Williams (Cambridge Univ., UK), provided an cxcclicnt case in point. Most secondary mctabolitcs arc made only for a &ort time in the devciopmcntal cynic ofthc organism. K. Chatcr (John lnncs Inst., Norwich, UK) described how, as more information bccomcs Ivaiiablc on the regulatory cascadck which control the csprcssion of antibiotic production, it wcms that the antibiotics arc made in rcsponsc to cellular regulatory effcctors, and not (as was once thought) bccausc the antibiotics thcmsclvcs arc the etk:ors. Thus it may bc dangerous to gcncraiizc and to assume that all such mctnbolitcs arose to satis@ a sin& global function. It is a giant evolutionary icap from primitive cells using thcsc structures as scaffolds for assembly ofprimitivc RNA, to some of tk exotic cffccts that sccondnry mctabolitcs arc now shown to have. Evolution - a thorny issue 130~5 connidcring the cvoiution of secondary metabolitcs ht-lp us to focus on their timction? T. CavaiicrSmith (Univ. British Columbia. Canada) dcmonstratcd that inspcccion of phyiogcnctic trcrs scc’ms uninformative in pointing to the gcncsis ofsccondary metabolism. and how it has subscqucntly been dispcrscd. Taking a physioiogict’s approach. it is cicar (L. Vining. D~lhousic Univ., Canada) th.u production of secondary mctaboiitcs scc‘nis to correlate with specif:c x0iogical n&es. For cxampic, rhc fiinmcl:tous bacteria arc very distant in cvuiutionary time Co111 rhr tiiamrntour fungi, but both occupy the salnc ecological niche and both arc prodigiously active in the biosynthesis of secondary mrtabolitcs. Species which arc more closely r&cc.: XJ cithcr the bacteria or tb~ tu:,kj, but occupy dificrcnt n’--ks, do not make thcsc products. kkbitat. it seems, Is much more in-,pcrtant than phyiogcny. This obserx+atioia is vital to the starch for new mcr;;boiites bccatL
it rcprccrntr one of the m$or goats of the pixrmaccutical industv. WC should not restrict our search for candidatc producer\: to Inicroor~~cliinnls: many hi&r lit? forms produce 1_condar?, mctabolitcs of potential intcrest. K. Rinrhart (Univ. Illinois, IL, indicated USA) that mark mxroorganismc product a wide range ofsccondary mctabolitrs. sonic of which arc not tijund in tcrrcstriai
past. ‘This compiicatcs enormously the analysis ofcvolutionary trees. but rhc data arc conristcnt with gene duplication followed by scparatc cvoiution of primary and secondary pathway-s. Rcmcmbcr too that what WC pcrcciw as the end point of a pathwa) may not bc the name compound that is driving the evolutionary sclcction ofthat pathway. D. Hopmood (Jc!!n Inncs Inst., Norwich, UK) pointed out that aincc we know so lit& about the natural roles ofthcse compounds iI? the producer organisms. it is possible that :n intcrmcdiatc ofthc pathwry could bc more import;rnt th,jn an end-product tu the bioio~~ of the orgmism. Wc should not bc too focused on the Sinai product of a pathway. bcc~msc who k~xxvs what intcrestmg phnrmacoiogicdl xciviticc some of the intcrrncdiatrc might have? IJcrxusc they 3rc 1n3de in minute ciuantitics. or r!lcir isolation has not bet-n punucd, they- have yet to bc rccognizcd as active in bioloical x-rccnk is it time to rc-csnminc this issue, in p3nllci \vitil the more usual cfiort of scrxniiir: ior novel mctrbolites from fresh .isotntcG At the moment, due to i_gnorancc. \vc could be panning tor goid and isolating the gold dust white throwing away the more valudbic nu@!ts. The
search for definition Sccondnn_ mctabolitcs arc nirr.rdv cst~hiishcd as ctktivc nnrimicrobials, irnmunosupprcss~nts. antihclrninthics. herbicide; and choicstcruiacmic drug. The x-cry nature of the subject means that a debate o: I c-x-oiutimnl-,d its consequences can, at times. bc drivcu more by prcjudicc than by scicntiiic fact. Howc*:x. thcrr was Sood agccmcnt among tbc sytnposium participants th.It. although they wcrc not es&y sure hnxv secondary mctnbrrlitcs had cv<>lvcd, they \\-crc very pieascd tbar it had bappcncd and thx mankind is the bcnekiary in terms ofbettcr hcuithcare. Throughout the mcctin$. 3 definition of-sccondat)rnct.lbol& 1~3s bring pursued. i’c4~rps the beut suggestion \cts: ~biocbcmirri pathIY’;I)‘swhich arc not ncccssary fix the vowth or reproduction of.m organtsm. but which cdn bc dcmonstrxcd gcnctically, pl”~iolo~icaiiy or biochcrnic~alh-‘.
146
the chemical stfuctufcs
havr
been
dcttxmincd and the biochemist? of rhr biosynthetic pathways studred. Compared with primaT metabolism, the c~~rymolo~y and regulation ofthe patlLways is stili ieis-well understood. However, now that molecular biology has opened a IWV door
on cl:“: problems,
the hope
is
tlm it will provide a unitkd view CC how expression of the gcncs which
make the tnetabolites is controlled and, fro111an understanding ufthcsc signals, the role ofthe mctnbolitcs for chc organisw m:?y bc made nmrc clcx. For the blotcchnolog indusof try, the cloning and rspfrasion genes CnGdiirg whole ix parrial
MessageAmplification Pkenotyping(MAPPing)*principles,practicean potential James W. Larrick Message amplification nhenotyaing (MAPPing)* is
a sensitive PoWerase
chain
reaction (PCRtbased technique for the rapid determination of the mRNA phenotype of small numbers of ceils. Isolated mRNA is reverse-transcribed (RT) into DNA, and then the DNA fragments corresponding to proteins or genes of interest are specifically amplified by PCR. The technique, also called RT-PCR, has wide application in biology and medicine, and comparism with bioassays demonstrates that MAPPing saves significant time and material. The teckique
shoirld be applicable
to analyse mRNAs of virtually any tissue or cell type. Wound healing, inflammation and cancer, a- well ns the normal processes of cellular growth and diffcrcntiation, arc co-ordinatcd by a complcs array of protein mcdiaton; cytokines, growth &ctors, cnzymcs and their rcccpton’-4. No single mediator is responsible for any particular biologjcal response; rather, a wide spectrum of cytokines and growth factors interact to detcrminc the fate of various ccl1 subpopulations. These mediators and the cells involved in these processes xc often obtainable for analysis only in small quantities. The dcvclopmcnt of scnsitivc tcchniyucs for the dctcction and quantification of mRNA is thcrcfore important to the cell biologist. The trcmcndous amplification obt;rinablc through PCR technology’ has contributed to the dcvclopmcnt of a method called message amplification phenotyp?ng (MAPPin@-+ (xc Fig. 1). Thr iechniquc incorporates a micro-procedure for isolating RNA, rcversc rran-
scnption oftotal cellular RNA to product cDNA, and enzymatic ampiitication of target DNA fragmcnr:, using PCR. The overall pattern of mcssengcr RNAs prcscnt in rmnll numbers of cells can therefore bc dcccrmincd siruply , without the complexity and time required for individual assays (c-g. bioassays, cnzymc immunoassay [HA] or radibimmunoassay [RIA]) for the transcribed proteins. The MAPPing technique has been used to detect and cotnparc the rclativc amounr~ of spccitic mRNAs from man:; ccllulrr sources (FCC
Table l), alld rcccnt advances permit quantification of individual mRNA spccics. This tcchniyuc should be appiicablc to cells from virtually any source. Esprcssion of cckaryotic mRNAs has traditionally been studied using the northcm b!ot technique’. Eowevcr, this method is inscnsitivc, requiring microgram quantities of purified polyadenylated RNA
[ poly(A)+ IINA], and cumbcrsornc, requiring the use ofspccihc probes for dctccting ml
1992.Elwer
Soence PubishersLtd(UK1