Ustilago maydis, the delightful blight

[]~EVIEWS 23 Pfeifer, G.P. et al. (1990) Proc. Natl Acad. Sci. USA 87, 8252-8256 24 Wolffe, A.P. and Brown, D.D. (1986) Cell 47, 217-227 25 Solomon, M.J. and Varshavsky, A. (1987) Mol. Cell. Biol. 7, 3822-3825 26 Bonne-Andrea, C., Wong, M.L. and Alberts, B.M. (1990) Nature 343, 719-726 27 Adams, R.L.P. (1990) Biochem.J. 265, 309-320 28 Gottesfeld, J. and Bloomer, L.S. (1982) Cell 28, 781-791 29 Holmquist, G.P. (1987) Am.J. Hum. Genet. 40, 151-173 30 Graves, J.A.M. (1987) Trends Genet. 3, 252-256 31 Migeon, B.R. (1990) Genet. Res. 56, 91-98 32 Svaren, J. and Chalkley, R. (1990) Trends Genet. 6, 52-56 33 DePamphilis, M.L. (1988) Cell 52, 635-638 34 Pfeifer, G.P., Tanguay, R.L., Steigerwald, S.D. and Riggs, A.D. (1990) GenesDev. 4, 1277-1287 35 Pfeifer, G.P. and Riggs, A.D. (1991) GenesDev. 5, 1102-1113

g s t i l a g o maydis or corn smut fungus belongs to the Basidiomycetes, a group of fungi that includes the c o m m o n m u s h r o o m and many plant pathogens, such as the smuts and rusts. U. maydis is pathogenic only on corn and its close relative teosinte - it causes stunting and induces the formation of tumors (or galls) in many different parts of the plant. U. maydis is dimorphic (Fig. 1): one of the forms is haploid and unicellular, divides by budding, and is nonpathogenic; the other form is filamentous and dikaryotic and requires the plant for its growth. This filamentous form is pathogenic and arises by fusion of two compatible haploid cells (see below). Dimorphism and m a n y of the steps in the life cycle are under the genetic control of two mating type or incompatibility loci, the a and b loci. Recent studies indicate that the a locus e n c o d e s c o m p o n e n t s of a p h e r o m o n e response p a t h w a y and that the

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Lewis, J. and Bird, A. (1991) FEBSLett. 285, 155-159 Wolffe, A.P. (1991)J. CellSci. 99, 201-206 Boyes, J. and Bird, A. (1991) Cell64, 1123-1134 Migeon, B.R., de Beur, S.J. and Axelman, J. (1989) Ex'p. Cell Res. 182, 597-609 Villarreal, L.P. (1991) Microbiol. Rev. 55, 512-542 Zhimulev, I.F., Belyaeva, E.S., Bolshakov, V.N. and Mal'ceva, N.I. (1989) Chromosoma 98, 378-387 Karpen, G.H. and Spradling, A.C. (1990) Cell 63, 97-107 Aparicio, O.M., Billington, B.L. and Gottschling, D.E. (1991) Cell66, 1279-1287 Gmnstein, M. (1990) Trends Genet. 6, 395-400 Selker, E.U. (1990) TrendsBiochem. Sci. 15, 103-107

A.D. RIGGS AND G.P. PFEIFER ARE IN THE DEPARTMENT OF BIOLOGY, BECKMAN RESEARCH INSTITUTE OF THE CITY OF

HoPI~ DUARTF,CA 91010, USA.

Ustilago maydis, the delightful blight FLORA BANUETr Recent studies of the corn smut fungus life cycle and its regulation by two mating type loci and otber genes provide a cornucopia of challenges in cell biology, genetics and protein structure. Thefungus can exist in two states: nonpathogenic and pathogenic. The change from one state to the other is accompanied by a change in morphology (yeast-like to fllamentous) and growth properties (saprophytic to parasitic). b locus e n c o d e s a gene regulatory protein. In addition to these loci, three other genes ( f u z l , f u z 2 and rtfl) have recently b e e n identified that are necessary for

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Ustilago maydis is a dimorphic fungus. (A) The unicellular haploid form with buds (arrow). (B) A single nucleus (stained with DAPI) is present in each haploid cell. This form is nonpathogenic and grows on a variety of media (i.e. it is saprophytic). (C) A filamentous dikaryon, whose tip cell contains two nuclei (DAPIstained, arrows in D). This form is pathogenic and requires the plant for growth (i.e. it is parasitic) and differentiation into spores competent to undergo meiosis. (E) Filaments in a mycelial mat. (F) The plate assay for filaments" (the fuzz reaction): when two haploid strains carrying different a and b alleles are mixed on medium containing charcoal41, a white fuzziness develops due to formation of dikaryotic filaments (the Fuz ÷ phenotype)7,9, which can grow on this medium for a limited time. Filaments are not formed if the strains contain identical a or identical b alleles (the Fuz- phenotype). This plate assay parallels tumor formation and permits the rapid determination of mating types. In (F) the horizontal strains are a2 bl (top) and a2 b2 and the vertical strains (left to right) are al bl, al b2 and al bll. T1G MAY 1992 VOL. 8 .XO. 5 ©1992 ElsevierScience PublishersLtd (UK)

I

~EyIEWS filamentous growth and other aspects of the life cycle and whose products might function downstream from a and b. The intellectual challenges posed by the life cycle of U. maydis are numerous and include understanding: (1) the exquisite regulation of karyogamy, a process that occurs not after cell fusion but instead much later in the life cycle; (2) h o w pheromones encoded by one of the mating type loci stimulate filamentous growth, which occurs after cell fusion; (3) h o w the fungal cell distinguishes identical from nonidentical alleles of a locus with 25 alleles (the b locus); the products of these alleles can form, in principle, 625 combinations, 600 of which can be argued to result in functional regulatory proteins; (4) h o w the plant endows the fungus with competence to undergo meiosis and h o w the fungus alters growth control of the host. U. maydis presents not only this intellectual smorgasbord but is itself a culinary delicacy: cobs that have been infected by U. maydis give rise to a delight known as huitlacoche (the ambrosia of the Aztecs).

Genetic control of the life cycle

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The haploid form of U maydis produces compact colonies on a variety of laboratory media 1, and is thus amenand rain I I ~ ~ able to manipulation by standard microbiological t and molecular genetic germinating teliospore teliospore Dikaryotic hyphal cells techniques 2-4. U maydis is thus an ex(2N) roundupto become perimentally accessible fungal plant teliospores. Karyogamy occurs. pathogen. Compatible haploid cells fuse (Fig. 2) to form a filamentous diFIG[] karyon that, in contrast to the haploid, is difficult to propagate on laboratory Life cycle of Ustilagomaydis. The interaction of U. maydiswith its host is a two-step media: its growth and differentiation process that results in disease development and completion of its life cycle. In the require infection of the plant. As the first step, infection results in the induction of tumors, the most characteristic filamentous dikaryon grows in the symptom of the disease. These tumors, which occur on all aerial plant parts, are plant, it induces the formation of presumably due to alteration of growth control of plant cells by fungal gene products. The fungus might produce plant hormones or metabolites that alter the tumors (Fig. 3). The fungal hyphae difhost hormonal balance, or alternatively the fungus might transfer a DNA segment to ferentiate within these tumors, where the plant cells. In the second step, the host apparently produces signals that trigger karyogamy takes place, resulting in fungal differentiation, culminating in production of sexually mature diploid spores the formation of diploid spores competent to undergo meiosis. Modified, with permission, from Ref. 42. (teliospores) (Fig. 2)~,5-7. H o w nuclear fusion is regulated is not known, but plant signals presumably play a crucial role in the Teliospore production and competence to undergo timing of this event. The teliospores, which are not meiosis require growth in the host; so far, attempts to capable of vegetative growth, completely fill the tumorinduce meiosis under laboratory conditions have failed. ous outgrowths (Fig. 3) and are readily spread by wind The two mating type or incompatibility loci, a and when the tumors crack open. The teliospores germinate b, govern this life cycle. For the pathogenic filamenon a plant or on nutrient medium and produce a short tous dikaryon to be produced, the haploid mating filament into which the diploid nucleus migrates and partners must have different alleles at both a and b undergoes meiosis, resulting in a short septated fila(for example, al bl plus a2 b2) (Fig. 1). Such strains ment consisting of four haploid cells (Fig. 2). These are said to be compatible. If they carry identical alleles haploid cells give rise to progeny haploid cells by at either locus, filamentous growth and tumor inducmitotic divisions, thus completing the life cycle s . tion do not occur. "I'IGMAY 1992 VOL. 8 NO. 5

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The mating type or incompatibility loci

FIGI~ Corn plants exhibiting symptoms of corn smut disease. Infection with U maydis results in strong induction of anthocyanin pigmentation (A) and induction of tumors, the most characteristic symptom of the disease (A, B). These tumors occur on leaves (A), cobs (B), and other plant parts. Infected cobs at an earlier stage (huitlacoche) are used in cooking. The infected kemels in (B) are full of sooty teliospores, from which the name smut is derived. Many features of the dikaryon can be studied in vegetative diploid strains, which can be readily obtained in U. maydis (by mating strains carrying complementing auxotrophic markers and selection for prototrophy) 1,9. Interestingly, these diploids can exhibit properties of either the haploid or the dikaryon, depending on the media used. On most media, these diploids divide by budding, like the haploid; in contrast, on media containing charcoall the diploid can form filaments like the dikaryon.

The a locus The a locus has two alleles, a l and a2 (Ref. 10), and has recently been cloned n,l~. The a l and a2 alleles comprise heterologous regions of DNA flanked by homologous regions. Mthough the a locus was expected to code for a regulatory protein, by analogy with many other mating Wpe loci 13, the a alleles instead appear to encode at least two components of a pheromone response pathway12: a putative pheromone and a pheromone receptor (Fig. 4). A small open reading frame (ORF) of approximately 40 amino acids, which contains a CAAX (Cys-aliphatic-aliphatic-X) motif at its carboxy terminus 12, probably encodes a pheromone precursor. The CAAX motif serves as the recognition substrate for addition of an isoprenoid moiety (famesyl or geranylgeranyl) to proteins 14-17, which may be necessary for membrane association. Several fungal pheromones are famesylated: a-factor of Saccharomyces cerevisiae 14, A- and a-tremerogens of Tremella mesenterica 18,19, and rhodotorucine A of Rhodosporidium toruloides2°. It was in the latter two relatives of U. maydis that carboxy-terminal modification with famesyl was discovered. Secretion of these fungal pheromones may occur via a nonconventional pathway involving a product analogous to the STE6 protein of yeast, a membrane-bound transporter 21. Each a allele also contains an ORF encoding a polypeptide that has seven putative transmembrane domains 12 and shows similarity to the receptor for the a-mating factor of S. cerevisiae (the STE3 gene product) =. The a l allele is proposed to code for the receptor for the a2 pheromone, and the a2 allele for the al pheromone receptor (Fig. 4). The'putative pheromones of U. maydis are thought to bind to the appropriate receptor and activate a signal transduction pathway that mediates cell fusion. This mode of action of the a locus would thus be analogous to the role of the yeast pheromones and receptors in sexual conjugation. Many components of the pheromone response pathway have been identified in S. cerevisiae, including four presumptive protein kinases and a transcription factor23. A U. maydis homologue of the S. cerevisiae STE7 gene (encoding one of the serine/ threonine protein kinases 24) has recently been identified (F. Banuett, unpublished) and may play a role in the mating factor response pathway or perhaps in response to plant signals that induce fungal differentiation. Other steps in these pathways might be coded by some of the f u z genes (see below). The a locus is also necessary to maintain filamentous growth9: diploid strains that are homozygous for a and heterozygous for b (alia1 bl/b2 or a2/a2 bl/b2) do not form filaments. The implication is that the a pheromones participate in an autocrine response: their continuous presence is required for stimulation of filamentous growth. Another possibility is that a gene not yet identified in the a locus is responsible for filamentous growth. The b locus The pioneering work of Rowell and DeVay a° led to the identification of the b locus and its multiple alleles

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and showed that any combial nation of different b alleles resuits in tumor formation. These mfal pral observations were confirmed j k I m and extended by Puhalla 25,26, who estimated that at least 25 b alleles exist in nature. It is in4.5 kb deed remarkable that any combination of two different alleles a2 elicits a similar degree of response. Such observations lead pra2 mfa2 to the challenging question of _-st, I m j k I .,. kN\\\\\\N, x] N\N] I the molecular mechanism by which the fungal cell distin8.0 kb guishes between identical and nonidentical alleles. The recent FIG~ cloning and sequencing of several of these alleles suggests The a locus encodes components of a pheromone response pathway. The a locus has m'o that monitoring of different b alleles whose heterologous DNA segments are drawn as large rectanglesU.12: al contains mfal (encoding al mating factor) and pral (encoding the receptor for the a2 mating factor); alleles involves the interaction a2 contains tufa2(encoding a2 mating factor) and pra2 (encoding the receptor for the al of polypeptides to create a mating factor). The a alleles might contain additional cell type-specific genes. The thin lines functional regulatory protein. with the letters j, k, 1, m indicate homologous DNA regions flanking the a alleles. The b locus is a complex locus consisting of two divergently transcribed genes designated bE and bW. Each an identical organization to that of bE (Fig. 5): the b allele thus has two separate genes: bl contains bW1 first 130 amino acids comprise a variable region (46% identity) and the remainder of the polypeptide is bE1; b2 contains bW2 bE2, etc. highly conserved (96% identity) and contains a The bE2 gene was cloned as a DNA segment that is homeodomain motif 31. The bE and bW polypeptides sufficient to confer filamentous growth and tumorinducing ability on a diploid strain homozygous for are dissimilar, except for the four invariant amino bl (bW1 bE1/bW1 bED, which normally is capable acids (WF-N-R) of the homeodomain and a few other only of yeast-like growth and is nonpathogenic 27,28. residues in this region. The presence of two genes Different bE alleles contain an ORF of identical length, encoding dissimilar homeodomain proteins at the encoding a product of 410 amino acids. These products multiallelic b locus is similar to the organization at differ in their first 110 amino acids (the variable region; 63% identity), 260 bp bE whereas the rest of the polypeptide is bW highly conserved (the constant region; 41 90% identity) 28,29 (Fig. 5). The constant region contains a motif 2s related to the homeodomain of multicellular eukaryotes, a region k n o w n to mediate sequence-specific DNA binding30. HD HD Additional similarities in this region are shared with yeast regulatory pro(3 ] II W///A N N c teins (al and 0t2 of S. cerevisiae; matPi 130 aa 120 aa of Schizosaccharomyces pombe) 28 that govern cell type specificity (Fig. 6). It constant variable variable constant was thus originally proposed 2~ that bE polypeptides interact to create a DNA410 aa binding regulatory protein that governs 626 aa expression of genes for filamentous FIG~I growth and tumor induction. Deletion analysis to determine the functional Organization of the multiallelic b locus. The b locus, the major pathogenicity role of this regulatory protein led to determinant of U maydis, consists of two genes, bEand bll>; encoding polypeptides the identification of the adjacent bW of 410 and 626 amino acids, respectively. (The bEgene may contain an intron near its 3' end, which when spliced would result in a polypeptide of 473 amino acids2S.) gene. The bE and bW polypeptides show similar organization (a variable region and a The bW gene contains an ORF enconstant region) but no similarity at the amino acid level, except in the coding a polypeptide of 626 amino homeodomain-related motif (HD) present in the constant region. It is thought that acids that is different in the four self-nonself monitoring in the dikaryotic cell occurs by interaction of bE and bW strains so far analysed: bW1, bW2, polypeptides contributed by different b alleles, and results in formation of a bW3 and bW4 alleles are found in bl, heteromultimeric protein that positively regulates genes for filamentous growth and b2, b3 and b4 strains, respectively 31. tumor inductionS1 and negatively regulates genes for saprophytic growth Remarkably, the bW polypeptide has (F. Banuett, unpublished). TIC MAr 1992 VOL. 8 NO. 5 17"7

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FIG[] The homeodomain motif of the bE and bW polypeptides encoded by the b locus. The homeodomain region of the bE1 and bW1 polypeptides encoded by the b l allele is compared with the Drosophila proteins Antennapedia (Antp)43 and engrailed (en) 43 and with the following fungal regulatory proteins that govern cell type specificity: A0cY1 of Schizophyllum c o m m u n e 39, al (Ref. 43) and cz2 (Ref. 43) of Saccharomyces cerevisiae, and matPi of Schizosaccharomyces pombe43. The four invariant amino acids (WF-N-R) in all homeodomain proteins of multicellular eukaryotes are indicated by asterisks. Residues that are identical to those of the bE or bW polypeptide are in black boxes. Alignment of amino acids is based on the alignment of en and 0t2 (Ref. 44). Helices 1, 2 and 3 refer to helices of the homeodomain; helix 3 is the recognition helix30,44. The # symbol in the bE1 polypeptide indicates a loop of 16 amino acids. the multiallelic mating type loci of two other Basidiosuch a dimer could determine whether DNA recogmycete fungi (see below). nition helices (which correspond to helix 3 of the The presence of different b alleles is a prerequisite homeodomain30) are properly positioned to bind DNA to trigger pathogenic development. Do different bE or and thus whether a functional protein is generated. To different bW polypeptides or both turn on filamentous understand h o w the subunits might interact, imagine growth? Analysis of b E and b W mutants (Table 1) has that the variable regions of the dimer assume a helical conformation and face each other in an antiparallel led to the hypothesis that the regulatory protein produced by the b locus is a heteromultimer of bE and fashion. In the case of polypeptide helices from the bW subunits, with the additional restriction that bW same allele (for example, b E 1 and b W 1 ) , charged and bE must be from different b alleles31. The funcresidues on one face of a helix are proposed to face tional bW-bE heteromultimer appears to be a positive 'complementary' residues on the other helix, that is, regulator of genes for filamentous growth and tumor amino acids of opposite charge; or bulky residues induction, a conclusion drawn from the observation might be opposite compact residues. Such an arrangethat both gene products are needed to form filaments ment would pack the two variable helices close toand to induce tumors (Table 1) 31 . Monomers of bE and gether and, it is argued, would lead to improper posbW might interact in solution or bind separately to itioning of the DNA recognition helices and prevent DNA and interact once b o u n d binding to DNA. If, on the to DNA; the following discussion other hand, the helices are from T/,m~. 1. Deletion analysis o f the b locus polypeptides from different assumes that they associate in alleles (for example b E 1 and solution to form a dimer that Fuz/Tum b W 2 ) , 'noncomplementary' resibinds to a specific target site. (1) b W 1 bE1 + b W 2 bE2 + Because any combination of dues (of identical charge or (2) b W 1 bE1 + b W 2 bE2+ two different b alleles works, similar bulkiness) might face (3) b W 2 bE2 ~ + b W 2 bE2each other. This would prevent we need to explain h o w 25 dif(4) b W 1 bE1- + b W 2 bE2ferent bE monomers and 25 the variable helices from as(5) b W 1 bE1 + b W 2 - bE2 + sociating too closely with each different bW monomers can (6) b W 2 " bE,? + b W 2 - bE2 other and would result in form, in principle, 600 functional (7) b W 1 ~ bE1 + b W 2 - bE2 proper positioning of the DNA species (for example, bWl-bE2, (8) b W 1 bE1- + b W 2 - bE2 + recognition helices so that they bW3-bE7) and 25 nonfunctional can bind to DNA. species (bWl-bE1, bW3-bE3, Fuz, filament formation on charcoal medium: A precedent for interaction Turn, tumor induction on corn plants; -, no etc.). Presumably the variable refilament formation or tumor induction; ÷, filagion is responsible for selfof two very different polypepment formation and tumor induction nonself recognition - it might tides to create a functional proMating of a b W 1 bE1- strain with a b W 2 - bE2 tein is found in the al and 0t2 mediate association of monostrain (line 8) results in filament formation polypeptides of yeast. These mers or activity of the multimer and tumor induction indistinguishable from formed 2s. polypeptides form a heteromating between b W 1 bE1 with b W 2 bE2 meric protein (al-cz2) 3233, What are the rules by which (line 1). The results in this Table indicate that which is responsible for the a functional protein is generated? the b locus regulatory protein is a heterospecialized properties of the a/cz Consider a specific model in multimer consisting of bE and bW subunits, which a dimer is formed via an diploid cell. Other examples in which bW and bE are from different of protein-protein interactions association domain in the conb alleles31. The b W and bE mutations are chromosomal deletions of these genes31. are provided by the leucine stant region; interactions bezipper and the helix-loop-helix tween the variable regions of T1G MAY1992 VOL. 8 NO. 5

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[I~EVIEWS proteins34-37. The diversity of b W a n d bE sequences in U. maydis should provide a great opportunity to study determinants of protein-protein interactions. The mating type loci of two other Basidiomycetes, Schizophyllum commune and Coprinus cinereus, share several features with the b locus of U maydis. These fungi contain two unlinked genetic determinants, A and B, that govern dikaryon maintenance and fruiting body formation. Each is multiallelic and controls a different pathway of dikaryon morphogenesis. In order to activate the A pathway, cells that fuse must carry different alleles at A (which has two components, Acz or A[3); to activate the B pathway they must differ at B 3~39. The S. commune A0c locus consists of two divergently transcribed genes ( z and y)39 (C. Novotny and R. Ullrich, pers. commun.), which encode dissimilar homeodomain proteins. It is thought that z and y polypeptides contributed by different alleles form a heteromultimeric regulatory protein that is capable of activating the A pathway (C. Novotny and R. Ullrich, pers. commun.), a situation remarkably similar to that of the b locus of U. maydis. The A~ and A[3 loci o f C cinereus contain at least four homeodomain proteins. These comprise two classes of dissimilar homeodomain proteins whose interactions are proposed to result in formation of a regulatory protein that activates the A pathway of sexual development (L. Casselton, pers. commun.). Thus, self-nonself recognition in S. commune and C. cinereus also appears to involve interaction of dissimilar polypeptides. The f u z and rtf genes Regulation of the U maydis life cycle thus involves two major genetic determinants, one of which encodes components of a pheromone response pathway, and the other encodes a regulatory protein. Genes more directly involved in filamentous growth and tumor induction should exist downstream of a and b. Three new genes (fuzl, f u z 2 and rtfl) were recently identified 4° in mutants defective in filament formation (Fuz- mutants). The mutants obtained, for example, in an a l bl strain could not form filaments after mating to an a2 b2 strain 40. The f u z l and f u z 2 genes are unlinked to a or b; rtfl is near but separable from b. In addition to being necessary for filament formation, these genes are also required for other processes4°: f u z l is needed for teliospore formation, normal tumor response, and the pheromone response pathway, while f u z 2 is necessary for germination of the teliospore. The f u z 2 gene may be needed for cell wall breakdown or it may be a cytoskeletal component necessary for formation of the short filament upon germination. In rtfl- mutants, the requirement for different b alleles for tumor induction is bypassed. Co-infection of plants with a l bl and a2 bl strains that are both defective in rtfl yields a tumor response identical to that following co-infection with wild-type a l bl and a2 b2 strains. The rtfl product may be an inhibitor of tumor formation whose synthesis is turned off in the dikaryon by the b regulatory protein, thereby allowing tumor development 4°. Elucidation of the role of these genes in cell fusion, filamentous growth and pathogenicity is likely to

QuesadUlas de huitlacoche 1 lb of huitlacoche 1/4 finely chopped onion 1 clove garlic finely chopped 1 sprig of epazote (Chenopodium ambrosioides) 1 sprig of cilantro 2 chillis poblanos, roasted and cut into strips salt to taste Cook the onion and the garlic in vegetable oil until they are soft but not brown. Add chilli strips, epazote, cilantro (finely chopped), the roughly chopped buitlacoche and salt, and cook until the mixture is soft. Put a bit of this mixture in the center of an uncooked tortilla. Fold the tortilla in half. Fry in hot oil until it browns. Serve immediately. Accompany with any of a number of sauces or with guacamole. In the absence of tortillas the filling can be used to stuff thin crepes, which can then be covered with cream.

contribute to a better knowledge of cell biological processes and pathogenicity in U maydis. These genes may also be found in other fungi and provide experimental handles for studying other pathogens. Having sampled the intellectual challenges posed by U maydis during the course of its life cycle, let us now sample a dish of huitlacoche (see Box), whose delicacy parallels that of its fellow Basidiomycete, the chanterelles, and its Ascomycete cousins, the morels and truffles.

Acknowledgements

I thank Ira Herskowitz for discussion and comments on this manuscript, and Regine Kahmann, Charles Now~tny, Robert Ullrich and Lorna Casselton and members of their laboratories for communicating unpublished results.

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TIG MAY 1 9 9 2 VOL. 8 NO. 5

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[]~EVIEWS 3 2 Goutte, C. and Johnson, A. (1988) Cell 52, 875-882 3 3 Dranginis, A.M. (1990) Nature 347, 682-685 3 4 Landschulz, W.H., Johnson, P.F. and McKnight, S.L. (1988) Science 240, 1759-1764

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35 O'Shea, E.K., Klemm, J.D., Kim, P.S. and Alber, T. (1991) Science 254, 539-544 36 Murre, C. el al. (1989) Cell 58, 537-544 37 Lassar, A.B. et al. (1991) Cel166, 305-315 3 8 Casselton, L.A. (1978) in The Filamentous Fungi (Vol. 3) (Smith, J.E. and Berry, D.R., eds), pp. 275-297, Arnold 3 9 Novotny, C.P. et al. (1991) in More Gene Manipulations in Fungi (Bennett, J.W. and Lasure, L.L., eds), pp. 234-257, Academic Press 40 Banuett, F. (1991) Proc. Natl Acad. Sci. USA 88, 3922-3926 41 Day, P. and Anagnostakis, S. (1971) Nature New Biol. 231, 19-20 42 Kenaga, C.B., Williams, E.B. and Green, R.J. (1971) Plant Disease Syllabus, Bait Publishers, Lafayette 43 Scott, M.P., Tamkun, J.W. and Hartzell, G.W., Ill (1989) Biochim. Biophys. Acta 989, 25--48 44 Wolberger, C. et al. (1991) Cell 67, 517-528

F. BANUEITIS IN THE DEPARTMENTOF BIOCHEMISTRYAND BIOPHY$1C~ SCHOOLOFMEDICINE, UNIVERSITYOF CALIFORNIA SAN FRANaSC~ SAN FRANaSC¢~ CA 94143-0448~ USA.

I

T h e initiation and progression of malignant disease are generally thought to involve the accumulation of mutations in several genes (proto-oncogenes) with roles in the control of cellular proliferation 1. While much progress has been made in the past several years in unraveling the complex interactions among proto-oncogene products and other cellular proteins, particularly in various signal transduction pathways, a detailed understanding of how mutations cause cancer remains to be elucidated. One reason for the intense recent interest in the retinoblastoma gene (RB1) on human chromosome 13 is that mutations in both alleles of this gene induce tumor formation in the retina. The apparent simplicity of this model system suggests that understanding the function of the RB1 gene may provide important insights into the regulation of cellular proliferation. Young children with a germ-line mutation in one RB1 allele have a 95% chance of developing a retinoblastoma tumor in their eyes; in most instances, multiple tumors develop, affecting both eyes 2. Germ-line mutation in RB1 also predisposes to a discrete set of other tumors, and approximately 10% of patients develop osteosarcomas or fibrosarcomas. Analysis of epidemiological and clinical data led Knudson to recognize in 1971 that initiation of retinoblastoma tumors was dependent on only two mutations, the first of which, in hereditary cases, is a germ-line RB1 mutation3. Nonhereditary, unilateral retinoblastoma tumors arise when two somatic mutations occur in the same retinal cell. The predisposition to retinoblastoma was linked to chromosome 13ql4,,and restriction fragment length polymorphism (RFLP) analysis of chromosomes in retinoblastoma tumors revealed homozygous loss of function in both alleles of the RB1 locus 4. Since the TIC, MAY 1 9 9 2 ~1992 Elsc~ icr Science Publishers Ltd (I'K)

The retin0blast0ma protein and cell cycle regulation P.A. HAMEL, B.L. GAIHE AND R.A. pHIl.liPS Although the precise function of the retinoblastoma gene product, p l lO P~1, remains unknown, recent data suggest that it plays a role in the control of ceUular proliferation by regulating transcription of genes required f o r a cell to enter or stay in a quiescent or GO state, or f o r progression through the G1 phase of the cell cycle. However, it is difficult to rationalize the expression of p I 10 T M in a wide range of tissues with the fact that mutations in the RBI gene initiate cancers in a limited number of tissues.

presence of the gene product prevents the formation of retinoblastoma tumors, the RB1 gene has been called a 'tumor suppressor gene' or a 'recessive oncogene'. In 1986 Dryja, Friend and Weinberg cloned a cDNA corresponding to the RB1 geneS,6. The 4.7 kb transcript detected by their cloned cDNA is derived from the 27 exons of the RB1 gene, which spans 180 kb on chromosome 13 and contains two very large introns, of 35 kb and 70 kb. Using the cloned cDNA, several investigators confirmed the Knudson hypothesis by showing that all retinoblastoma tumors had mutations in both RB1 alleles ('-1°. Individuals with heritable retinoblastoma had a germ-line mutation in the RB1 gene, affecting either the coding region9 or the promoter region n. The open reading frame of 928 amino acids (Fig. 1) predicted a protein of 110 kDa, but sequence analysis provided no clues to the function of RB1. Analysis of VOL. 8 NO. 5

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