Molecular biology of self-incompatibility in plants

reviews Molecular biology of selfincompatibility in plants

TIG - - September 1986

In many species distn~outed in almost halfthe familie~of flowering plants, seif4e"rtdization is blocked by a genetically controlled mechanism termed self-incompatibility; that is, the pistil Mikhail E. Nasrallah and June B. Nasrallah of the flower (comprising the Self-i~"dy re@omes, gcnegmUy determinedby alleles at the,S locus, p~evmt ovary, which contains the female self.fertilimtion in many species of flowen'ngplants. Recent biochemicaland Bmetic gametes or ovules, and the studies on families with spompkyticor eametopkyticcontrol of incompatibili~ s ~ e s t stigma and style, which receive that S alleles encode sped~ glycoprotei~ expressedin ~ pis61 and polltm. Selfpollen and allow growth of the sequences are consm~d wilhin families. In Brassica (spo~h~ic pollen tube towards the ovary), control) differ¢~ S allelesshow ¢lnn~ homalo~, and may differby a numberof ~,~nall rejects pollen which comes from replacementsand rearmr~or~ts. the same planL As outbreeding mechanisms, self-incompatfoility systems must have influenced the breeding surface of the stigma, with the result that no structure, the effidency of sexual reproduction and the incompatfole pollen tubes grow through into the style. evolution of both monocotyledonousand dicotyledonous

plants. The mechanism by which the diploid pistil distinguishes between self and unrelated pollen is not well understood. Se~-incompatibilityhas been extensively reviewedt-s and so an exhaustive review w~ not be attempted here. Ra~her, we wal concentrate on recent advances made towards a molecular analysis of the process, and the implicationsof those developments for our understanding of the structure, expression and evolution of self-incompatibilitygenes. The genetics of seff-menmpatibility In several families of flowering plants, the control of incompatibility can be attributed to a single genetic locus, designated S. Multiple alleles of the S locus can be identified in wild populations and some species are capable of expressing well over 200 distinct alleles. As with other allelomorphs, any two S alleles may be carried by a given diploid plant. Incompa~ility occurs if the alleles expressed in pollen and pistil are identical Seifincompatfoilitysystems are classified as being heteromorplfic, where cross-compatible plants have visible differences in floral morphology, or homomorphic, where no such differences are discernible. Homomorphic systems have been classified ~n turn into two groups, gametophytic and sporophytic, on the basis ofthe genetic control of pollen phenotype (Fig. 1). Gametopl~tic incompagbil~

In gametoph~c systems, exemplified by Solanaceae and Rosaceae, the incompa~oilityphenotype of a pollen grain is determined by its own haploid genotype. Fertilization is prevented if the S allele carded by the pollen is identical to one of the S alleles of the recipient plant (Fig. 1). In general, one-locus gametophytic systems, such as those found in the Solanaceae, are characterized by pollen tube inhibitionduring growth in the transmitting tissue of the style. Incompatible pollen germinates and the pollen tubes grow into the pistil, but at different sites in the style they swell and sometimes burst. More complex gametophytic systems have also been described. For example, in the grass family, Gramineae, it has been concluded that the control of i n c o n ~ a ~ is determined by two polyallelicloci instead of one. The alleles at the two loci act in a complementary fashion, each pair determining one specificity, with identical alleles at each locus in pollenand stigma being necessary for rejection. In these systems, inhibitionoccurs at the o xg~. ~ v ~

~

~

e.v.. ~

o~-

~ ~

Sporop~tic incompatibili~ In certain families, including Cruciferae (Brassicaceae) and exemplified by Brassica, incompatibility is

determined by a sporophytic system. The behaviour of the pollen is determined by the interaction of the two alleles that are borne by the nucleus of the diploidparent plant (sporophyte) that produces the pollen (Fig. 1). In sporophytic systems, as in the two-!ocus gametophytic systems, inhibitionoccurs at the surface of the stigma (Fig. 2). InBrassica for example, withinminutes after the initialcontact between the pollen or pollen tube and the papilkr cells on the outer surface of the stigma, a morphologicalresponse is elidted that prevents normal pollen tube growtit Pollen grains usually fail to germinate, or more rarely, germinate to produce pollen tubes that coil at the surface of the papillar cells and are unable to invade the surface layer of stigma cells. Althoughmultige.nesystems with sDorophyticcontrol have been reported°, a single S locus is the rule amongst most members of the Cruciferae. Complicated interactions between the alleles are often observed. The two different alleles in a heterozygote may be co-dominant, they may interact competitively and lead to mutual weakening, or one allele may be dominant to the other. Furthermore, the dominancerelationship for a particular pair of alleles may differ between stigma and the pollenproducing anther of the same plant. It is interesting that interactions between the S alleles do not occur in the diploid style of species with gametophytic control, suggesting some differences in the mode of action of the Sgene from that operating in sporophytic systems. Nevertheless, it has often been suggested that sporophytic and gametophytic systems rosy in fact be related, differing mainly in the timing or precise localizationofS locus gene activity in the anther. For example, in plants with sporophytic control, S-gene activity in the anther during pollen production could lead to products from the dominantallele or, in the case of codominance, from both alleles of the parent plant passing into all the pollen grains. In plants with gametophytic control the S gene might not be activated until after meiosis so that in each pollengrain only the singleS allele carried by that grain would be expressed. The relationsldp of sporophytic and gar:~tophytic forms of incompatibilitywith heteromorphic systems is even less certa~ In heteromorphic incompatibility, which has been foundin 23 families, genes determining a number of features of floral morphology, such as the 239

TIG-- S@t~r 1986

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relative lengths of stamen and styles, are closely linked events have been ascribed to mutations at the S locus, with the genetic elements responsible for pollen givingrise to so-called self-fertileSf mutants. Mutations rejection1. Heteromorphic systems apparently have to self-compatibilitycan affectthe poll~mdone, the pistil only two alleles of their incompatibility-determining done, or both the pollen and the lds~il. On the other genes, rather than the polyallelic series characteristic hand, self-compatibilityconditionedby mutations at nonof the more common gametophytic and sporophytic dlelic modifier genes has been described in both homomorphic systems. Interestingly, linkage to genes sporophytic and gametophytic systems 1. In addition, in determining floral morphologyand colour has also been Brmsica it has been shown that self-compatibility can reported for the S loci of a number of species exhibiting result from the competitive interaction of certain homomorphic incompatibility1. Other conserved link- S-allele combinations. ages have also been reported in gametophyticsystems; they include tight linkage between one locus of the Identification and properties of ~re!ated two-factor incompatibility system of rye and a peroxi- molecules dase genes, and linkage to peroxidase-1 in Nicotiava Spedes of the gametophytic type (Oenot/w~ and (tobacco)7 and in Lyc~ersicon (tomato)s. Petunia) were first used in pioneering applications of Attempts to induce new S-allele specificities by serological procedures to detect S-specific proteins 11,1~. mutagenic treatments have been unsuccessful. How- It has proved easier to identify S-related molectlles in the ever, studies in gametophyticsystems have shown that style or stigma than in pollen grains, dthough S-related new specificities are produced at highfrequencies under molecules must dearly be present in both pistil and conditions of forced inbreeding9'1°. Classes of mutations pollen to elicit the incompatibility response. The rote of leading to complete self-compatibility have also been the anther and pollen will be discussed in a later section. described. In gametophytic systems, certain of these The sporophyticdly controlled Brassica system has (e) Gametophylic control Pollen parent genotype S1Sz

I

Pollen genotype and phenotype S~(o) or $3 (e)

/ Male gametophyte

~

~

Pollen Pollen tube

~~;

(b) Sporophytic control

Examples

cotiana conI LNiycopersi Petunia Prunus Oenothera

Solanaceae Rosaceae Onagraceae

Pollen parent genotype $1S3

I

Pollen genotype $I or $3 Pollen phenotype (with co-dominance) $1$3

Stioma

Brassi ca I Raphanus

Cruciferae

Style Pistil Ovary Ovule - -

Fig. 1. Geneticcontrolofpollen-stigma interaction. (a) Gametop~tic [email protected] t~ parent SISa/soneof two genoO~s, St or $3. Sl canfert///ze S~53orS~4, butnotSxSa.SscanfertilizeSxSzor $2S4,butnotS~. Incompat/b/epol/end~/ops a shoripollen tubebeforebeingink.'ted in the sole (Sol=naceae,Rosaceae)or sterna (Onagraceae). (b) Sporophyt;ccontrol. Pollenfrom /rarest plant S~Ss can developtmllen tubes and fm~i~ S~$4 but mot S~Saor SzS3, i.e. t~ tmllen germtype is S~ or ,% but its kscompa~ phenoOpe/sStSa. In ~/s m=W/e (Brassica) the ~ldes sin= co.dominance.

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Fig. 2. Cov~r~le (a) and ~ l e

Co)pollimuio~ in Brassica..p, poll~: pt, ~llts~tube:pc, papillarcell.

recently been found to be very suitable for immunochemical studies on S.gene expression in the stigma. The S alleles in Brassica were shown to produce, in stigma cells, S-allele specilic antigens, the segregation of which correlated perfectly with the segregation of S alleles in the F1 and Fz generations°. These antigens were then showi-, to correspond to particular protein bands on p o l y a ~ d e gels14. More recently, Srelated molecules have been detected in Brassica1sin and Raphanus Is by isoelectric focusing 0EF) of stigmatic or stylar extracts. In a further study of B. o/eracea, 12 S homozygotes were analysed and found to have polymorphic S-allele specific molecules xT. In the game.t.,~h~c systems analysed to .da~, including .J' P m n.m ,

Nicotiama~o,~s,~9, Lycopcvsic~

o

and Petu-

n/az°, S-allele associated proteins have also been detected by IEF, but only for a few different alleles. All of the S-related molecules detected so far are glycoproteins with alkalineisoelectric points. However a number of differences can be discerned between the S-Iocns specific glycoproteins (SLSGs) of sporophyfic systems and the S-associated glycoproteins of gametophytic systems. The size range of SLSGs (55-65 kDa) is different from that of S-associated glycoproteins (28-32 kDa). In addition, the SLSGs of Brassica are heterogeneous in size within a given genotypeTM,and it is known that the degree of glycosylafion can vary Widelyz]. In some genotypes, the SLSGs also show charge heterogeneity. In contrast, the S-associated glycoproteins found in gametophytic systems appear generally to be homogeneous in charge and size within a given genotype. The glycoprotein associated with the $2 allele of Nicotia~ has been analysed and found to contain approximately 25% carbohydrate composed of fucose, arabinose, xytose, ugnlnlmgmmose,galactose, glucose and N-acetylglucosamine Is' 9. Several lines of evidence suggest that the glycoproteins play an important role in incompatibifity. For

sporophytic systems, data have been accumulated on SLSGs in.Raphanus and two Brassica species. (1) The inheritance of the glycoproteins correlates perfectly with the segregation of the corresponding S alleles in genetic crosses involving many Fzpopulations derived from a number of different S homozygotes and analysed in different laboratories, indicating that the gene responsible for this polymorphism must lie at or close to the S locusls'='m. (2) The 81ycoproteins are found in stigmas but not in

styles, ovaries or seedlings~4. (3) The concentration of these molecules is ~ected by S-allele dosage~. (4) Their loca~tion in the surface papillarcells of the stigma is consistent with that expected of molecules involved in the highly localized surface reaction of incompatibility in Brass/cau. (5) The developmental regulation of the concentradons of these molecules correlates well with the developmentally controlled expression of self-incompatibility. During flower development in Brassica, the young buds are initiallyself-compatible and become selfincompetible only one or two days before the anthers mature (anthesis). The immature self-compatible stigmas are fully receptive to pollen, thus permitlbtg the production and maintenance of S homozygotes by fe"rtdizationwith mature pollen from genetically identical plants. Low levels of SLSG can be detected in young self-compatible buds by immunochemical methods~ by sensitive protein stainingl~ ~ d by in~-orporation of labelled amino acids in vivo~. The S , ~ to selfincompatibility has been correlated with incr~ses in the amount and rate of synthesis of SLSGs. (6) The analysis of self-compatibYe mutant swains resulting from the action of a modilie~ gene unlinked to the S locus has demonstrated that a critical concentration of SLSG is required for self-incompa~ility in 241

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TIC,

Brassica. In these strains, selfcompatibilityis accompanied by a decreased productiond SLSG, whichnever attains the high concentrations characteristic of the normal self-incompatible

~"

~

stigmas 26,27.

The evidence that the S-associated glycoproteins in gametophytic systems are involved in self-incompatibilityis not as extensive and the details differ. The proteins co-segregate with the corresponding S allele1°'19. T~,ey a=-egenerally only fo~d in pistil tissue, although~ surprisingly, in Prun=s they have been detected in the medium of suspension cultures derived from leaf and stem tissue 17. In N/cot/aria, where inhibitionoccurs in the style, S-associated glycoproteins are detected ~ along i the pistil with the highest concentrations in the stigma and the upper portion of the style~9. Un- Fig. 3. R e s ~ froglike the situation in sporophytic mint patterns of three Brassica S.aUele systems such as Brassica, diff.~rent homozygotes probed with where inhibitionimmediatelyfol- sel)-i~o~tibility cDNA lows pollen-stigma contact, the done,andsho~oin8S-ge~ogrowingpollentubesof N/co~=~ t~s~b~orphS'ms. therefore appear to withstand growth in high concentrations d S-associated glycoprotein before being inhibited further downthe length of the style. Finally,although the onset of the self-incompatibilityresponse during flower development in Nicotiana parallels that described above forBrassica, S-associated glycoproteinsare not detected inimmature self-compatiblegreen-bud styles, and appear only in self-incompatiblebuds. The gone encoding these molecules therefore appears to be expressed over a very short time period duringflowerdevelopmentin Nicotiana. Molecular cloning of sequences encoding an SLSG in Brassic~ The strategy used for cloningsequences encoding an SLSG in Brassica oleraceawas based on the fact that in some SWdins,approximately 5% of protein synthesis in the stigma one day before anthesis is devoted to the production of SLSG24. A cDNA library was constructed using mRNA from stigmas prepared one day before anthesis22. One of the clones obtained, pBOS5, containinga 1.3 kb insert, appears to encode an SLSG on the basis of the followingcriteria. ii ....

t-"

(1) It corresponds to an approximately 2 kb mRNA species whichis of quite high abundancein the stigma but is not found in leaf and seedling tissues. (2) During the development of the stigma, the transcriptional activity of the genes represented by pBOS5 closely parallels the synthesis of the SLSG. (3) Polypeptides synthesized inE. coilcells carrying a fusion of an open reading frame derived from pBOS5 with a I~-galactosidase gone were found to react wi~ antibodies raised against authentic SLSG. (4) In situ hybridizationof frozen pistil sections to 3HlabelledpBOS5 DNA demonstrates that the homologons transcripts are exclusively localized in the surface 242

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1986

papillarcells of the stigma (Nasralhh etal., unpublished). The sequence analysis of pBOS5 identified a long (900 bp) 3' untranslated region. There is a 1.1 kbp open reading frame, sufficient to code for a polypeptide consisting of over 300 a~dno adds. Sequence information is available for 50% of the polypeptide, and is consistent with the reported properties of native purified SLSG21. Genomic organizationofthe 8-locus region in Brassic~ The analysis d the sequences in the Brassica genome homologous to pBOS5 was I~rticul~ly revealing. pBOS5 sequences hybridized strongly to all 15 S-allele homozygotes analysed, indicatingthat there is substantial homology amongst the different S alleles. Extensive polymorphisms in restriction endonuclease-generated fragments of Brassica genomic DNA were also revealed (Fig. 3). These polymorphisms segregate predsely with the S locus in F3 populations, indicatingthat the genomic fragments corresponding to these clones are at the S locus or very tightly linked to it, and supporting the contention that the SLSGs are the direct translational products of the ~ locus. The large number of bands hybridizingwith pBOS5 in each track d Fig. 3 suggests that the Brassica genome must contain a number of different DNA sequences homologous to SLSG cDNA, and the genetic data show that at least the majority d these sequences are clustered in the genetic vicinity d the S locus. Such an organization supports a previously suggested mechanism for the generation of new S alleles by recombination involving tandem sets of related sequenceszs. Thus S alleles may differ not by changes in a single codon but by several replacements and small rearrangements. This might also explain the failure to generate new functional S specificities experimentally. For example, clustering d partially related sequences would facilitate processes d unequal crossing-over or d gone conversion which would allow reshuffling of the sequences and could thereby eliminate any new mutation that had been introduced experimentally.

Molecular cloning ofsequences encoding an S-associatedglycoprotein inNicotiana The approach used for cloning the Sz-associated glycoprotein in the gametophytically controlled N~ot~na a/ata was based on determining the amino acid sequence of the N-terminus of this protein19. A cDNA library was prepared from mRNA isolated from mature self-incompatible styles of plants with the genotype $2S3. Differential screening of the library with probes prepared from nfftNA obtained from mature styles, from inunature green-bud styles and from ovaries identified clones that hybridized only to mature style probes. From this population of positive clones, the desired recombinants were identified by hybridization to a synthetic oligonucleotide corresponding to the N-terminus of the Sz-associated glycoprotein. The sequence analysis of the cDNAinsert identifiedthe compl~~e~ . ~ o acid sequence of the mature $2-associated glycoprotein in addition to a 30-aminoacid putative signalpeptide. The corresponding 940 bp message was not detected in green-bud styles, but f~und in mature styles and, unexpectedly, in ovaries bearir,g the $2 allele. Hybridization histochemistry experiments localized the $2 transcripts to the

reviews

TIG - - September 1986

transmitting tissue of the style. In contrast to the observation in the Brmsim system that the pBOS5 probe hybridized strongly to mRNA from a number of different S homozygotes, onlyweak (< 1%) hybfi~,ation was found inN. a/ata betwee~ the cDNAprobe for the S~ ~ele and mRNA from styles bearing the Sz or Ss alleles. This suggests that inN, a/ata the different S alleles I~ve either drastically different expression levels or very different transcripts. 5-gene e:q~ression in anther and lmllen An important question concerning the pollen-pistil interaction in self-incompat~illW is what is the role of the pollen? What S-gene products are expressed in the pollen, are they identical to the S-relatedglycoproteins in the stigma or style, and what is the timing d their expression during production and germination of the poller2 These questions are fundamental for understanding the molecular medmnlsm ofincompan~oilityand the differert~s between the sporophytie and gametophytic systems. Althoughmost early studies focused on the pollen, little information concerning the biochemical properties of pollen in~mpatibility a n ~ s such as those, reported by Lewisxx and Linskens ~z is available. No protein associated with S genotype has as yet been found in Nicotiana pollen, for example TM. In Brass~a, however, components homologous to stigma SLSG, but

incompatibilitygenes, such as that shown for the S locus region of Brassim (perlmps reflecting the multiplidty of S alleles and the probable differential expression of the locus in stigma and pollen) will be the rule for all selfincompatibility systems, or a c~haracteristic only of species with sporophytic control. Prospects The molecular cloning of sequences encoding giycoprote'ms apparently involved in self-incompatibility is a significant development in the study of this phenomenon at the molec~br level The structure, organization and expression of the self-incompatibility genes can now be investigated. Analysis of the pollen-pistil interaction at the level of the interacting molecules themselves will follow the ctmracterization and purificationof the pollen determinants of incompatibility. Most critical however, is the task of proving conclusively t ~ t these sequences do in fact originate from the S Iotas. The S locus has recently been mapped in Lycopcr~onS; the location, on the genetic map, of the sequences encoding S-associated glycoproteoins in solanaceous plants can therefore be determined immediately and ILMmge to the S locus verifie& However, defini~,e proof in both the Brassica and the Nico~zna systems willhave to await the demonstration that transformation with the cloned sequences can change the incompatibilityspecifidty of a plant. By careful selectionof S allelesin donor and redpient, transfonnants modified in their self-incompal~oility specificity should be readily identifiable. Such experiments are feasible s~nce cell and tissue culture, plant regeneration, transformation with Ti-plasmid vectors and by electroporation are possible in both of the systems.

occmdng in much lower concentrations, have been detected on native and western blots of anther and pollen extracts probed with anti-SLSG antibodies2°. S-genotype associated glycoproteins have also been resolved by elect]'ophoresis of extracts from pollen and mature anthers~°, but the S-allele spedfidty of these molecules remains to be determined in segregating Fz populations. The detection, in anther tissue, of a ndCNA homologous to the SI,SG-encoding sequences is also promising (Nasrnllnh and Nas~llnl~, unpublished). Refereuces NeUancourt, D. (1977) l ~ / t y im Ang/osperms Comparison of the sequences expressed in stigma and I de (Monographs on Theoretical and AppliedGenetics VoL 3), m,d~er is therefore now possible, and should allow the Sprier-VeinS m.~hanism of pollen recognitionto be elucidated and the 2 Heslop.Hmison,J. (1975)A~. Rev.PlantPhysioL26, 403-425 coordinate expression of one gene in the two tissues 3 Knox, R. B. (1984)inE~jc~ofPlmflPkysiology(VoL 17) (Linkse~ IL F. and HeslopHarrison,J., eds), pp. 508-608, involved in self-incompatibilityto be investigated. Spr~er-Vemg Evolutionary relationships Hybridization of the N/cot/a~z S2-associated done to stylar RNA from another closely related solanaceous genus Lycopersicon, and of the Brassica SLSG done to genomic DNA from five genera of the Cmciferae have been demonstrated. Thus, S-allele products appear to be quite highly conserved within gametophytically controlled and sporophytically controlled families. No homology is apparent, however, between the deduced sequence of the Nicoliana S~-assodated protein and the partial sequence of the Brassica SLSG. If, as this suggests, self-incompatibilityis polyphyletic in origin, the recent progress made in the molecular analysis of self-incompatibility in each system will not be easily translated to the other system. Nevertheless, in view of the similarities noted earlier between gametophytic and sporophyfic systems at the genetic and operational levels, some basic features of the organization of the self-incompa~bility genes might be expected to be similar in the two systems. Data on the genomic sequences homologousto the Nicoliana $9 done are not yet avagable. It is therefore not possible at present to determine whether a complex organization of the self-

4 Lundqvist,/L (1975)Prec. R. Soc.LondonB 188, 233-245 5 Lewis,D. (1979)N.Z.]. Bot 17, 637-644 6 Wdcke, G. and Webli~ P. (1985) Tkem,. AppL Goner. 71,

289-291 7 Labroche,P., Pdrier-Hamon,S. and Pemes,J. (1983)T/~or. Appt C,e~t 65, 163-170 8 Tanksley,S. D. andLoaiza-Flgueroa,F. (1985)Proc.Nat/Ac¢~ Sci. USA S2, 5093-5096 9 de Nettancourt,D., Ecoclm~ R., Perquin,M. D. G., van der Drift, T. and Westerhof, ~ (1971) Tkeov. AppI. Greet 41, 120-129 10 Kheyr-Pour,,eLandPemes,J. (1986)inB/o~/mo/o~andEco/o~ ofPoUm (Mulcahy,D. L, BergunbdMulcahy,G. andOttaviano,

E., eds), pp. 191-196,Si~Bger-Verlag

11 Lewis,D. (1952)Proc. R. Soc.LondonB 140, 127-135 /2 Linskens,H. F. (1960)7..Bot. 48, 128-135 13 Nasragah,M. E. andWallace,D. H. (1967)Hered/ty22, 519-527 14 Nasragab,M.E., Barber,J. T. andWagace,D. H.(1970)Hcred#y

5,23-27

15 Hinata,IT. and1¢~o, T. (1978)Hered//y41, 93-100 16 Nasr~lb~ J. B. and Nasmlbh, M. E. (1984)E R ~ t / a 40,

279-281

17 Man,S. L, Raft,J. andClarke,A. E. (1982)P/m~156,505-516 18 Clarke,A. E., Anderson,M. A., Bacic,T., ~ P. J. andMan, S. L. (1985)J.CeUScL SuppL 2, 261-285 19 Anderson, M. A. et ¢1. (1986)Nature321, 38-44

20 Kamboj,!¢. K. andJackson,J. F. (1986)Tkeor.Appl.Genet 71, 815-819 21 lCmbio,T. and Hinata,K. (1982)Gend~ 100,641-647 243

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TIG - - S e p t e m ~ 1986

22 Nasrallah,J. B., Kao, T-H., Goldberg, M. L. andNasra]lah, M. E. (1985) Nature 318, 263-267 23 Nasrallah,J. B. and Nasrallah, M. E. (1986) in Biotechnoio~ and Crop Improvementand Protect/on (Day, P. R., ed.), pp. 83-89, The British Crop Protection Council 24 Nasrallah,J. B., Doney, R. C. andNasrallah, M. E. (1985)P/amta 165, 100-107 25 Sedgley, M. (1974) Heredity 33, 412-.416 26 Nasrallah, M. E. (1974) Genet/cs 76, 45-50 27 Hinata, IC and Okasald, K. (1986) in Biotecknologya~lEcologv of Pollen (Mulcahy,D. L., BergamjniMulcahy,G. and Ottaviano, E., eds), pp. 185-190, Sp~inger-Verlag

28 Chadeswort~D. andChadesworth,B. (1979)Hered~43,41-55 29 Nasr~h, M. E. and Na~ah, J. B. (1986)B/otechno/ow Ecolo~ of Pollen (Mulcahy,D. L., P~zrgan~dMulcahy,G. and Ottaviano,E., eds), pp. 197-201,Springer-Vedag ,70 Gaude,T. and Dumas,C. (1986)in Bio~hsolo~ and £colo~ oJ Pollen(Mu!cahy,D. L., BergaminiMulcahy,G. m~dOttavia~,E., e&), pp. 209-214,Springer-Verlag M. E. N aerallah and]. B. NasraUah are at the Section ofPlant B ioloo, Plant ScienceBuilding, Co~nellUniversity, Ithaca, N Y 14853, USA.

The activation of cellular oncogenes by retroviral insertion

Retroviruses can induce tumors in several different ways. Transformingviruses have transduced host cell proto-oncogenes and express these genes in a modified form, such that target cells swiftly become tumorigenicz'2. Generally, transforming viruses Roel Nusse have sacrificed some viral information in the process of transReplication-competent retroviruses can induce a variety of tumors by inserlienal duction of cellular sequences, activation of cellular oncogenes. Transposon tagging techniques have uncovered many novel cellulargenes implicated in tumorigenesis. Activatien of thesegenes can occur by and are hence dependent on repinsertion of viral promote~, transc~tional enhancement over large distances, or the lication-competent helper viruses generation of novel chimeric pro~'ns. for propagation. Most replicating retroviruses are unable to transform cells directly, but they are not completely sis by ALV become understandable, but the stage was innocuous; many of them can cause tumors in suscep- set for the subsequent unmaskingof the c-myc and c-abl tible animals, albeit after long incubation periods com- genes at chromosomal breakpoints in non-viral tumors. pared with their oncogene-carrying derivatives. Another rewarding highlight for tumor virologists was In recent years, the mechanism underlyingoncogen- the identification of the ras oncogenes, again originally esis by slowlyoncogenic viruses has been elucidated to a found in retroviruses, as the culprits in cellular transforlarge extent. The implications of these findings have marion after transfection of tumor DNA into NIH/3"~ been wide-ranging: from the first demonstration that cells. These and other findings showed that the search cellular proto-oncogenes could actually contribute to for genes implicated in viral oncogenesis could also lead tumorigenesis, to the discovery of a whole series of us to the genetic events underlying non-viral forms of novel oncogenes that had not been encountered before cancer 1,2. in viral form. Other genes activated by proviral insertion Early indications and the activation of c-myc Meanwhile, the large variety of animaltumors induced A first g'~,p~e of the mechanism of viral oncogenesis by slowly oncogenic retroviruses was explored to find without viral oncogenes was obtained when restriction novel insertion sites and genes. Established cell lines enzyme analysis of tumor DNA showed that proviral from tumors provided additional sources of material. DNA was integrated in a clonalfashion, i.e. tun~orswere Retroviruses can i,-,ducemany different tumors, ranging descend~ts of a single infected cell. It was realized that from lymphoid proliferative diseases to mammary ,'~arproviral insertions could cause mutations in the host cell cinomas and neplu'oblastomas. In most of these tumors, DNA, not onlyrecessive mutations due to disruption of a integrated proviral DNA can be found, and used as a tag gene, but also integrations resulting in dominant mum. to ident~y the integration domainby molecular cloningof tions, as a consequence of c/s-activation of cellular viral DNA-host cell DNA junction fragments. The sequences. The strategy for identifying the relevant presence of proviruses in the same region of the cellular cellular ge~s was straightforward and analogous to genome in multiple, independent tumors is then taken as transposon t~ggingtechniques used by Drosophila gen- evidence for the presence of a cellular gene whose eticists: proviral DNA was used as a_probe to isolate activation has given the cell a growth advantage. The flanking DNA from tumor cell libraries~. This approach argument is based on the widely held but formally proved to be successful (see below), but the first unproven assumption that proviruses integrate at nudiscovery of a gene activated by an in.fred provirus merous, if not random sites in the cellular DNAS. A was actually made when Hayward, Neel and AsLTin common integration site therefore indicates selection of conjectured that cellular proto-oncogenes could be one particular cell on its way to tumorigenicity out ,of a among the targets for proviral insertion. They simply large population of infected but otherwise normal cells. tested their hypothesis by screening virus-induced The prediction that a relevant cellular oncogene is tumors for disruption of restriction fragments of known present in the integration domain can be tested by oncogenes, and found that a large majority of avian searching for transcriptional activity, structm-ai analysis leukosis virus (ALV)-h~lducedbursal lymphomas con- of the gene and biological assays. rained a provirus just ~stream from the c-myc gene4. By followingthis strategy, many different integration Their discovery wa~ a key event in the history of donminshave been identified, and in some of them, novel oncogenetics. Not only :rid the mechanism of oucogene- putative oncogenes have been found. The mouse 244 © 1986,ElsevierSciencePubeshersB.V.,Amstenbm 0168-~ . 2 0 0