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Vegetative incompatibility in filamentous fungi: het genes begin to talk
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Self-nonself recognition between somatic cells is common to all living organisms. It is exemplified in higher eukaryotes by graft rejection, but also occurs in lower eukaryotes such as protists and fungi 1. The mechanisms involved in somatic incompatibility are highly diverse. In animals, the recognition of somatic cells is mediated by direct cellular interactions and/or humoral responses that have been extensively studied. However, in most other organisms, mechanisms of incompatibility are not well understood, although they are all associated with at least one genetic difference between incompatible cells. In filamentous fungi, anastomosis between somatic cells from different strains is controlled by a process described as vegetative or heterokaryon incompatibility, which occurs very frequently when two different strains fuse. The genetics of vegetative incompatibility has been studied in many fungal species z and recent advances in recombinant DNA technology in these organisms have provided an opportunity to investigate this process at the molecular level. Mechanisms and roles of vegetative incompatibility Filamentous fungi are haploid organisms that grow as mycelia: that is, networks of multinucleated cells known as hyphae or filaments (Fig. 1). Within these mycelia, there are frequent anastomoses between filaments; such associations also occur when two mycelia grow close to each other. Fusion between hyphae from different strains leads to the formation of heterokaryotic filaments that contain a mixture of the nuclei of both parental strains in a common cytoplasm. It is frequently impossible to obtain stable heterokaryotic strains by fusing different wild-type isolates from the same species. This phenomenon has been described as vegetative or heterokaryon incompatibility. In many species, incompatibility between strains can be detected by the presence of a barrageS, an abnormal contact in the region where the incompatible mycelia fuse (Fig, 2). Incompatibility can also be detected using mutant markers to force heterokaryosis between two strains 4. When the strains are incompatible, either no heterokaryon is formed at all or if heterokaryons do grow, they do so very poorly and have an abnormal phenotype. From morphological, cytological and genetic observations it was deduced that failure to form stable heterokaryons can be mediated by different mechanisms. In most cases, the hyphae of incompatible strains can fuse but the heterokaryotic cells are rapidly destroyed by a lytic and degenerative reactions (Fig. 3). A similar lethal reaction has also been described in Myxomycetes after plasmodia have fuse#. In other cases, even though the filaments themselves cannot fuse, stable heterokaryons can be obtained by protopL~st fusion; this suggests that the cell wall can modulate cell recognition TM. The instability of heterokaryons can sometimes be the consequence of a postfusion event, such as the specific loss of one of the two parental nuclei9. The diversity of the phenotypic expression of incompatibility suggests that several different molecular mechanisms may be involved. The role of vegetative incompatibility in the biology of fungal populations is still unclear because the
Vegetative incompatibility in filamentous fungi: bet genes begin to talk JOELBEGUERET,B~I'IUCE 'r~CQ AND comm~ oAvt Somatic or vegetative incompatibility is widespread itt fllamentous fungg It prevents the coexistence of g ~ different nuclei wtthitt a common cytoplasnt Ck~ng the bet gettes that coRtrolthis process has beemachieved in several species. This has provided essential informatiott on thefmgtton of the genes in. the biology offungi and has also led to theformulattcm of models that may explaiB similar phenomeu in other organisms.
quantitative importance of heterokaryosis in nature is unknown. In Basidiomycetes and some other fungi in which specialized sexual organs do not differentiate, heterokaryosis is the first step in the sexual phase and is under the control of mating-type (mr) genes. The role of heterokaryosis in other fungi is less certain. The heterokaryotic state can provide some of the advantages of diploidy, such as the masking of recessive, deleterious alleles and an increased ability to adapt to environmental change 1°. Heterokaryosis also provides an opportunity for somatic genetic recombination, which is particularly important in imperfect fungi that have lost their sexuality. If heterokaryon formation is required for genetic recombination, then vegetative incompatibility obviously limits outbreeding and genetic polymorphism within the species. Another possible role of vegetative incompatibility in nature has been revealed by analysing the transfer of cytoplasmic factors in some fungal populations. In the chestnut blight ascomycete Cryphonectria parastttca, hypovirulence is related to the presence of a virus-like double-stranded RNA1], which can be transmitted through populations by heterokaryosis between strains. However, the efficiency of transfer is highly reduced between incompatible strains 12. The limitation of this horizontal transfer may be effective not only under laboratory conditions but also in natural populations. Biocontrol of this pathogen by spreading hypovimlent strains has been more effective in Europe than in North America. This difference has been tentatively related to differences in the number of incompatibility groups in the two geographical areas 13. There is evidence from several other species that the efficiency of the horizontal transfer of cytoplasmic genetic elements such as mitochondria, plasmids and stable RNA is reduced between incompatible strains btas. This may be an important means of protecting strains of natural populations against invasion by harmful cytoplasmic genetic elements. Genetics of vegetative incompatibility Genetic control of vegetative incompatibility has been well studied in several species of Ascomycetes and always results from genetic differences at one or
TIG DECEMBER1994 VOL. 10 No. 12
]REVIEWS Perithecium
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Karyogarny~ . Diploid nucleus ,,)tt, n+n-2n ~ /'(.~~ / /" ) _ ""., ~ (+) / ,~~, ft~ - ) Ascogoniurn i ~ ~ 4'/ n
""',,,,,,. ,/ Protoperitheolum n+n (dikaryon) hntm 1, The life cycle of Podospora anserfna, Each mycelium differentiates male and female sexual organs but no asexual repro. ductive organ, P, anserfna is a pseudohomothallicascomycetewith two mating types, mat+and mat-, After t~rtilizattonattd meiosis, each peritheclum contains about a hundred ascl, each of which encloses four blnucleated ascopores that can be propagated, As the mating-typelocus is far from the centromere, the two nuclei of each ascospore carry the opposite mating types, Germination of binucleated ascospores produce myceliathat can be self-fertilized.However, in a few asct, one binudeated spore is replaced by two smaller unlnucleated spores. These ascospores contain either a mat+ or mat- locus. They produce mycelia that must be cross. fertilized to achieve the sexual cycle. This is the situation that has been represented in the figure. more so-called bet loci, For compatibility to occur, the alleles at all the her loci must be identical; a single difference at one bet locus leads to what is referred to as heterogenic incompatibilityt6, Genetic analysis of wild-type isolates has revealed that the number of bet loci is generally high: eleven in Neurospora crassa; nine in Podospora anserfna; eight in Aspergiilus ntdulans; and at least five in C, parasittca t7-20, The existence of numerous bet loci in imperfect fungi was deduced from tile high number of vegetative compatibility groups present in these species. Since such fungi are often pathogens, these compatibility groups are useful marRers to study population diversity and dynamics 2t, Genes present at the bet loci can be involved in allelic or nonallelic interactions. In allelic systems, incompatibility is triggered by alternate alleles present at a single bet locus. In most cases, only two alternative alleles have been identified in natural isolates; however,
multiailelic betloci have been described in A, ntdulans2Z. In nonallelic interactions, such as those that occur in P. anserina and C, parasitica, a specific combination of alleles at two separate loci results in incompatibility, When nonallelic genes are involved, the offspring of crosses between incompatible strains include individuals in which the two antagonistic genes are combined in a single haploid nucleus, which leads to a lethal phenotype. The growth of these 'self-incompatible' strains is inhibited soon after ascospore germination when the cells are destroyed by a lytic reaction tg. Vegetative incompatibility genes generally do not interfere with sexual reproduction. However, in some Ascomycetes like N. crassa or Ascobolus immersus, mt alleles have a dual function. Mating only occurs between strains with genotypes that differ at the mt locus; however, these alternative mt alleles also function as bet genes during the vegetative phase. In N. crassa,
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REVIEWS mating function and vegetative incompatibility cannot be dissociated by recombination 17. In P. anserina and some other species, the fertility of crosses between some incompatible strains is reduced or abolished, suggesting the existence of an incompatibility reaction when the sexual cells fuse TM. Mutations that relieve incompatibility have been described. In N. crassa, most mutations at the mt locus abolish both fertility and vegetative incompatibility. When mutant loci were sequenced it was found that they contained nucleotide insertions or deletions within the open reading frame that resulted in the expression of truncated polypeptides. The polypeptides encoded by the mating-type genes mt a-1 and mt A-1 are responsible for both mating functions and vegetative incompatibility. Although a mutant containing a missense mutation that changes an arginine residue at position 258 in MT(x-1 to a serine is insensitive to incompatibility, it is still fertile, indicating that incompatibility resulting from an interaction between a and A is not required for the completion of the sexual phase 23. A similar conclusion can be drawn from mutations at the tol locus in Neurospora, which abolish vegetative incompatibility in A/a interactions but do not affect mating functions. Incompatibility resulting from interactions between other bet genes is not suppressed by tol mutations tT. In Neurospora, a large set of intra- and extragenic mutations that confer tolerance between incompatible strains has recently been identified by spontaneous and insertional mutagenesis. Suppressor mutants are compatible in spite of genetic differences at o n e o r m o r e bet loci 24. In P. anserina, mutations that permit the growth of lethal self-incompatible strains have been extensively characterized, These mutations occur either at one of the two antagonistic her loci, leading to inactive mutant alleles 25, or at distinct loci in suppressor (or modifier) rood gene# 6. In this species, strains containing rood mutations display defective pmtoperithecia formation and pigmentation, which suggests that rood genes have an essential function in the life cycle of the fungus as well as eliciting vegetative incompatibility27,2s.
In N. crassa, chromosome walking and RFLP analyses have been used to clone alleles of the bet-c and her-6 loci (G. Kuldau and M. Smith, pers. commun.). Several bet genes and suppressors of incompatibility have also been cloned from strains containing insertional mutations z't.
Properties of bet gene products The mt locus of N. crassa The fwst bet genes for which molecular information became available were A and a from N. crassa. These two mt alleles were found to be idiomorphs. The mt a-1 and mt A-1 genes encode dissimilar polypeptides
that are predicted to be transcriptional regulators23,32. Although mating depends upon the ability of the MT a-1 polypeptide to bind a specific DNA sequence, vegetative incompatibility is not affected by mutations that suppress this DNA binding33. Therefore, mt a-1 and mt A-1 may function by a biochemically distinct mechanism in mating and vegetative incompatibility. The bets locus of P. anserfna
Two incompatible alleles, bets and bet-S, have been described at the bets locus in P. anserina. They encode a protein of 289 amino acids that has no similarity to any previously identified protein. Strains in which the bets locus have been inactivated by gene disruption have a neutral incompatibility phenotype and are compatible with strains containing bets or bet-S. This confirms that the polypeptide encoded by this locus is responsible for triggering vegetative incompatibility and cell death when hyphae of bets and betS strains fuse. The mutant strains show a wild-type phenotype: their growth, differentiation of male and female reproductive organs, and fertility are not affected by inactivation of the bets locus. Therefore, expression of this locus is not essential for cell viability and completion of the life cycle of the fungu# 't. The possibility that inactivation of bets could be complemented by an homologous gene was not ruled out.
Clonl~ hot series The biochemical basis of vegetative incompatibility has not yet been elucidated. However, specific expression of proteolytic enzymes and phenoloxidases during the incompatibility reaction has been described in P. anserina, and models for a role of incompatibility genes in the life cycle of the fungus have been proposed 29. The recent cloning of bet genes has thrown light on their function and the molecular mechanism of the incompatibility response. Three bet loci, bets (Ref. 30), bet-c (Ref. 31) and het-e (S. Saupe, B. Turcq and J. B~gueret, unpublished), have now been cloned in P. anserina. One allele of each locus was cloned from cosmid libraries after the allele had been expressed in a recipient strain containing a neutral allele at the locus under study. The transformants that expressed the relevant bet genes were identified by the presence of a bat~ge reaction with strains containing incompatible her genes. Other alleles of these loci were isolated from genomic libraries by hybridization with the cloned genes as probes, or by PCR.
Fmt~ 2. Barrage between incompatiblestrainsof Podospora anserina. The strains were grown on solid corn meal agar. A contact between two incompatible strains is shown by the solid black arrow. The thick, dark myceliumline results fi'om abundant regeneration of apical hyphae after the death of heterokaryoticcells. The other arrow shows a contact between compatible strains.
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FtGtam3. A view of the incompatibilityreaction at the cellular level in Podosporaanserlna. Top: Two hyphae of incompatible strains have just fused at the position markedby the arrow. Bottom:The same hyphae were examined 20 rain after the fusion. Note the high content of vacuoles in the cytoplasm,which is the first symptomof cell death. (Kindlyprovided
by J, Beisson,) Polypeptides encoded by the alternate alleles het-s and bets differ by 14 amino acids, The molecular basis of allele specificity has been investigated to determine how many and which amino acid differences are necessary to trigger incompatibility, This was achieved by constructing chimaeric bet.a-.bet.S genes or modifying codons by site-specific mutagenesis. The incompatibility phenotype conferred by the new alleles was determined by transforming a recipient strain containing an inactive bets allele, and then testing the transformants with bets and bets strains, It was found that the amino acid at position 33 is proline in the polypeptide encoded by bets but histidine in that encoded by betS, A mutation in bets that converts this histidine to proline also converts bets to an allele that confers bets specificity. So, it has been proved that a single amino acid difference at this position in the protein encoded by the bets locus is sufficient to elk'it incompatibility between two strains35. The bet-e and bet-c genes of P. anserina Different alleles of the bet-c and bet-e loci have also been cloned. These loci are multiaUelic and are involved in nonallelic incompatibility, The bet-e locus contains an open reading frame encoding a polypeptide of 1356 amino acids that shows two interesting structural features (S. Saupe, B. Turcq and J. B6gueret,
unpublished). The carboxy-termina! region of the protein contains repeats of 42 amino acids homologous to those found in all [3 subunits of trimeric G proteins36. These repeats are also found in various proteins with different functions, and may be involved in proteinprotein interactions37. The number of these repeats varies from 3 to 12 according to the allele present at the bet-e locus. Such a variation in the repeat number has not been reported for other proteins of this type. The amino-terminal region contains amino acid stretches that match GTP-binding consensus sequences of GTPases. If an essential amino acid in the GTP-binding domain is mutated, a neutral allele that has lost its reactivity in incompatibility is produced, suggesting that the GTP-binding activity of the Het-E polypeptide is essential to trigger the lethal reaction. The polypeptide encoded by the bet-e locus thus shows structural features of ct and J3 subunits of trimeric G proteins, and may define a new class of proteins involved in the transduction of cellular signals. Four wild-type alleles of the bet-c locus that display different incompatibility specificities against the different alleles of the antagonistic het-e locus have been isolated. They encode a protein of 208 amino acids similar to a protein from the pig brain that catalyses the exchange of glycolipids between cellular membranes. The proteins encoded by these different alleles differ by between 1 and 15 amino acids (S. Saupe, B. Turcq and J. B(~gueret, unpublished). As for the bets locus, limited sequence differences at the bet-c locus confer differential specificity in incompatibility. Inactivation of the het-c locus by gene disruption drastically affects ascospore production. In self crosses of the gene-disrupted strain, perithecia contain many aborted asci and the few remaining ascospores are highly heterogeneous in size, suggesting that the distribution of nuclei during the postmeiotic division is abnormal3t. The properties of the bet-c mutant suggest that a gene involved in vegetative incompatibility is essential for the life cycle of the fungus.
Conclusion The function of vegetative incompatibility in natural populations of fungi is still an open question, although its widespread occurrence suggests that it is important in the biology of these organisms. Different hypotheses have been proposed for the role of bet genes in the population biology of fungiz. As mentioned above, incompatibility limits horizontal infection by extrachromosomal elements that could be deleterious. It has also been suggested that the presence of bet genes creates a basis for evolution by limiting outbreeding and favouring the evolution of isolated groups within a specie#. If prevention of heterokaryosis is beneficial in natural populations, bet genes may have been selected and evolved essentially to limit heterokaryon formation. The properties of genes present at cloned bet loci in P. arisen'ha and at the mt locus in N. crassa suggest another biological role for vegetative incompatibility. It is possible that bet genes have primary cellular functions and that the polypeptides encoded by these genes are active as homo- or heteromeric protein complexes according to whether they are involved in allelic or
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nonallelic incompatibility, reAllelic incompatibility spectively. Since gene evolution in wild-type populations Strain genotype Complexes formed between hetgene products has led to sequence divergence, the coexpression of het-s incompatible genes could pross duce abnormal protein complexes that cause lethal disorders in metabolism or genetic exhot-S pression. Indeed, as described SS above, there are several known Heterokaryon examples of a single amino acid difference in polypeptides ss SS sS encoded by bet genes having a significant effect on their ability Nonallelicincomp~ibili~ to elicit incompatibility through interaction with other allelic or hehChehe nonallelic genes. Ce Thus, it is possible that mutations can create new alleles that elicit an incompatibility he~che~E reaction through specific genetic cE interactions. This hypothesis is Heterokaryon supported by genetic analyses het-Chet.e/het-chet.E [ ~ WAD ~ I~ in P. anserina, in which vegCe ce cE CE etative incompatibility can arise from an interaction between at least two mutated genes that Fmum 4. Models for poison complexes formed by interactions between the products of are different from all other bet incompatible bet genes. In a heterokaryoticcell containing two allelicincompatiblegenes, genes identified in wild-type three differentcomplexes could be formed between the products of the genes. The function isolates~. This suggests that of the heteromericcomplex would be deleterious and dominant over the normal function the number of genes potentially performed by homomeric complexes. When two nonallelic incompatiblegenes such as involved in vegetative incom- hel-Cand het-Eare coexpressed within the heterokaryoticcell, the complex formed by the products of the antagonisticgenes would also be poisonous. patibility may be higher than (Modified from Ref. 41,) that detected by genetic analysis of wild-type isolates, We propose an hypothesis to account for vegetative Acknowledgements We thank A. Groppi for helping with the figures. incompatibility in which abnomml complexes between the products of incompatible genes are lethal to the cell, This 'poison complex' model would then be References I Esser, K. and Blaich, R, (1973) Adv. Genet. 17, 107-152 analogous to that proposed in yeast39 and Drosophaa 4o 2 Glass, N,L and Kuldau, G.A. (1992) Annu. Rev. to explain nonallelic noncomplementation between Phytopathol. 30, 201-224 mutations in different genes encoding components of 3 Rizet, G. (1952) Rev, Cytol. Btol. Veget. 13, 51--92 the cytoskeleton, This hypothesis for allelic and non4 Gamjobst, L and Wilson, J.F. (1956) Proc. NatlAcad. $ci. allelic incompatibility is summarized in Fig. 4. The USA 42, 613-618 model is similar to that proposed by Steams and 5 Beisson-Schecroun,J. (1962) Ann. Genet. 4, 3--50 Botstein 4t to explain allele-specific noncomplemen6 Caflile,J.M. and Dee, J. (1967) Nature 215, 832--834 tation between the tub1 and tub2 mutations in yeast. 7 Dales, R.B. and Croft,J.H. (1977) FEMSMicrobiol. Left. 1, However, for bet genes, poison complexes would be 201-203 the consequence of the coexpression of natural variant 8 Typas, M.A. (1983) J. Gen. Microbiol. 129, 3043-3056 genes in heterokaryotic cells. This hypothesis offers an 9 Pittenger, T.H. and Browner, T.G. (1961) Genetics46, 1645-1663 explanation for the abnormal growth or lethality of hybrid offspring produced by interacial crosses or ?,O Jinks, J.L. (1952) Proc. R. $oc. London Ser. B 140, 83-99 crosses between related species. Abnormal hybrids fre- I I Nuss, D.L. and Koltin, Y. (1990) Annu. Rev. Pbytopathol. 28, 37-58 quendy occur in #ants and animals42; these have been 12 Anagnostakis, S.L. (1982) Science215, ~ 7 1 ascribed to the presence in parents of complementaw lethal genes that could be similar to her genes 13 Anagnostakis,S.L., Hau, B. and Kranz.J. (1986) Plant Dis. 70, 536--538 in fungi, 14 Caten, C.E. (1972)./. Gen. Microbiol. 72. 221-229 Characterization of her genes from other fungi is 15 Del~ts, F., Yang, X. and Grifflths, A.J. (1994) Curt. Geoet. now under way. Such studies will hopefully provide 26, 113-119 information to answer some general questions about 16 Esser, K. (1971) Mol. Gen. Genet. 110, 86-100 the molecular basis of prevention of heterokaryosis, the 17 Perkins, D.D. (1988) Fungal Genet. News35, 44--46 mechanisms of cell death, and also about the function /8 Bemet,J. (1965) Ann. SoL Nat. Bot. 6, 611-768 19 Jinks, J.L. and Grindle, M. (1963) Heredity 18. 407.-.411 of these genes in individuals and populations. TIG DECEMBER1994 VOL. 10 NO. 12
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REVIEWS 20 Anagnostalds, S.L. (1982) Genetics 102, 25-28 21 Leslie,J.F. (1993) Annu. Rev. Pbytopathol. 31, 127-150 22 Dales, R.B., Moorhouse, J. and Croft, J.H. (1993) Heredity 70, 537-543 23 Staben, C. and Yanofsk'y, C. (1990) Proc. Naa Acad. Sci. USA87, 4917--4921 24 Arganoza, M.T., Ohrnberger, J., Min, J. and Akins, R.A. (1994) Genetics 137, 731-742 25 B~gueret, J. (1969) C. R. Acad. Sci. Paris 269, 458.-461 26 Belcour, L. and Bernet, J. (1969) C. R. Acad. Sct. Paris 269, 712-714 27 Boucherie, H. and Bemet, J. (1980) Genetics96, 399--411 28 Durrens, P. and Bemet, J. (1982) Curr. Genet. 5, 181-186 29 Bemet, J. (1992) Heredity 68, 79--87 30 Turcq, B., Denayrolles, M. and B~gueret, J. (1990) Curr. Genet. 17, 297-303 .5/ Saupe, S., Descamps, C., Turcq, B. and B~gueret, J. (1994) Proc. Naa Acad. Sci. USA 91, 5927-5931 32 Glass, N.L., Grotelueschen, J. and Metzenberg, R.L. (1990) Proc. Natl Acad. Sci. USA 87, 4912-4916 33 Philley, M.L. and Staben, C. (1994) Genetics 137, 715-722
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34 Turcq, B., Deleu, C., Denayrolles, M. and B6gueret, J. (1991) Mol. Gen. Genet. 228, 265-269 .55 Deleu, C., Clav6, C. and B~gueret, J. (1993) Genetics 135, 45-52 .56 van der Voom, L. and Ploegh, H.L. (1992) FEBS Lett. 307, 131-134 .57 Duronio, R.J., Gordon, J.I. and Boguski, M.S. (1992) Proteins 13, 41-56 .58 Delettre, Y.M. and Bemet, J. (1976) Mol. Gen. Genet. 144, 191-197 .59 Vinh, D.B.N. et aL (1993) Genetics 135, 275-286 40 Hays, T.S. et al. (1989) Mol. Cell. Biol. 9, 875--884 41 Stearns, T. and Botstein, D. (1988) Genetics 119, 249-260 42 Stebbins, G.L. (1958) Adv. Genet. 9, 147-162
.I. B~GUERLrl;, a TURLT~ AND C. CLAV~ ARE IN THE LABORATOliIE DE G~N~QUE ~T BIOLOGI~ M O L ~ L ~ nES CHA~PIGNON~ UPR CNRS 9 0 2 ~ UNn~tSn~ nR B o l m ~ u X !!, AVENUEDES FA~LTES 33400, FRANC~.
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Physician to the Gene Pool: Genetic Lessonsand Other Stories by James v. Neel John Wiley & Sons, 1994, $24.95/£18,95 hbk (457 pages) ISBN 0 471 30844 7 The flyleaf describes this book as an 'enthralling memoir by a founding father of human medical genetics which is more than just a travelogue and which challenges the received genetic wisdom of the day'. It is in fact a tour de force, an exceptional book written by an exceptional man. Jim Neel writes as he speaks, clearly with careful marshalling of the facts backed by deep thought and leading to forceful argument and logical conclusions, and all spiced with anecdotes reflecting a puckish sense of humour. He is indeed one of the pioneers in human genetics and his 'travelogue' traces his interests, frustrations and achievements over a half-century during which enormous advances in our understanding of human genetics have been made, an understanding to which he himself has made many contributions. The early part of the book is a fascinating account of the young scientist who took his initial PhD training as a Drosophila geneticist under Curt Stern and who 'agonized over the decision to turn my back on the hard genetics of Drosophila and enter the soft and minted [at that time by the eugenics movement] field of human genetics'. This he did by first taking a medical degree, which was followed by an 18-month military posting
to the atom-bombed cities of Hiroshima and Nagasaki, and thence to a post at the University of Michigan where he has been based ever since. Neel's original posting to Japan was to assist a group set up to advise the National Academy of Sciences on a possible research programme into delayed effects of the atomic bombings on radiation-exposed survivors, He ended up being responsible for the design and organization of the long-term genetic follow-up studies and continues his interest in the programme to this day. The enormous efforts of the American and Japanese scientists involved have provided us with by far the most comprehensive data on the effects of ionizing radiations in producing mutations in the germ cells of exposed humans. This work shows that we are not as sensitive to radiation-induced mutations as was once thought: in both mouse and humans, the chronic radiation dose needed to double the rate of spontaneous background mutation in germ cells is around 4 Sv, a dose some two orders of magnitude greater than that received from normal background radiation over the human reproductive lifetime. With his interests and expertise Neel was, not surprisingly, called upon as a member of a number of committees TIG DECEMBER1994 VOL. 10 NO. 12
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dealing with the assessment of genetic risk. He writes amusingly of confrontations in committee between Sewali Wright and HJ. (Joe) Muller, and comments scathingly on the regulatory authorities, particularly with regard to radioactive waste depositories with the proposed 'risk management bearing precious little relation to the risk assessment'. The relatively high frequency of marriages between first cousins amongst the Japanese provided Neel with an excellent opportunity to study the effects of inbreeding on mortality, morbidity and fertility, studies that he later extended in Brazil. His interest in the human gene pool, which had been engendered by his early studies on sickle cell anaemia and thalassaemia and nurtured in Japan, led Neel to mount a series of field trips into the depths of the Brazilian forests. The aim was to study one of the few remaining primitive populations of the wodd in terms of mutation, selection and population structure. Neel's descriptions of the organization required for these forays and accounts of life (and death) in the Amerindian villages are vivid portraits. These studies provided a wealth of information on genetic differences between different Yanonama villages for both common and rare polymorphisms and much is made of this 'microdifferentiation' in discussing human evolution. In his travels, Neei has pursued genes associated with sickle cell disease in Africa, mutations in Japan and gene pools in South America, and in this book 'looks at the current human genetic condition
Self-nonself recognition between somatic cells is common to all living organisms. It is exemplified in higher eukaryotes by graft rejection, but also occurs in lower eukaryotes such as protists and fungi 1. The mechanisms involved in somatic incompatibility are highly diverse. In animals, the recognition of somatic cells is mediated by direct cellular interactions and/or humoral responses that have been extensively studied. However, in most other organisms, mechanisms of incompatibility are not well understood, although they are all associated with at least one genetic difference between incompatible cells. In filamentous fungi, anastomosis between somatic cells from different strains is controlled by a process described as vegetative or heterokaryon incompatibility, which occurs very frequently when two different strains fuse. The genetics of vegetative incompatibility has been studied in many fungal species z and recent advances in recombinant DNA technology in these organisms have provided an opportunity to investigate this process at the molecular level. Mechanisms and roles of vegetative incompatibility Filamentous fungi are haploid organisms that grow as mycelia: that is, networks of multinucleated cells known as hyphae or filaments (Fig. 1). Within these mycelia, there are frequent anastomoses between filaments; such associations also occur when two mycelia grow close to each other. Fusion between hyphae from different strains leads to the formation of heterokaryotic filaments that contain a mixture of the nuclei of both parental strains in a common cytoplasm. It is frequently impossible to obtain stable heterokaryotic strains by fusing different wild-type isolates from the same species. This phenomenon has been described as vegetative or heterokaryon incompatibility. In many species, incompatibility between strains can be detected by the presence of a barrageS, an abnormal contact in the region where the incompatible mycelia fuse (Fig, 2). Incompatibility can also be detected using mutant markers to force heterokaryosis between two strains 4. When the strains are incompatible, either no heterokaryon is formed at all or if heterokaryons do grow, they do so very poorly and have an abnormal phenotype. From morphological, cytological and genetic observations it was deduced that failure to form stable heterokaryons can be mediated by different mechanisms. In most cases, the hyphae of incompatible strains can fuse but the heterokaryotic cells are rapidly destroyed by a lytic and degenerative reactions (Fig. 3). A similar lethal reaction has also been described in Myxomycetes after plasmodia have fuse#. In other cases, even though the filaments themselves cannot fuse, stable heterokaryons can be obtained by protopL~st fusion; this suggests that the cell wall can modulate cell recognition TM. The instability of heterokaryons can sometimes be the consequence of a postfusion event, such as the specific loss of one of the two parental nuclei9. The diversity of the phenotypic expression of incompatibility suggests that several different molecular mechanisms may be involved. The role of vegetative incompatibility in the biology of fungal populations is still unclear because the
Vegetative incompatibility in filamentous fungi: bet genes begin to talk JOELBEGUERET,B~I'IUCE 'r~CQ AND comm~ oAvt Somatic or vegetative incompatibility is widespread itt fllamentous fungg It prevents the coexistence of g ~ different nuclei wtthitt a common cytoplasnt Ck~ng the bet gettes that coRtrolthis process has beemachieved in several species. This has provided essential informatiott on thefmgtton of the genes in. the biology offungi and has also led to theformulattcm of models that may explaiB similar phenomeu in other organisms.
quantitative importance of heterokaryosis in nature is unknown. In Basidiomycetes and some other fungi in which specialized sexual organs do not differentiate, heterokaryosis is the first step in the sexual phase and is under the control of mating-type (mr) genes. The role of heterokaryosis in other fungi is less certain. The heterokaryotic state can provide some of the advantages of diploidy, such as the masking of recessive, deleterious alleles and an increased ability to adapt to environmental change 1°. Heterokaryosis also provides an opportunity for somatic genetic recombination, which is particularly important in imperfect fungi that have lost their sexuality. If heterokaryon formation is required for genetic recombination, then vegetative incompatibility obviously limits outbreeding and genetic polymorphism within the species. Another possible role of vegetative incompatibility in nature has been revealed by analysing the transfer of cytoplasmic factors in some fungal populations. In the chestnut blight ascomycete Cryphonectria parastttca, hypovirulence is related to the presence of a virus-like double-stranded RNA1], which can be transmitted through populations by heterokaryosis between strains. However, the efficiency of transfer is highly reduced between incompatible strains 12. The limitation of this horizontal transfer may be effective not only under laboratory conditions but also in natural populations. Biocontrol of this pathogen by spreading hypovimlent strains has been more effective in Europe than in North America. This difference has been tentatively related to differences in the number of incompatibility groups in the two geographical areas 13. There is evidence from several other species that the efficiency of the horizontal transfer of cytoplasmic genetic elements such as mitochondria, plasmids and stable RNA is reduced between incompatible strains btas. This may be an important means of protecting strains of natural populations against invasion by harmful cytoplasmic genetic elements. Genetics of vegetative incompatibility Genetic control of vegetative incompatibility has been well studied in several species of Ascomycetes and always results from genetic differences at one or
TIG DECEMBER1994 VOL. 10 No. 12
]REVIEWS Perithecium
Uninucleated (+) asoosporogermination
Uninucleated (-) ascosporegermination
;
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(-) Mycelium
i~'~ `cr°c°nloiumMeiosis~ ~
(+'Mlcr°con~.a~~¢~~
Fe ""z "
Fertilization J (~.
N~,~) (.) ~ Ascogonium
~
n
~
Karyogarny~ . Diploid nucleus ,,)tt, n+n-2n ~ /'(.~~ / /" ) _ ""., ~ (+) / ,~~, ft~ - ) Ascogoniurn i ~ ~ 4'/ n
""',,,,,,. ,/ Protoperitheolum n+n (dikaryon) hntm 1, The life cycle of Podospora anserfna, Each mycelium differentiates male and female sexual organs but no asexual repro. ductive organ, P, anserfna is a pseudohomothallicascomycetewith two mating types, mat+and mat-, After t~rtilizattonattd meiosis, each peritheclum contains about a hundred ascl, each of which encloses four blnucleated ascopores that can be propagated, As the mating-typelocus is far from the centromere, the two nuclei of each ascospore carry the opposite mating types, Germination of binucleated ascospores produce myceliathat can be self-fertilized.However, in a few asct, one binudeated spore is replaced by two smaller unlnucleated spores. These ascospores contain either a mat+ or mat- locus. They produce mycelia that must be cross. fertilized to achieve the sexual cycle. This is the situation that has been represented in the figure. more so-called bet loci, For compatibility to occur, the alleles at all the her loci must be identical; a single difference at one bet locus leads to what is referred to as heterogenic incompatibilityt6, Genetic analysis of wild-type isolates has revealed that the number of bet loci is generally high: eleven in Neurospora crassa; nine in Podospora anserfna; eight in Aspergiilus ntdulans; and at least five in C, parasittca t7-20, The existence of numerous bet loci in imperfect fungi was deduced from tile high number of vegetative compatibility groups present in these species. Since such fungi are often pathogens, these compatibility groups are useful marRers to study population diversity and dynamics 2t, Genes present at the bet loci can be involved in allelic or nonallelic interactions. In allelic systems, incompatibility is triggered by alternate alleles present at a single bet locus. In most cases, only two alternative alleles have been identified in natural isolates; however,
multiailelic betloci have been described in A, ntdulans2Z. In nonallelic interactions, such as those that occur in P. anserina and C, parasitica, a specific combination of alleles at two separate loci results in incompatibility, When nonallelic genes are involved, the offspring of crosses between incompatible strains include individuals in which the two antagonistic genes are combined in a single haploid nucleus, which leads to a lethal phenotype. The growth of these 'self-incompatible' strains is inhibited soon after ascospore germination when the cells are destroyed by a lytic reaction tg. Vegetative incompatibility genes generally do not interfere with sexual reproduction. However, in some Ascomycetes like N. crassa or Ascobolus immersus, mt alleles have a dual function. Mating only occurs between strains with genotypes that differ at the mt locus; however, these alternative mt alleles also function as bet genes during the vegetative phase. In N. crassa,
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REVIEWS mating function and vegetative incompatibility cannot be dissociated by recombination 17. In P. anserina and some other species, the fertility of crosses between some incompatible strains is reduced or abolished, suggesting the existence of an incompatibility reaction when the sexual cells fuse TM. Mutations that relieve incompatibility have been described. In N. crassa, most mutations at the mt locus abolish both fertility and vegetative incompatibility. When mutant loci were sequenced it was found that they contained nucleotide insertions or deletions within the open reading frame that resulted in the expression of truncated polypeptides. The polypeptides encoded by the mating-type genes mt a-1 and mt A-1 are responsible for both mating functions and vegetative incompatibility. Although a mutant containing a missense mutation that changes an arginine residue at position 258 in MT(x-1 to a serine is insensitive to incompatibility, it is still fertile, indicating that incompatibility resulting from an interaction between a and A is not required for the completion of the sexual phase 23. A similar conclusion can be drawn from mutations at the tol locus in Neurospora, which abolish vegetative incompatibility in A/a interactions but do not affect mating functions. Incompatibility resulting from interactions between other bet genes is not suppressed by tol mutations tT. In Neurospora, a large set of intra- and extragenic mutations that confer tolerance between incompatible strains has recently been identified by spontaneous and insertional mutagenesis. Suppressor mutants are compatible in spite of genetic differences at o n e o r m o r e bet loci 24. In P. anserina, mutations that permit the growth of lethal self-incompatible strains have been extensively characterized, These mutations occur either at one of the two antagonistic her loci, leading to inactive mutant alleles 25, or at distinct loci in suppressor (or modifier) rood gene# 6. In this species, strains containing rood mutations display defective pmtoperithecia formation and pigmentation, which suggests that rood genes have an essential function in the life cycle of the fungus as well as eliciting vegetative incompatibility27,2s.
In N. crassa, chromosome walking and RFLP analyses have been used to clone alleles of the bet-c and her-6 loci (G. Kuldau and M. Smith, pers. commun.). Several bet genes and suppressors of incompatibility have also been cloned from strains containing insertional mutations z't.
Properties of bet gene products The mt locus of N. crassa The fwst bet genes for which molecular information became available were A and a from N. crassa. These two mt alleles were found to be idiomorphs. The mt a-1 and mt A-1 genes encode dissimilar polypeptides
that are predicted to be transcriptional regulators23,32. Although mating depends upon the ability of the MT a-1 polypeptide to bind a specific DNA sequence, vegetative incompatibility is not affected by mutations that suppress this DNA binding33. Therefore, mt a-1 and mt A-1 may function by a biochemically distinct mechanism in mating and vegetative incompatibility. The bets locus of P. anserfna
Two incompatible alleles, bets and bet-S, have been described at the bets locus in P. anserina. They encode a protein of 289 amino acids that has no similarity to any previously identified protein. Strains in which the bets locus have been inactivated by gene disruption have a neutral incompatibility phenotype and are compatible with strains containing bets or bet-S. This confirms that the polypeptide encoded by this locus is responsible for triggering vegetative incompatibility and cell death when hyphae of bets and betS strains fuse. The mutant strains show a wild-type phenotype: their growth, differentiation of male and female reproductive organs, and fertility are not affected by inactivation of the bets locus. Therefore, expression of this locus is not essential for cell viability and completion of the life cycle of the fungu# 't. The possibility that inactivation of bets could be complemented by an homologous gene was not ruled out.
Clonl~ hot series The biochemical basis of vegetative incompatibility has not yet been elucidated. However, specific expression of proteolytic enzymes and phenoloxidases during the incompatibility reaction has been described in P. anserina, and models for a role of incompatibility genes in the life cycle of the fungus have been proposed 29. The recent cloning of bet genes has thrown light on their function and the molecular mechanism of the incompatibility response. Three bet loci, bets (Ref. 30), bet-c (Ref. 31) and het-e (S. Saupe, B. Turcq and J. B~gueret, unpublished), have now been cloned in P. anserina. One allele of each locus was cloned from cosmid libraries after the allele had been expressed in a recipient strain containing a neutral allele at the locus under study. The transformants that expressed the relevant bet genes were identified by the presence of a bat~ge reaction with strains containing incompatible her genes. Other alleles of these loci were isolated from genomic libraries by hybridization with the cloned genes as probes, or by PCR.
Fmt~ 2. Barrage between incompatiblestrainsof Podospora anserina. The strains were grown on solid corn meal agar. A contact between two incompatible strains is shown by the solid black arrow. The thick, dark myceliumline results fi'om abundant regeneration of apical hyphae after the death of heterokaryoticcells. The other arrow shows a contact between compatible strains.
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FtGtam3. A view of the incompatibilityreaction at the cellular level in Podosporaanserlna. Top: Two hyphae of incompatible strains have just fused at the position markedby the arrow. Bottom:The same hyphae were examined 20 rain after the fusion. Note the high content of vacuoles in the cytoplasm,which is the first symptomof cell death. (Kindlyprovided
by J, Beisson,) Polypeptides encoded by the alternate alleles het-s and bets differ by 14 amino acids, The molecular basis of allele specificity has been investigated to determine how many and which amino acid differences are necessary to trigger incompatibility, This was achieved by constructing chimaeric bet.a-.bet.S genes or modifying codons by site-specific mutagenesis. The incompatibility phenotype conferred by the new alleles was determined by transforming a recipient strain containing an inactive bets allele, and then testing the transformants with bets and bets strains, It was found that the amino acid at position 33 is proline in the polypeptide encoded by bets but histidine in that encoded by betS, A mutation in bets that converts this histidine to proline also converts bets to an allele that confers bets specificity. So, it has been proved that a single amino acid difference at this position in the protein encoded by the bets locus is sufficient to elk'it incompatibility between two strains35. The bet-e and bet-c genes of P. anserina Different alleles of the bet-c and bet-e loci have also been cloned. These loci are multiaUelic and are involved in nonallelic incompatibility, The bet-e locus contains an open reading frame encoding a polypeptide of 1356 amino acids that shows two interesting structural features (S. Saupe, B. Turcq and J. B6gueret,
unpublished). The carboxy-termina! region of the protein contains repeats of 42 amino acids homologous to those found in all [3 subunits of trimeric G proteins36. These repeats are also found in various proteins with different functions, and may be involved in proteinprotein interactions37. The number of these repeats varies from 3 to 12 according to the allele present at the bet-e locus. Such a variation in the repeat number has not been reported for other proteins of this type. The amino-terminal region contains amino acid stretches that match GTP-binding consensus sequences of GTPases. If an essential amino acid in the GTP-binding domain is mutated, a neutral allele that has lost its reactivity in incompatibility is produced, suggesting that the GTP-binding activity of the Het-E polypeptide is essential to trigger the lethal reaction. The polypeptide encoded by the bet-e locus thus shows structural features of ct and J3 subunits of trimeric G proteins, and may define a new class of proteins involved in the transduction of cellular signals. Four wild-type alleles of the bet-c locus that display different incompatibility specificities against the different alleles of the antagonistic het-e locus have been isolated. They encode a protein of 208 amino acids similar to a protein from the pig brain that catalyses the exchange of glycolipids between cellular membranes. The proteins encoded by these different alleles differ by between 1 and 15 amino acids (S. Saupe, B. Turcq and J. B(~gueret, unpublished). As for the bets locus, limited sequence differences at the bet-c locus confer differential specificity in incompatibility. Inactivation of the het-c locus by gene disruption drastically affects ascospore production. In self crosses of the gene-disrupted strain, perithecia contain many aborted asci and the few remaining ascospores are highly heterogeneous in size, suggesting that the distribution of nuclei during the postmeiotic division is abnormal3t. The properties of the bet-c mutant suggest that a gene involved in vegetative incompatibility is essential for the life cycle of the fungus.
Conclusion The function of vegetative incompatibility in natural populations of fungi is still an open question, although its widespread occurrence suggests that it is important in the biology of these organisms. Different hypotheses have been proposed for the role of bet genes in the population biology of fungiz. As mentioned above, incompatibility limits horizontal infection by extrachromosomal elements that could be deleterious. It has also been suggested that the presence of bet genes creates a basis for evolution by limiting outbreeding and favouring the evolution of isolated groups within a specie#. If prevention of heterokaryosis is beneficial in natural populations, bet genes may have been selected and evolved essentially to limit heterokaryon formation. The properties of genes present at cloned bet loci in P. arisen'ha and at the mt locus in N. crassa suggest another biological role for vegetative incompatibility. It is possible that bet genes have primary cellular functions and that the polypeptides encoded by these genes are active as homo- or heteromeric protein complexes according to whether they are involved in allelic or
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REVIEWS
nonallelic incompatibility, reAllelic incompatibility spectively. Since gene evolution in wild-type populations Strain genotype Complexes formed between hetgene products has led to sequence divergence, the coexpression of het-s incompatible genes could pross duce abnormal protein complexes that cause lethal disorders in metabolism or genetic exhot-S pression. Indeed, as described SS above, there are several known Heterokaryon examples of a single amino acid difference in polypeptides ss SS sS encoded by bet genes having a significant effect on their ability Nonallelicincomp~ibili~ to elicit incompatibility through interaction with other allelic or hehChehe nonallelic genes. Ce Thus, it is possible that mutations can create new alleles that elicit an incompatibility he~che~E reaction through specific genetic cE interactions. This hypothesis is Heterokaryon supported by genetic analyses het-Chet.e/het-chet.E [ ~ WAD ~ I~ in P. anserina, in which vegCe ce cE CE etative incompatibility can arise from an interaction between at least two mutated genes that Fmum 4. Models for poison complexes formed by interactions between the products of are different from all other bet incompatible bet genes. In a heterokaryoticcell containing two allelicincompatiblegenes, genes identified in wild-type three differentcomplexes could be formed between the products of the genes. The function isolates~. This suggests that of the heteromericcomplex would be deleterious and dominant over the normal function the number of genes potentially performed by homomeric complexes. When two nonallelic incompatiblegenes such as involved in vegetative incom- hel-Cand het-Eare coexpressed within the heterokaryoticcell, the complex formed by the products of the antagonisticgenes would also be poisonous. patibility may be higher than (Modified from Ref. 41,) that detected by genetic analysis of wild-type isolates, We propose an hypothesis to account for vegetative Acknowledgements We thank A. Groppi for helping with the figures. incompatibility in which abnomml complexes between the products of incompatible genes are lethal to the cell, This 'poison complex' model would then be References I Esser, K. and Blaich, R, (1973) Adv. Genet. 17, 107-152 analogous to that proposed in yeast39 and Drosophaa 4o 2 Glass, N,L and Kuldau, G.A. (1992) Annu. Rev. to explain nonallelic noncomplementation between Phytopathol. 30, 201-224 mutations in different genes encoding components of 3 Rizet, G. (1952) Rev, Cytol. Btol. Veget. 13, 51--92 the cytoskeleton, This hypothesis for allelic and non4 Gamjobst, L and Wilson, J.F. (1956) Proc. NatlAcad. $ci. allelic incompatibility is summarized in Fig. 4. The USA 42, 613-618 model is similar to that proposed by Steams and 5 Beisson-Schecroun,J. (1962) Ann. Genet. 4, 3--50 Botstein 4t to explain allele-specific noncomplemen6 Caflile,J.M. and Dee, J. (1967) Nature 215, 832--834 tation between the tub1 and tub2 mutations in yeast. 7 Dales, R.B. and Croft,J.H. (1977) FEMSMicrobiol. Left. 1, However, for bet genes, poison complexes would be 201-203 the consequence of the coexpression of natural variant 8 Typas, M.A. (1983) J. Gen. Microbiol. 129, 3043-3056 genes in heterokaryotic cells. This hypothesis offers an 9 Pittenger, T.H. and Browner, T.G. (1961) Genetics46, 1645-1663 explanation for the abnormal growth or lethality of hybrid offspring produced by interacial crosses or ?,O Jinks, J.L. (1952) Proc. R. $oc. London Ser. B 140, 83-99 crosses between related species. Abnormal hybrids fre- I I Nuss, D.L. and Koltin, Y. (1990) Annu. Rev. Pbytopathol. 28, 37-58 quendy occur in #ants and animals42; these have been 12 Anagnostakis, S.L. (1982) Science215, ~ 7 1 ascribed to the presence in parents of complementaw lethal genes that could be similar to her genes 13 Anagnostakis,S.L., Hau, B. and Kranz.J. (1986) Plant Dis. 70, 536--538 in fungi, 14 Caten, C.E. (1972)./. Gen. Microbiol. 72. 221-229 Characterization of her genes from other fungi is 15 Del~ts, F., Yang, X. and Grifflths, A.J. (1994) Curt. Geoet. now under way. Such studies will hopefully provide 26, 113-119 information to answer some general questions about 16 Esser, K. (1971) Mol. Gen. Genet. 110, 86-100 the molecular basis of prevention of heterokaryosis, the 17 Perkins, D.D. (1988) Fungal Genet. News35, 44--46 mechanisms of cell death, and also about the function /8 Bemet,J. (1965) Ann. SoL Nat. Bot. 6, 611-768 19 Jinks, J.L. and Grindle, M. (1963) Heredity 18. 407.-.411 of these genes in individuals and populations. TIG DECEMBER1994 VOL. 10 NO. 12
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REVIEWS 20 Anagnostalds, S.L. (1982) Genetics 102, 25-28 21 Leslie,J.F. (1993) Annu. Rev. Pbytopathol. 31, 127-150 22 Dales, R.B., Moorhouse, J. and Croft, J.H. (1993) Heredity 70, 537-543 23 Staben, C. and Yanofsk'y, C. (1990) Proc. Naa Acad. Sci. USA87, 4917--4921 24 Arganoza, M.T., Ohrnberger, J., Min, J. and Akins, R.A. (1994) Genetics 137, 731-742 25 B~gueret, J. (1969) C. R. Acad. Sci. Paris 269, 458.-461 26 Belcour, L. and Bernet, J. (1969) C. R. Acad. Sct. Paris 269, 712-714 27 Boucherie, H. and Bemet, J. (1980) Genetics96, 399--411 28 Durrens, P. and Bemet, J. (1982) Curr. Genet. 5, 181-186 29 Bemet, J. (1992) Heredity 68, 79--87 30 Turcq, B., Denayrolles, M. and B~gueret, J. (1990) Curr. Genet. 17, 297-303 .5/ Saupe, S., Descamps, C., Turcq, B. and B~gueret, J. (1994) Proc. Naa Acad. Sci. USA 91, 5927-5931 32 Glass, N.L., Grotelueschen, J. and Metzenberg, R.L. (1990) Proc. Natl Acad. Sci. USA 87, 4912-4916 33 Philley, M.L. and Staben, C. (1994) Genetics 137, 715-722
BOOK
34 Turcq, B., Deleu, C., Denayrolles, M. and B6gueret, J. (1991) Mol. Gen. Genet. 228, 265-269 .55 Deleu, C., Clav6, C. and B~gueret, J. (1993) Genetics 135, 45-52 .56 van der Voom, L. and Ploegh, H.L. (1992) FEBS Lett. 307, 131-134 .57 Duronio, R.J., Gordon, J.I. and Boguski, M.S. (1992) Proteins 13, 41-56 .58 Delettre, Y.M. and Bemet, J. (1976) Mol. Gen. Genet. 144, 191-197 .59 Vinh, D.B.N. et aL (1993) Genetics 135, 275-286 40 Hays, T.S. et al. (1989) Mol. Cell. Biol. 9, 875--884 41 Stearns, T. and Botstein, D. (1988) Genetics 119, 249-260 42 Stebbins, G.L. (1958) Adv. Genet. 9, 147-162
.I. B~GUERLrl;, a TURLT~ AND C. CLAV~ ARE IN THE LABORATOliIE DE G~N~QUE ~T BIOLOGI~ M O L ~ L ~ nES CHA~PIGNON~ UPR CNRS 9 0 2 ~ UNn~tSn~ nR B o l m ~ u X !!, AVENUEDES FA~LTES 33400, FRANC~.
REVIEWS
Physician to the Gene Pool: Genetic Lessonsand Other Stories by James v. Neel John Wiley & Sons, 1994, $24.95/£18,95 hbk (457 pages) ISBN 0 471 30844 7 The flyleaf describes this book as an 'enthralling memoir by a founding father of human medical genetics which is more than just a travelogue and which challenges the received genetic wisdom of the day'. It is in fact a tour de force, an exceptional book written by an exceptional man. Jim Neel writes as he speaks, clearly with careful marshalling of the facts backed by deep thought and leading to forceful argument and logical conclusions, and all spiced with anecdotes reflecting a puckish sense of humour. He is indeed one of the pioneers in human genetics and his 'travelogue' traces his interests, frustrations and achievements over a half-century during which enormous advances in our understanding of human genetics have been made, an understanding to which he himself has made many contributions. The early part of the book is a fascinating account of the young scientist who took his initial PhD training as a Drosophila geneticist under Curt Stern and who 'agonized over the decision to turn my back on the hard genetics of Drosophila and enter the soft and minted [at that time by the eugenics movement] field of human genetics'. This he did by first taking a medical degree, which was followed by an 18-month military posting
to the atom-bombed cities of Hiroshima and Nagasaki, and thence to a post at the University of Michigan where he has been based ever since. Neel's original posting to Japan was to assist a group set up to advise the National Academy of Sciences on a possible research programme into delayed effects of the atomic bombings on radiation-exposed survivors, He ended up being responsible for the design and organization of the long-term genetic follow-up studies and continues his interest in the programme to this day. The enormous efforts of the American and Japanese scientists involved have provided us with by far the most comprehensive data on the effects of ionizing radiations in producing mutations in the germ cells of exposed humans. This work shows that we are not as sensitive to radiation-induced mutations as was once thought: in both mouse and humans, the chronic radiation dose needed to double the rate of spontaneous background mutation in germ cells is around 4 Sv, a dose some two orders of magnitude greater than that received from normal background radiation over the human reproductive lifetime. With his interests and expertise Neel was, not surprisingly, called upon as a member of a number of committees TIG DECEMBER1994 VOL. 10 NO. 12
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dealing with the assessment of genetic risk. He writes amusingly of confrontations in committee between Sewali Wright and HJ. (Joe) Muller, and comments scathingly on the regulatory authorities, particularly with regard to radioactive waste depositories with the proposed 'risk management bearing precious little relation to the risk assessment'. The relatively high frequency of marriages between first cousins amongst the Japanese provided Neel with an excellent opportunity to study the effects of inbreeding on mortality, morbidity and fertility, studies that he later extended in Brazil. His interest in the human gene pool, which had been engendered by his early studies on sickle cell anaemia and thalassaemia and nurtured in Japan, led Neel to mount a series of field trips into the depths of the Brazilian forests. The aim was to study one of the few remaining primitive populations of the wodd in terms of mutation, selection and population structure. Neel's descriptions of the organization required for these forays and accounts of life (and death) in the Amerindian villages are vivid portraits. These studies provided a wealth of information on genetic differences between different Yanonama villages for both common and rare polymorphisms and much is made of this 'microdifferentiation' in discussing human evolution. In his travels, Neei has pursued genes associated with sickle cell disease in Africa, mutations in Japan and gene pools in South America, and in this book 'looks at the current human genetic condition