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The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus.

The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins... Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press The combination of dissimilar alleles of the AoL and A/8 gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus Ursula Kiies/ Wendy V.J. Richardson/ Anna M. Tymon, Effie S. Mutasa, Berthold Gottgens/ Stefan Gaubatz, Andreas Gregoriades, and Lorna A. Casselton^'^ School of Biological Sciences, Queen Mary and Westfield College, London El 4NS, UK The A mating-type factor is one of two gene complexes that allows mating cells of the mushroom Coprinus cinereus to recognize self from nonself and to regulate a pathway of sexual development that leads to meiosis and sporulation. We have identified seven A genes separated into two subcomplexes corresponding to the classical Aa and A/3 loci. Four genes, one a and three p, all coding for proteins with a homeo domain-related motif, determine A-factor specificity; their allelic forms are so different in sequence that they do not cross-hybridize. It requires only one of these four genes to be heteroallelic in a cell to trigger A-regulated sexual development, and it is the different combinations of their alleles that generate the multiple A factors found in nature. The other three genes cause no change in cell morphology and may regulate the activity of the four specificity genes. [Key Words: Mating type; Coprinus-, sexual development; sexual compatibility; homeo domain motifs] Received October 28, 1991; revised version accepted February 13, 1992. The mushroom Coprinus cinereus has a typical hy- eventually fuse and undergo meiosis in special cells on menomycete life cycle with two functionally distinct the undersurface of the mushroom (Casselton 1978). mycelial stages. A single sexual spore germinates to give The genes that determine monokaryon compatibility a uninuclcate-celled mycelium, the monokaryon, which and regulate sexual development are the mating-type is sexually sterile but produces abundant asexual spores, genes. Two species have been used as experimental mod­ the oidia. For sexual reproduction, two compatible els to study hymenomycete mating-type gene function, monokaryons must mate to give the fertile binucleate- C. cinereus (Casselton 1978) and Schizophyllum com­ celled mycelium, the dikaryon, which no longer pro­ mune (Raper 1983). From classic genetics it was estab­ duces oidia, but given the right temperature and light lished that in both fungi there were two unlinked genetic conditions, differentiates the fruiting bodies. Hyphal fu­ determinants that were called the A and B mating-type sion is sufficient for mating and initiates the develop­ factors (see Raper 1966). These have multiple specifici­ mental pathway outlined in Figure 1. Nuclei are ex­ ties (designated Al, A2, A3, etc., and Bl, B2, 33, etc.) changed, and the donor nucleus in each case migrates that must be different for dikaryon formation. As indi­ through the established cells of the recipient hyphae, cated in Figure 1, A and B independently regulate differ­ triggering the breakdown of cell septa until it reaches the ent steps in dikaryon morphogenesis; the A factor de­ hyphal tip cell. A complex mode of cell division then scribed in this paper governs the synchronized division occurs that involves formation of a specialized structure of the mated nuclei and the formation of the clamp cell, at each septum, the clamp connection, through which whereas clamp cell fusion and nuclear migration require one nucleus must pass. The clamp ensures the equal different B factors. There is no barrier to fusion of in­ distribution of both genetically different nuclei to each compatible hyphae; and when only one factor is differ­ daughter cell, because it is these two nuclei that will ent, this leads to the isolated operation of the part of the sequence it regulates. When only A is different, unfused clamp cells develop (Sweizynski and Day 1960). In C. cinereus and 5. commune, it was shown by classic ge­ 'Present address: Department of Plant Sciences, University of Oxford, netics that the A factor derives its multiple specificities Oxford OXl 3RB, UK. ^Corresponding author. from two closely linked multiallelic genes termed a and GENES & DEVELOPMENT 6:568-577 © 1992 by Cold Spring Harbor Laboratory ISSN 0890-9369/92 $3.00 568 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Coprinus A mating-type gene complexes UNINUCLEATE CELLED MONOKARYONS ne w regulatory protein from polypeptides coded by het- IZIEZIIIED -^ /-- c ° ^ 0 ~T eroalleles of these genes. Results i » t * ° * Transcript analysis identifies seven h-factor genes NUCLEAR EXCHANGE In our initial analysis of the cloned AAl factor, we iden­ O ^ • • -r^~f tified two genes that were both able to elicit A-regulated sexual development (formation of unfused clamp cells; RECIPROCAL NUCLEAR MIGRATION Fig. 1) whe n introduced into host cells wit h different A factors. Because we had shown that O.I ma p unit s repre­ sent s a physica l distance of 10 k b (Mutasa et al. 1990), w e expected the a and /3 loci to be —7.0 kb apart, which A GENES woul d correspond to the 0.07 map units determined by recombinatio n analysis (Day 1960). Th e two genes iden­ tified were unexpectedly close together, however, and only 1.0 kb apart. A particularly puzzling feature was tha t the genes were embedded in 9.0 kb of A42-specific DN A sequence that was shown to code for three differ­ CLAMP CELL FORMATION CLAMP CELL en t transcripts (Mutasa et al. 1990). Thes e two facts sug­ FUSION gested that the A factor might be a larger complex of genes tha n predicted by genetic analysis and that we had n\ n\ , ,/ o • ^ 0 • ^ o • identified only one of the A loci. ) " BINUCLEATE CELLED DIKARYON T o look for other A genes, a detailed restriction map of th e DNA sequence containing AAl was constructed. Figure 1. Regulation of dikaryon formation by the A and B Starting from the position of the genes identified previ­ mating-type genes of the mushroom C. cinereus. ously, a sequence of 40 kb was examined for transcripts. A series of probes was chosen to cover this region and used to identify A42 factor-specific mRNAs in Northern blots of poly(A) RNA isolated from an A42 monokaryon. Tw o discrete clusters of genes, one wit h two transcripts /3 (Papazian 1951; Raper et al. 1958, I960; Day 1960). a and one with five transcripts, were identified, which and /3 are functionally indistinguishable, every Aafi al­ were both shown by transformation to promote clamp lele combination is unique (i.e., aipi, aip2, a2(il, and cell development (see below). These clusters were sepa­ a2/32 all have different specificities), and potentially rated by a 7.0-kb sequence from which no transcripts large number s of A factors can be generated by recombi­ could be detected (Fig. 2)—a distance corresponding ex­ natio n between a few alleles at each locus. The two A actly to that predicted to separate the a and j3 loc i (see loci of C. cinereus are very closely linked (0.07 map above). units), and we have been able to isolate an entire A factor on a single cosmid clone (Mutasa et al. 1990). Th e A factor is flanked by two closely linked genes pab-1 and ade-8 (Day I960); the A42 factor was isolated Th e multiallelic nature of the mushroo m mating-type by a chromosome walk from pab-1. By correlating the genes presents us with a fascinating problem in molec­ ular recognition. On the one hand, there is self-nonself genetic map with the physical map shown in Figure 2, recognition in determining compatibility and, on the w e can distinguish the two-gene cluster closer to pab-1 othe r hand, the regulation of commo n sets of genes that as the a locus and the five-gene cluster as the /8 locus (Fig. 3). The two a genes, designated al and a2, have determin e the differences between mated and unmated transcript s of 2.6 and 2.1 kb, respectively, and are sepa­ cells. Th e fact that there is n o barrier to fusion of incom­ patible hyphae means that self-nonself recognition oc­ rated by I.O kb of noncoding sequence. At the )3 locus , curs intracellularly. In this paper w e describe th e molec­ (31, ii2, and j83 wit h transcripts of 2.5, 2.2, and 2.45 kb, ular analysis of a cloned A factor arbitrarily designated respectively, are th e three genes identified by Mutasa et al. (1990). /32 and )82 are separated by 1.0 kb of noncoding A42. We identified seven genes, unevenly distributed sequenc e from /33. /34 has a 2.5-kb transcript and ^5 a int o tw o subcomplexes corresponding to the a an d p loci. We show that specificity resides in four of these genes, smaller and relatively more abundant 1.1-kb transcript. one a and three /3, and that the decision to initiat e A-reg- Th e direction of transcription in each case was deter­ ulate d development is a direct consequence of heteroal- mine d by selective strand labeling of probes and con­ firmed by sequencing the 3 ' ends of cloned cDNAs (for lelism of one or more of these genes. The presence of details, see Materials and methods). /31 and )35 are tran­ home o domain motifs in the proteins coded by the four specificity genes leads us to suggest that they code for scribed in the opposite direction to th e other five genes; transcriptio n factors and that the cell senses a compati­ th e /34 and j85 transcript s overlap by 25 bases at their 3 ' ble A interaction by its ability to produce at least one ends. Because the A42 genes are the first A-factor genes GENES & DEVELOPMENT 569 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Kues et al. a-Complex p-Complex Homologous Hole y 7.0 kb = 0.07 map units ^ H ES E BECS BHSE BE S H S H SHHE P S H B BESH H E B S ' ' ' I I I I u I I I I I I I I I I I I I II I I I I I 1 4 _8 ^ _ 11 4 9 10 1 2 ^o 11 2 3 4 8 9 10 11 10' 10' kb 2.1 2.2 2,45 2.5 1.1 2.6 2.5 Figure 2. Physical map and corresponding transcript analysis of the A42 mating-type region. Restriction sites relevant to this analysis are abbreviated as follows: (B) BamHl; |C) Clal; (C) £coRI; (H) Hindlll; (P) Pstl; (S) Sail. The chromosomal positions of the fragments used as hybridization probes in Northern blots (either subcloned in pBluescnpt or gel-purified fragments; see Materials and methods) are shovi^n belov^? the map. Numbers refer to the corresponding autoradiographs that show the transcripts detected by each of these probes. The arrows give the sizes and positions of transcripts with reference to the physical map and direction of transcription defined by selective strand labeling of pBluescript subclones. (10', 10") An example of using labeled single strands of plasmids pESM6 and pUK8 to differentiate two overlapping transcripts. The relative positions of the two clusters of transcripts identified as the a and fi complexes are indicated above the map. of C. cinereus characterized at the molecular level, we against Sail-cut pUK plasmids (Fig. 4A). By cuttin g pUK6 have given each gene the allele designation 1 (i.e., al-1, wit h Sail and £coRI, /34-1 and /35-1 were separated as a2-l, etc., in Fig. 3). close to the point of overlap as possible, and the frag­ ment s generated were tested for cross-hybridization in a separate experiment (Fig. 4B). The genes of the A factor share no detectable We could detect no cross-hybridization between any of sequence similarity th e genes, suggesting that they are very different in nu­ cleotide sequence. Th e lack of functional differentiation between a and j3, show n by classic studies, suggested that one locus might be a duplication of the other (Raper 1966). Althoug h we A mating-type specificity can be determined no w show that there are mor e than two genes, the same by four genes argumen t could apply because of th e obvious similarities in transcript sizes. If th e genes have a commo n evolution Th e introduction of a compatible A factor into a haploid w e could expect to detect some sequence similarity and hos t by transformation has the same effect as a compat­ accordingly have looked for cross-hybridization. Five of ible mating; it elicits >1-regulated clamp cell develop­ th e A42 genes have been cloned separately as Sail frag­ men t (Mutasa et al. 1990; May et al. 1991). Each of the ment s (pUKl-5; Fig. 4); a sixth Sail fragment cloned in A4 2 genes was tested separately to see whether it was pUK6 contains the overlapping p4 and 135 genes. Each of sufficient to activate A-regulated development in five th e plasmids was used separately as hybridization probe different A-factor backgrounds, A42 (control), A3, AS, 570 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Coprinus A mating-type gene complexes A4 2 a p pab-1 ade-8 —a 0.07 map units 0.5 1.3 (50 kb) Homologous Hole \ p-Comple x (i1-1 (3 2-1 p3-1 p 4-1 p 5-1 7 kb 2.1 kb 2.5 kb 2.2 kb 2.5 kb 1.1 kb Figure 3. Correlation of the genetic map, based on recombination data of Day (1960), and the physical map derived from this study. a2-l, 131-1, ^2-1, and p4-l (stippled boxes) are the i4-factor specificity genes that have alleles with unique sequence and that promote A-regulated sexual development when introduced by transformation into a host cell with a different allele of the gene. /33-1 (open box) is a nonspecificity gene with unique allele sequences, all and ^5-1 [solid boxes) are nonspecificity genes that have common sequences in all A factors tested (Fig. 6; Table 1). Sizes of transcripts and direction of transcription (Fig. 2) are indicated below the genes. A6, and A43. N o genetic data were available to indicate whethe r any of these factors share the same a or /3 gene —' CM r^ r o m m S complex; Day's (1963) analysis showed only that ^ 5 and A6 have different a and (3 alleles. T o select transformants, a cotransformation system — pBluescript KS' based on tryptophan auxotrophy was used. Cotransfor­ matio n occurs with 30-60% efficiency in C. cinereus (Mellon et al. 1987; Casselton and de la Fuente Herce [33 (J2 1989), bu t not all transforming DNA is expressed (Mel­ lon and Casselton 1988). Trp'^ transformants were screened for clamp cell development. Where a negative result was recorded, 50—100 transformants were checked. The results of these tests are summarized in (3 4 + [3 5 a 2 a 1 Table 1. Four genes, a2, pi, 132, and (34, individually elic­ ited A-regulated clamp cell development when intro­ duced into two hosts having either the A5 factor or the A3 factor. The obvious morphologic change caused by introductio n of these four A42 genes into an A 5 host is illustrate d in Figure 5. a2-l did not promote clamp cell developmen t in an A 6 host, but did in th e A43 host. )84-i failed to induce clamp cell development in both the A 6 and the A43 hosts. As expected, none of the A42 genes elicited clamp cell development when introduced into th e A42 host. The al-1, p3-l, and (35-1 gene s caused no •pBluescript KS' detectabl e phenotypic change in any of the hosts. Be­ cause w e kno w the precise location of the 3 ' ends of the transcript s of each gene, we are confident that the frag­ ment s used for transformation (Sail fragments of 4.2, 3.55, and 4.8 kb) are large enough to contain the entire Figure 4. Cross-hybridization analysis of A42 factor genes. [A] Plasmids pUKl-6 were cut with Sail to release the subcloned coding sequence together with —500 bp of 5'-flanking genes, and each plasmid was then used individually as probe to sequence. Thi s transformation analysis has allowed u s to the same Southern filter, pi, p2, etc., refer to the respective distinguis h at least two functionally distinct classes of genes present in each lane and to the corresponding autoradio- genes i n th e A factor. Th e genes that promote clamp cell graph obtained using the cloned gene as hybridization probe. [B] developmen t clearly determine A-factor specificity, and pUK6 cut with Sail and EcoRl to release j84 and j85 on separate we now refer to these as the specificity genes. fragments and corresponding hybridization data. GENES & DEVELOPMENT 571 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Kiies et al. Table 1. Identification of the four A42-factOT specificity th e A factors we have tested share th e sam e a or /3 allele s genes that promote A-regulated clamp cell development by (Day 1963), but our hybridization and transformation DNA-mediated transformation into hosts with different data indicate that AS and A4 2 share the same a specific­ A factors ity. Thes e studies demonstrat e that these tw o factors can also share alleles of a gene withi n the )3 comple x and still a Comple x /3-Complex hav e a different overall specificity. AM gene al-1 a2-l ^1-1 ^2- 1 fi3-l fi4-l 135-1 Ther e are tw o genes, one in each cluster, that appear to be the same in all A factors, al and j85. As far as we can Host A factor detect, there are no differences in the level of cross-hy­ A 3 - + + - + - bridizatio n between al genes in the different DNAs. For + + + AS - + - - /35, the corresponding gene in the AS factor was barely + + A6 - ~ - ~ - + + detectable, suggesting that there is some sequence vari­ A4 3 - + - ~ - AM - ~ ~ ~ - ~ - atio n among its alleles. We have, however, distinguished tw o classes of genes by this analysis: unique sequence Clamp cell development identifies a specificity gene ( + ); (~) genes [a2, /3i, /32, /33, and /34) and common sequence failure of the specificity gene to promote clamp cells in this genes [al and j85). host resulting from the fact that the host A factor has the same allele of the gene. (-) The gene did not promote clamp cell Th e 7-kb region that separates the a and /3 gen e com­ development in any host. plexes (see Fig. 2) was shown to be homologous in all strain s (Fig. 6), as expected by the reciprocal recombina­ tio n that occurs between a and jS (Day 1960). We have called this region the homologous hole of th e A factor. It Most A genes have alleles with unique sequence An unexpected feature of the fSl and /32 genes described previously was the lack of cross-hybridization between cloned genes and their alleles in other A factors (Mutasa et al. 1990). Thi s analysis has no w been extended to look for similarity between alleles of all seven genes and has allowed us to identify shared alleles in different A fac­ tors. This is of special interest to the specificity genes. Genomi c DNAs of five strains having the A factors 1 0|j.m A42 (control), A3, A5, A6, and A43 were digested with Hindlll and probed separately with each of the A41 genes, as show n in Figure 6. T o avoid any possible flank­ ing sequence homology, cloned cDNAs were used as probes. If we assume that all A factors have the same a 2-1 [5 1-1 genes, we can conclude that five of these genes, a2, (31, ^2, j33, and /34, have alleles with unique sequence. a2-l failed to hybridize to a corresponding A gene in A3 , AS, and A43 DN A but hybridized strongly to A6 DNA. Sig­ nificantly, a2-l failed to induce clamp cell development in an A6 host (Table 1), therefore, we conclude that AAl and A6 share the a2-l allele. Similarly, /34-2 did not cross-hybridize to A3 and AS DNA but did hybridize strongly to A6 and A43 DNA. /84-2 failed to induce clam p cell development in an A6 and an A43 host, indi­ cating that A42, A6, and A43 share the fi4-l allele. It is p2-J (5 4-1 interestin g to note that there is a restriction polymor­ phis m withi n the /34-J gene. Th e j8i-i allele showed faint hybridizatio n to corresponding alleles in all other factors tested, but homology was restricted to the 3 ' ends of the genes. The strong hybridization illustrated in Figure 6 wa s only detected when the probe was a short 0.3-kb EcoKl-Pstl fragment from the 3 ' end of pl-1 cDNA. ^2-1 failed to hybridize to a corresponding /32 gene in any other factor, and )33-l hybridized weakly to the allele in A4 3 (the result of some homology at the 3 ' end; data not Figure 5. A-regulated clamp cell development elicited in an A5 shown). host cell by transformation with A42 specificity genes. The un- From classic genetics we can expect different A factors transformed A5 monokaryon has only simple septa [top]. Trans- to share alleles of genes at either the a or the j8 locus . It formants with a2-l, pi-1, ^2-1, and /34-i [below] all produce is no t know n from recombination studies whethe r any of unfused clamp cells at each septum. GENES &. DEVELOPMENT Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Coprinus A mating-type gene complexes a 2 Homologous Hole 3' P 1 o j CO C\J CO T t -^ CO ur> CO Ti- -^ CO i n CO < < < < < < < < < < 7.1 - 4.3 - 3.0 - 1.9 - P4 P5 CM CO CM CO •<t -^ CD LO CO • * Tt CD i n CO < < < < < < < < < < 3.0- 0.8 - Figure 6. Genomic analysis of five different A factors. cDNAs corresponding to each of the ^442 genes were used to probe Hindlll-cut genomic DNA (5 fig) of monokaryons having the A factors indicated for each lane on the autoradiographs illustrated. Size markers (in kb) indicate the Hindlll fragments of the A42 factor. The respective hybridization pattern obtained for each probe is indicated by the gene designation above the autoradiograph. The results of using two different probes are illustrated for pi, (/3I and 3' j3i) to show the common 3' sequence in all of its alleles. The noncoding sequence between the a and /3 complexes was investigated using two genomic Hindlll fragments of 4.3 and 1.9 kb cloned in pAMT6 and pAMT7. The two Hindlll-BamHI fragments of pAMT7 (see physical map in Fig. 2) gave the same hybridization pattern as the whole plasmid (panel 3, top) confirming a length of —7.0 kb of homologous DNA constituting the homologous hole. is homologous because all A factors share this DN A se­ determine s compatibility in mating by regulating a de­ quenc e and a hole , from the point of view that we cannot velopmenta l sequence that leads to fruit body develop­ ment , meiosis, and sporulation. We have identified detect any transcripts from it. seven genes separated by 7 k b of noncoding homologous DN A sequence into two subcomplexes that correspond The A-factor specificity gene proteins contain a homeo to the a and j8 loci identified by classic recombination domain-related motif analysis. Th e organization of these genes is summarized Althoug h the complete sequences of the four A42 spec­ in Figure 3 . We have used sequence similarity and func­ ificity genes are no t yet available, preliminary data show tion as criteria for characterizing the A genes, and this tha t all encode proteins containing a putative DNA- has enabled us to distinguish the three classes repre­ binding domain, a home o domain-related motif (Scott et sented by th e different boxes in Figure 3 . Four genes, one al. 1989). The sequences of these domains have been a and three (3 (stippled boxes), determine A-factor spec­ compared with those present in the al and al mating- ificity. Th e alleles of each gene have uniqu e sequences. It type proteins of Saccharomyces cerevisiae (Fig. 7). In the is only whe n the same allele is presen t in tw o different A (x2-l and j82-2 proteins, the home o domain is more sim­ factors that we can detect cross-hybridization (Fig. 6). ilar to that of al , and in the pi-1 and ^-1 proteins it is Transformatio n data suggest that it requires only one of mor e similar to that of a2. thes e genes to be mad e heteroallelic in a cell to promote A-regulated sexual development (i.e., a2-l, not present Discussion i n A3 , A5 , or A43 , triggers clamp cell developmen t when introduce d into host cells with any one of these A fac­ Th e genes we have described constitute an A mating- tors). A third gene with unique sequence alleles (open typ e factor of th e mushroo m C. cinereus, a complex that GENES & DEVELOPMENT 573 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Kiies et al. RQIEVWFQNH R R A R K E LEKYFEYNAY P C. cinereus a2-l LEKYFEYNAY P R Q I E V W F Q N H R R A R K E C. cinereus fi2-l LEj Q VlFjR RKQSLNSK E KlE I T P L [QJV R |V W F| I [N. Ri S K K m S. cerevisiae al WLKDNWYNPYP S SRKDIDAWFIDARRRIGW N C. cinereus [il-l S R K D I D A W F I D A RITI R I G W N C. cinereus (H-l WLKDNWYNPYP S " L IS U 11 T1 1 I> VVJF A K|_NJI E[N_ S R|I | Q l | K N|_WJ V S N R R R R K E K T S. cerevisiae <i2 P Y * * * Figure 7. Comparison of homeo domain-related motifs of A41 specificity gene proteins of C. cinereus and those of the al and al mating-type proteins of 5. cerevisiae (Shepherd et al. 1984). The most invariant amino acids in the homeo domain, WF-N-R, which are found in the recognition helix (Scott et al. 1989), are marked by asterisks (*). box; Fig. 3) an d two genes that are shared in all A factors alleles lack any detectable homology, but heteroallelism tested (solid boxes; Fig. 3) have been detected only by of this gene alone is insufficient to promot e y4-regulated sexual development. With so man y specificity genes and thei r transcripts; their functions are unknown. their apparent redundancy, it is possible that some of their alleles are nonfunctional. We do not know, as yet, Function of the h-factor genes th e function of the other two genes. It is tempting to speculate that they may have a role in post-translational A compatible mating requires that the A factors of the activation, as we have been unable to detect any devel­ tw o mating cells have different specificities. We have opmenta l regulation of A-gene transcription. show n by DNA-mediated transformation that there are four genes in the AAl factor that determine this speci­ ficity, al, 131, /32, and /34. Preliminary sequence data Determination of h-factor specificity sho w that these encode putative DNA-binding proteins, each with a home o domain-related motif (Fig. 7), which Thi s study shows that it requires only one of the four suggests that they are transcription factors. The role of A-factor specificity genes to be made heteroallelic for fungal mating-type proteins as transcription factors has A-regulated sexual development to proceed. We con­ been elegantly demonstrated in S. cerevisiae (for review, clude that nonself recognition and developmental regu­ see Herskowitz 1989), and increasing evidence suggests lation are jointly governed by the ability of a cell to syn­ tha t this is also true in other species. Th e three polypep­ thesize polypeptides coded by heteroalleles of these tides coded by the S. cerevisiae al, al , and al genes all genes. It is the n logical to suggest that heteroallelic poly­ bind DN A sequences upstream of regulated genes to ac­ peptides can associate to form novel regulatory proteins tivat e or repress a- and a-specific haploid cell functions no t present in unmate d cells. Thi s idea is similar to mod­ or as an al-a 2 dimer to repress haploid functions in a els proposed by Schulz et al. (1990) to explain the recog­ diploid cell (see Herskowitz 1989). It is particularly rel­ nitio n function of the h gene of the hemibasidiomycete evan t that a l and al polypeptides have a home o domain- smu t fungus U. maydis. A precedent for the interaction related motif (Shepherd et al. 1984; Scott et al. 1989). A of different mating-type polypeptides is provided by S. similar motif is found in the matl-Pl mating-type pro­ cerevisiae, where the al-a2 dimer is a new regulatory tei n of Schizosaccharomyces pombe (Kelly et al. 1988) protei n that can only be formed in mated cells (see Her­ and in the multiallelic b gene polypeptides of Ustilago skowit z 1989; Dranginis 1990) . It may be significant maydis (Schulz et al. 1990). Preliminary sequence data tha t a l and a 2 have dissimilar home o domain s (Shepherd for Aa gene alleles of S. commune show that these al­ et al. 1984). Two of the homeo domains in the C. ci­ leles also encode proteins with homeo domain-related nereus AAl mating-type proteins {a2-l and j82-i) are motifs (Ullrich et al. 1991). mor e similar to that of al , and two [pi-1 and (34-1) are mor e similar to that of al (Fig. 7) . Thi s could suggest Transcrip t analysis has enabled us to identify three tha t the interaction that triggers sexual development in othe r genes associated with the specificity genes. The C. cinereus is between nonallelic homeo domain pro­ tight clusters tha t these genes form strongly suggest that teins . Our transformation experiments do not rule out the y are part of the A-factor complex, al and )85 are the thi s possibility because of th e large numbe r of specificity flanking genes and have a DN A sequence that appears to genes present in each factor. be at least partly homologous in the different A factors examined, but there is n o homologou s flanking sequence Th e problem of polypeptide recognition in basidio- betwee n these genes and the adjacent specificity genes mycete s is more complex than in S. cerevisiae because a2 and j84. j84 and j85 are transcribed in opposite direc­ ther e are multipl e alleles, no t just two alleles at th e mat­ tion s with overlapping 3 ' ends, and several long tran­ ing-type locus. In U. maydis there are —25 alleles of the scripts of al, only identified by cDN A cloning (U. Kues, b gene, which controls both mating compatibility and unpubl.), overlap the 5' end of the a2 gene. Th e jSS gene formation of the pathogenic dikaryon (Rowell and is particularl y puzzling: As wit h the specificity genes, its DeVay 1954; Puhalla 1968). Thes e alleles are largely ho- 57 4 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Coprinus A mating-type gene complexes mologou s in sequence, and alleles encode polypeptides revealed one specificity gene at the a locus and two genes in addition to (i4-l at the /3 locu s (U. Kiies et al., wit h only a variable amino-terminal domain (Kronstad unpubl.). As expected from the hybridization data (Fig. and Leong 1990; Schulz et al. 1990). In both hymeno- 6), all of these alleles are different from those present in mycete s studied, C. cinereus shown here and S. com­ A42 . Because bot h sets of specificity genes trigger A-reg- mune (Giasson et al. 1989), alleles of the A mating-type ulate d development in an A3 host, we can conclude that genes do not cross-hybridize, indicating very different at least two of the (3 specificity genes are multiallelic. nucleotid e sequences. This does not preclude some sim­ Thi s study shows that the A mating-type factor is far ilarity at the protein level, as shown by preliminary se­ mor e complex than predicted by classic genetics. With quenc e data for alleles of the same mating-type gene of S. four different genes, all contributin g a uniqu e specificity, commune (Ullrich et al. 1991). Allelic genes are different our analysis provides an insight into how such large enoug h not to cross-hybridize, but their proteins may number s of different A factors have developed in nature. still have conserved regions necessary for polypeptide recognition. This leads to an important problem, high­ lighted in our study. With four specificity genes, appar­ Materials and methods entl y identical in function, how are polypeptides brought together by mating distinguished from those already C. cinereus strains include AAlBAl: JV6 wild type; LN118 ade-2 presen t in the unmate d cell? It seems likely that some of trp-1.1,1.6; A6B6: H9 wild type; LT2 trp-1.1,1.6; A5B6: H5 wild th e specificity genes are redundant. It will require us to type; FA2222 trp-1.1,1.6; A3S1: C692 ade-3; 218 trp-l.1,1.6; A43fi43: OK130 ade-8; ATS ade-8 trp-1.1,1.6. Media and meth­ generate null mutants to demonstrate this, a difficult ods for culturing C. cinereus have been described by Lewis tas k in C. cinereus because homologous gene replace­ (1961), with modifications summarized by Mutasa et al. (1990). men t by transforming DNA is extremely rare (Binningcr The transformation procedure was based on that of Binninger et etal . 1991). al. (1987), as modified by Casselton and de la Fuente Herce (1989). Plasmid pCclOOl, containing the C. cinereus trp-1 gene (Binninger et al. 1987), and pDBl, containing the C. cinereus Multiple A-factor specificities trp-3 gene (D.M. Burrows, T.J. Elliott, and L.A. Casselton, un­ Day (1963) identified 31 A factors in a sample of 33 in­ publ.), were used for cotransformation experiments with trp dependen t isolates of C. cinereus. From this analysis it is auxotrophic C. cinereus strains. Photographs of clamp cells were taken with a Zeiss photomicroscope using phase contrast. predicted that >I60 A factors exist (Raper 1966). We have shown that four genes determine specificity and tha t different A factors can share their alleles. If all allele DNA manipulations combination s arc possible, only four alleles of each gene are required to generate 256 A factors. It is unlikely, Genomic DNAs of C. cinereus strains were prepared either by the method of Wu et al. (1983) or by the small-scale method of however, that these genes are easily reassociated in na­ Zolan and Pukkila (1986). Routine cloning and plasmid ampli­ ture . Separation of the a2 gene into a separate subcom- fication was in Escherichia coli strains XL-1 Blue \recA\, lac , plex allows this to be rccombined with all specificity endAl, gyM96, thi, hsdRll, supEAA, relAl, {¥' proAB, lacl'^, genes in the j3 complex , but the organization within the lacZ^Ml5, TnlO)l (Stratagene) or DH5a (F , endAl, hsdRll /3 comple x suggests that its genes arc rarely rccombined. (r^ , m^ * ), .sup£44, thi , recAl, gyM96, reMl, ^^QMacTMlS] No t only do alleles of th e genes lack sequence similarity, (BRL) using standard DNA techniques (Sambrook et al. 1989). bu t flanking sequences are also nonhomologous (Mutasa et al. 1990). Reciprocal recombination is possible only if a region of homology is presented by a shared p allele. Plasmids constructs with C. cinereus DNA Th e only shared alleles detected in this study were /34-i pECl, pEC12, and pEC16 are recombinant clones from a cosmid and ^5-1. Because of their positions at the border of the library constructed in Lorist2 and contain parts or the whole complex, recombination within these genes would not sequence of the A42 mating-type factor (Mutasa et al. 1990). recombin e the other specificity genes to generate new A Gel-purified fragments or restriction digests of these cosmid factors. clones were subcloned into the polylinker of either pBlue- script(KS-) or pUC13 (pBG-series) and used for restriction and Because of the close linkage of a and j3, th e only esti­ hybridization analysis to map the A42 a and /3 loci and to com­ mate s of allele numbers at each locus are from a small plete the restriction map given earlier (Mutasa et al. 1990). survey of 10 A factors studied by Day (1963), whic h iden­ (Compare Fig. 2 to localize subcloned fragments especially men­ tified four a and five to seven p alleles. Unfortunately, tioned for transformation, hybridization, and sequencing exper­ only tw o of th e characterized factors (A5 and A6) are still iments.) available. Day's studies lead us to expect at least four pBluescript(KS-) subclones of genomic fragments used for Co­ alleles of the a2 specificity gene. A molecular approach prinus transformations pAMTl contains ^1-1 on a 3.0-kb wil l be required to determine whether the three (3 spec­ Hindlll fragment, and pESMl contains 132-1 on a l.S-BamHl- ificity genes are also multiallelic. The A43 factor of C. Sall fragment (Mutasa et al. 1990). pUKl, pUK2, and pUK4- cinereus used in this study has been cloned by May et al. pUK6 have genes al-1, a2-l, ^2-1, (33-1, and )35-2 on Sail frag­ (1991). Th e investigators provide evidence for three spec­ ments of lengths 4.2, 2.1, 3.4, 3.55, and 4.8 kb, respectively. ificity genes in this factor. We have shown here tha t A43 pUK6 contains /35-2, together with an inactive truncated copy of and A42 share the allele of one of these [(34-1) (Fig. 6). )34-l on a 4.8-kb Sail fragment. pESM2 contains the complete Better resolution of AA3 by transformation analysis has /34-J gene on a 4.0-kb BamHl fragment. GENES & DEVELOPMENT 575 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Kiies et al. Subclones used for Noithein analyses pAMTl, pUKl , pESM2, Gel-isolated DNA fragments or plasmid clones for Southern and the following plasmid subclones were used for Northern blots were labeled with [a-'^^P]CTP using a nick-translatio n kit analyses. pUK7 contains a 2.9-kb £coRI-HindIII fragment con­ (BRL). High-activity hybridization probes for RNA blots were taining mos t of th e a2-l gene. pAMT 6 and pAMT 7 contain 4.0- generated by labeling double-stranded DNA (dsDNA) or single- and 1.9-kb Hindlll fragments, respectively, which identify the stranded DNA (ssDNA) with [a-^^PjCTP (>3000 Ci/mmole, noncodin g sequence between the a and /3 genes. pUK3 has a Amersham ) using a random primer kit (Boehringer). dsDNA 3.5-kb Sail fragment containing the fil-l gene. A 0.5-kb Hindlll probes used for Norther n analyses, indicated in Figure 2 bu t not and a 0.4-kb HindIII-£coRI fragment with the 5' end of gene described above, were gel-purified restriction fragments of the /32-i was subcloned after partial restriction of pCE12 to give cosmids or pUC13 and pBluescript(KS - ) subclones. To deter­ pBGl . pBG2 contains a 2.9-kb EcoRl-HindlU fragment with the min e transcript directions, single-stranded DNA s were prepared 3 ' end of gene 132-1, an d pBG3 contains a 2.5-kb Hindlll-EcoRl from pBluescript(KS-) subclones using the helper phage fragment with most of the /33-I gene sequence. pESM3 is a M13K07 (Sambrook et al. 1989), and labeled strands were gen­ subclone of pBG2 containing a 1.2-kb Pstl-Sall fragment. erated using the Klenow fragment of £. coli DNA polymerase I pESM4, pESM5, pESM6, and pUK8 carry a 1.3-kb BamHl- and the M13 reverse primer (Ley et al. 1982; Sambrook et al. Hindlll fragment, a 1.0-kb Hindlll fragment, a 1.7-kb Hindlll- 1989). BamHl fragment, and a 0.7-kb HindIII-£coRI fragment, respec­ tively, each with parts of the /34-i gene sequence. pUK9 has a Construction of a cDNA library 1.0-kb £coRI-B<3mHI fragment carrying part of /35-1. pLAC l was isolated from a plasmid genomic library of C. A XgtlO cDNA library was contructed with mRNA isolated cinereus strain H9 constructed by Pukkila et al. (1984), using from wild-type strain JV6 and the cDNA synthesis system plus th e A42 a2-l gene as hybridization probe. pLACl contains the and cDNA cloning system-X.gtlO from Amersham (RPN 1256 tw o genes of the A6 a locus . Because A6 and ^442 have the same and RPN 1257) using the recommended £. coli strain NM514 alleles of these two genes, fragments from pLACl subcloned {hsdRSl4 (r^ m,, ) argH, galE, galX, StrA, lycB7* (Hfl^)] as int o pBluescript(KS - ) could be used as gene probes in Northern host. The library was screened with pBluescript(KS-) sub­ hybridizations . pHH5 and pHH7 contain the 5' and 3 ' ends of clones. Screening of 10"^ t o lO'" plaque s was needed to detect one th e al-1 gene cloned on 2.4-kb and 1.1-kb £coRI fragments, copy of each gene. respectively. pUKlO contains part of the a2-l gene on a 2.0-kb Clal-BamHl fragment. Sequencing Subclones used for Southern analysis pUKl-pUK6 were used All sequencing was performed with dsDNA of pBlue- to detect homology between different ^4 2 genes. cDNA s for all script(KS - ) or pUC13 subclones, "'''S-labeled ATP, the T7 poly­ A-gene transcripts were subcloned from \gtlO into pBluc- merase kit of Pharmacia, and either the M13 universal or re­ script(KS- ) with either the £coRI or the BamHl sites of the verse primers. Home o domain motifs (Fig. 7) were deduced from \gtl O adapter. These were used to detect homoallclic genes in partial sequence data of cDN A clones. Transcription directions genomic DNA digests. The length of the homologous hole was of all genes were confirmed by sequence analysis. cDN A 3 ' ends defined using gcl-elutcd fragments of pAMT6 and pAMT7. were located within specific genomic fragments identified by th e transcript mapping analysis. The results of these analyses RNA isolation can be summarized with respect to the gene, the plasmid con­ struct, sequence, and relative position on the restriction map Strain JV6 was grown in liquid minimal medium, and total show n in Figure 2: al-1, pHH7, TCAGAATCCA'TTCTC- RNA was prepared using the guanidinium thiocyanate-CsCl ACGGG , 0.25 kb upstream of the BamHl site; a2-l, step gradient procedure (Glisin et al. 1974; Sambrook et al. pAMT6A£coRI, TTGCATTTCA'AGCACATTTC , 0.12 kb up­ 1989), wit h the modification suggested by Chirgwi n et al. (1979) strea m of the £coRI site; (31-1, pAMTl, ATCCAGCATA*C- of resuspending the pellet in 7.5 M guanidiniu m hydrochloride TATCGACCA , 0.3 kb upstream of the Hindlll site; p2-l, after the ultracentrifugation step. Standard extractions were pESMl , CCGATCGCTA*GTTGCAATAC , 0.2 kb downstream from 10 gram s wet weight mycelium ground in 10 m l of extrac­ of the Pstl site; fi3-l, pBG3, TCTGCCTCCA-GAGTGGCAAA, tion buffer with a polytron homogenizer (Kinematica PTlO/35). 0.5 kb upstream of the £coRI site; (M-1 and /35-2, pUK8 mRN A was selected using oligo(dT) cellulose (Pharmacia) as C AAATAC AA G *' TA G AC AAGC ATTTGGGTTCCTTT C A * ^- described by Sambrook et al. (1989). For Northern blotting, ATCCTCGTAG , 0.05 kb from the £coRI site. Th e asterisk in mRN A (5 jJLg) was denatured at 55°C for 15 min with 17.5% each case indicates the polyadenylation site. *' marks the po­ formaldehyde and 50% formamide and electrophoresed on a 1 % sition of the pA site of the 135-1 gene transcript in the opposite agarose gel containing 17.5% formaldehyde. An RNA ladder of DN A strand; *^ indicate s the end of the ^4-1 transcript. 0.24—9.5 kb (BRL) was used to determine transcript sizes. Acknowledgments Nucleic acid blots and hybridizations Gels prepared for Southern or Northern analyses were blotted We thank Flora Banuett for her critical reading of the manu­ ont o Hybond- N membranes (Amersham) in 20 x SSC (3 M NaCl , script and her valuable discussion, and Georgiana May for the 0.3 M sodiu m citrate). For Southern blots, hybridizations were cloned A43 factor. This work was supported by Science and performed overnight at 65°C as described by Mello n et al. (1987). Engineering Research Council grants GR/E90175 and GR/ Hybridization s of Norther n blots and washes were performed as F78781 to L.A.C. and studentship to A.M.T., and a Gatsb y Char­ recommende d for Hybond-N by the manufacturers. For autora­ itable Foundation Sainsbury studentship to W.V.J.R. diography, filters were exposed to Fuji X-ray film. Filters for Th e publication costs of this article were defrayed in part by bot h Southern and Northern analyses were stripped (see man­ paymen t of page charges. This article mus t therefore be hereby ufacturers ' instructions) and reused to allow direct size compar­ marke d "advertisement" in accordance with 18 USC section isons. 1734 solely to indicate this fact. GENES & DEVELOPMENT 576 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Copiinus A mating-type gene complexes Pukkila , P.J., B.M. Yashar, and D.M. Binninger. 1984. Analysis References of meiotic development in Copiinus cineieus. Controlling Binninger, D.M., C. Skrzynia, P.J. Pukkila, and L.A. Casselton. event s in meiosis. Symp. Soc. Exp. Biol. 38: 177-194. 1987. DN A mediated transformation of the basidiomycete Raper, C.A. 1983. Control s for development and differentiation Copiinus cinereus. EMBO /. 6: 835-840. of th e dikaryon in Basdiomycetes. In Secondary metabolism Binninger, D.M., L. LeChevanton, C. Skrzynia, CD . Shubkin, and diffeientiation in fungi (ed. J.W. Bennet t and A. Ciegler), and P.}. Pukkila. 1991. Targete d transformation in Copiinus pp. 195-238. Marcell Dekker, New York. cineieus. Mol. Gen. Genet. 227: 245-251. Raper J.R., M.G. Baxter, and A.H. Ellingboe. 1960. Th e genetic Casselton, L.A. 1978. Dikaryon formation in higher basidio- structur e of the incompatibility factors of Schizophyllum mycetes . In The filamentous fungi (ed. J.E. Smith and D.R. commune: The A factor. Pioc. Natl. Acad. Sci. 46: 833-842. Berry), vol. 3, pp. 275-297 . Edward Arnold, London, UK. Raper, J.R. 1966. Genetics of sexuality in highei fungi. The Ron­ Casselton, L.A. and A. de la Fuente Herce. 1989. Heterologous ald Press Company, New York. gene expression in the basidiomycete fungus Copiinus ci­ Raper J.R., M.G. Baxter, and R.B. Middleton . 1958. Th e genetic neieus. Cuii. Genet. 16: 35-40 . structur e of the incompatibility factors in Schizophyllum Chirgwin, J.M., A.E. Przybyla, R.J. MacDonald , and W.J. Rutter. commune. Pioc. Natl. Acad. Sci. 44: 889-900. 1979. Isolation of biologically active ribonucleic acid from Rowell, J.B. and J.E. DeVay. 1954. Genetic s of Ustilago zeae in sources enriched in ribonuclease. Biochemistiy 18: 5294 - relatio n to basic problems of its pathogenicity. Phytopathol­ 5299. ogy 44:356-362. Day, P.R. 1960. The structure of the A mating type locus in Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Moleculai Copiinus lagopus. Genetics 45: 641-650. cloning: A laboiatoiy manual. Cold Spring Harbor Labora­ . 1963. Th e structure of the A mating type factor in Co­ tory Press, Cold Spring Harbor, New York. piinus lagopus: Wild alleles. Genet. Res. Camb. 4: 323-325 . Schulz, B., F. Banuett, M. Dahl, R. Schlesinger, W. Schafer, T. Dranginis, A.M. 1990. Binding of yeast al and a 2 as a heterodi- Martin , I. Herskowitz , and R. Kahmann . 1990. Th e b allele of me r to the operator DN A of a haploid-specific gene. Natuie U. maydis, whose combinations program pathogenic devel­ 347: 682-685 . opment , code for polypeptides containing a homeodomain- related motif. Cell 60: 295-306. Giasson, L., C.A. Specht, C. Milgrim, C.P. Novotny, and R.C. Ullrich . 1989. Cloning and comparison of Aa mating-type Scott, M.P., J.W. Tamkum, and G.W. Hartzell III. 1989. The alleles of the basidiomycete Schizophyllum commune. Mol. structur e and function of the homeodomain. Biochim. Bio- Gen. Genet. 218: 72-77. phys. Acta 989: 25-48 . Glisin, V.R., R. Crkvenjakov, and C. Byus. 1974. Ribonucleic Shepherd, J.C.W., W. McGinnis, A.E. Carrasco, E.M. DeRober- acid isolated by caesium chloride centrifugation. Biochem­ tis, and W.J. Gehring. 1984. Fly and frog homeo domains istiy 13: 2633-2637. show homologies with yeast mating type regulatory pro­ Herskowitz , I. 1989. A regulatory hierarchy for cell specialisa­ teins . Natuie 310: 70-71. tion in yeast. Natuie 342: 749-757. Sweizynski, K.M. and P.R. Day. 1960. Heterokaryon formation Kelly, M., J. Burke, M. Smith, A. Klar, and D. Beach. 1988. Four in Copiinus lagopus. Genet. Res. Camb. 1: 114-128. mating-typ e genes control sexual differentiation in the fis­ Ullrich, R.C, C.A. Specht, M.M. Stankis, H. Yang, L. Giasson, sion yeast. £AlBO /. 7: 1537-1547. and C.P. Novotny. 1991. Molecular biology of mating-type Kronstad, J.W. and S.A. Leong. 1990. Th e b mating-type locus of determinatio n in Schizophyllum commune. In Genetic en- Ustilago maydis contains variable and constant regions. gineeiing, piinciples and methods (ed. J.K. Setlow), vol. 13, Genes &) Dev. 4: 1384-1395. pp. 279-306. Plenum Press, New York. Lewis, D . 1961. Genetica l analysis of methionin e suppressors in Wu, M., J. Cassidy, and P.J. Pukkila. 1983. Polymorphisms in Copiinus. Genet. Res. 2: 141-155. DN A of Copiinus cineieus. Cuii. Genet. 77: 385-392 . Ley, T.J., N.P. Anagnou, G. Pepe, and A.W. Nienhuis. 1982. Zolan, M.E. and P.J. Pukkila. 1986. 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Papazian, H.P. 1951. Th e incompatibility factors and a related gene in Schizophyllum commune. Genetics 36: 441-459. Puhalla, J.E. 1968. Compatibilit y reactions on solid mediu m and interstrai n inhibition in Ustilago maydis. Genetics 60: 461 - GENES & DEVELOPMENT 577 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus. U Kües, W V Richardson, A M Tymon, et al. Genes Dev. 1992, 6: Access the most recent version at doi:10.1101/gad.6.4.568 This article cites 30 articles, 8 of which can be accessed free at: References http://genesdev.cshlp.org/content/6/4/568.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. 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The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus.

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Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press The combination of dissimilar alleles of the AoL and A/8 gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus Ursula Kiies/ Wendy V.J. Richardson/ Anna M. Tymon, Effie S. Mutasa, Berthold Gottgens/ Stefan Gaubatz, Andreas Gregoriades, and Lorna A. Casselton^'^ School of Biological Sciences, Queen Mary and Westfield College, London El 4NS, UK The A mating-type factor is one of two gene complexes that allows mating cells of the mushroom Coprinus cinereus to recognize self from nonself and to regulate a pathway of sexual development that leads to meiosis and sporulation. We have identified seven A genes separated into two subcomplexes corresponding to the classical Aa and A/3 loci. Four genes, one a and three p, all coding for proteins with a homeo domain-related motif, determine A-factor specificity; their allelic forms are so different in sequence that they do not cross-hybridize. It requires only one of these four genes to be heteroallelic in a cell to trigger A-regulated sexual development, and it is the different combinations of their alleles that generate the multiple A factors found in nature. The other three genes cause no change in cell morphology and may regulate the activity of the four specificity genes. [Key Words: Mating type; Coprinus-, sexual development; sexual compatibility; homeo domain motifs] Received October 28, 1991; revised version accepted February 13, 1992. The mushroom Coprinus cinereus has a typical hy- eventually fuse and undergo meiosis in special cells on menomycete life cycle with two functionally distinct the undersurface of the mushroom (Casselton 1978). mycelial stages. A single sexual spore germinates to give The genes that determine monokaryon compatibility a uninuclcate-celled mycelium, the monokaryon, which and regulate sexual development are the mating-type is sexually sterile but produces abundant asexual spores, genes. Two species have been used as experimental mod­ the oidia. For sexual reproduction, two compatible els to study hymenomycete mating-type gene function, monokaryons must mate to give the fertile binucleate- C. cinereus (Casselton 1978) and Schizophyllum com­ celled mycelium, the dikaryon, which no longer pro­ mune (Raper 1983). From classic genetics it was estab­ duces oidia, but given the right temperature and light lished that in both fungi there were two unlinked genetic conditions, differentiates the fruiting bodies. Hyphal fu­ determinants that were called the A and B mating-type sion is sufficient for mating and initiates the develop­ factors (see Raper 1966). These have multiple specifici­ mental pathway outlined in Figure 1. Nuclei are ex­ ties (designated Al, A2, A3, etc., and Bl, B2, 33, etc.) changed, and the donor nucleus in each case migrates that must be different for dikaryon formation. As indi­ through the established cells of the recipient hyphae, cated in Figure 1, A and B independently regulate differ­ triggering the breakdown of cell septa until it reaches the ent steps in dikaryon morphogenesis; the A factor de­ hyphal tip cell. A complex mode of cell division then scribed in this paper governs the synchronized division occurs that involves formation of a specialized structure of the mated nuclei and the formation of the clamp cell, at each septum, the clamp connection, through which whereas clamp cell fusion and nuclear migration require one nucleus must pass. The clamp ensures the equal different B factors. There is no barrier to fusion of in­ distribution of both genetically different nuclei to each compatible hyphae; and when only one factor is differ­ daughter cell, because it is these two nuclei that will ent, this leads to the isolated operation of the part of the sequence it regulates. When only A is different, unfused clamp cells develop (Sweizynski and Day 1960). In C. cinereus and 5. commune, it was shown by classic ge­ 'Present address: Department of Plant Sciences, University of Oxford, netics that the A factor derives its multiple specificities Oxford OXl 3RB, UK. ^Corresponding author. from two closely linked multiallelic genes termed a and GENES & DEVELOPMENT 6:568-577 © 1992 by Cold Spring Harbor Laboratory ISSN 0890-9369/92 $3.00 568 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Coprinus A mating-type gene complexes UNINUCLEATE CELLED MONOKARYONS ne w regulatory protein from polypeptides coded by het- IZIEZIIIED -^ /-- c ° ^ 0 ~T eroalleles of these genes. Results i » t * ° * Transcript analysis identifies seven h-factor genes NUCLEAR EXCHANGE In our initial analysis of the cloned AAl factor, we iden­ O ^ • • -r^~f tified two genes that were both able to elicit A-regulated sexual development (formation of unfused clamp cells; RECIPROCAL NUCLEAR MIGRATION Fig. 1) whe n introduced into host cells wit h different A factors. Because we had shown that O.I ma p unit s repre­ sent s a physica l distance of 10 k b (Mutasa et al. 1990), w e expected the a and /3 loci to be —7.0 kb apart, which A GENES woul d correspond to the 0.07 map units determined by recombinatio n analysis (Day 1960). Th e two genes iden­ tified were unexpectedly close together, however, and only 1.0 kb apart. A particularly puzzling feature was tha t the genes were embedded in 9.0 kb of A42-specific DN A sequence that was shown to code for three differ­ CLAMP CELL FORMATION CLAMP CELL en t transcripts (Mutasa et al. 1990). Thes e two facts sug­ FUSION gested that the A factor might be a larger complex of genes tha n predicted by genetic analysis and that we had n\ n\ , ,/ o • ^ 0 • ^ o • identified only one of the A loci. ) " BINUCLEATE CELLED DIKARYON T o look for other A genes, a detailed restriction map of th e DNA sequence containing AAl was constructed. Figure 1. Regulation of dikaryon formation by the A and B Starting from the position of the genes identified previ­ mating-type genes of the mushroom C. cinereus. ously, a sequence of 40 kb was examined for transcripts. A series of probes was chosen to cover this region and used to identify A42 factor-specific mRNAs in Northern blots of poly(A) RNA isolated from an A42 monokaryon. Tw o discrete clusters of genes, one wit h two transcripts /3 (Papazian 1951; Raper et al. 1958, I960; Day 1960). a and one with five transcripts, were identified, which and /3 are functionally indistinguishable, every Aafi al­ were both shown by transformation to promote clamp lele combination is unique (i.e., aipi, aip2, a2(il, and cell development (see below). These clusters were sepa­ a2/32 all have different specificities), and potentially rated by a 7.0-kb sequence from which no transcripts large number s of A factors can be generated by recombi­ could be detected (Fig. 2)—a distance corresponding ex­ natio n between a few alleles at each locus. The two A actly to that predicted to separate the a and j3 loc i (see loci of C. cinereus are very closely linked (0.07 map above). units), and we have been able to isolate an entire A factor on a single cosmid clone (Mutasa et al. 1990). Th e A factor is flanked by two closely linked genes pab-1 and ade-8 (Day I960); the A42 factor was isolated Th e multiallelic nature of the mushroo m mating-type by a chromosome walk from pab-1. By correlating the genes presents us with a fascinating problem in molec­ ular recognition. On the one hand, there is self-nonself genetic map with the physical map shown in Figure 2, recognition in determining compatibility and, on the w e can distinguish the two-gene cluster closer to pab-1 othe r hand, the regulation of commo n sets of genes that as the a locus and the five-gene cluster as the /8 locus (Fig. 3). The two a genes, designated al and a2, have determin e the differences between mated and unmated transcript s of 2.6 and 2.1 kb, respectively, and are sepa­ cells. Th e fact that there is n o barrier to fusion of incom­ patible hyphae means that self-nonself recognition oc­ rated by I.O kb of noncoding sequence. At the )3 locus , curs intracellularly. In this paper w e describe th e molec­ (31, ii2, and j83 wit h transcripts of 2.5, 2.2, and 2.45 kb, ular analysis of a cloned A factor arbitrarily designated respectively, are th e three genes identified by Mutasa et al. (1990). /32 and )82 are separated by 1.0 kb of noncoding A42. We identified seven genes, unevenly distributed sequenc e from /33. /34 has a 2.5-kb transcript and ^5 a int o tw o subcomplexes corresponding to the a an d p loci. We show that specificity resides in four of these genes, smaller and relatively more abundant 1.1-kb transcript. one a and three /3, and that the decision to initiat e A-reg- Th e direction of transcription in each case was deter­ ulate d development is a direct consequence of heteroal- mine d by selective strand labeling of probes and con­ firmed by sequencing the 3 ' ends of cloned cDNAs (for lelism of one or more of these genes. The presence of details, see Materials and methods). /31 and )35 are tran­ home o domain motifs in the proteins coded by the four specificity genes leads us to suggest that they code for scribed in the opposite direction to th e other five genes; transcriptio n factors and that the cell senses a compati­ th e /34 and j85 transcript s overlap by 25 bases at their 3 ' ble A interaction by its ability to produce at least one ends. Because the A42 genes are the first A-factor genes GENES & DEVELOPMENT 569 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Kues et al. a-Complex p-Complex Homologous Hole y 7.0 kb = 0.07 map units ^ H ES E BECS BHSE BE S H S H SHHE P S H B BESH H E B S ' ' ' I I I I u I I I I I I I I I I I I I II I I I I I 1 4 _8 ^ _ 11 4 9 10 1 2 ^o 11 2 3 4 8 9 10 11 10' 10' kb 2.1 2.2 2,45 2.5 1.1 2.6 2.5 Figure 2. Physical map and corresponding transcript analysis of the A42 mating-type region. Restriction sites relevant to this analysis are abbreviated as follows: (B) BamHl; |C) Clal; (C) £coRI; (H) Hindlll; (P) Pstl; (S) Sail. The chromosomal positions of the fragments used as hybridization probes in Northern blots (either subcloned in pBluescnpt or gel-purified fragments; see Materials and methods) are shovi^n belov^? the map. Numbers refer to the corresponding autoradiographs that show the transcripts detected by each of these probes. The arrows give the sizes and positions of transcripts with reference to the physical map and direction of transcription defined by selective strand labeling of pBluescript subclones. (10', 10") An example of using labeled single strands of plasmids pESM6 and pUK8 to differentiate two overlapping transcripts. The relative positions of the two clusters of transcripts identified as the a and fi complexes are indicated above the map. of C. cinereus characterized at the molecular level, we against Sail-cut pUK plasmids (Fig. 4A). By cuttin g pUK6 have given each gene the allele designation 1 (i.e., al-1, wit h Sail and £coRI, /34-1 and /35-1 were separated as a2-l, etc., in Fig. 3). close to the point of overlap as possible, and the frag­ ment s generated were tested for cross-hybridization in a separate experiment (Fig. 4B). The genes of the A factor share no detectable We could detect no cross-hybridization between any of sequence similarity th e genes, suggesting that they are very different in nu­ cleotide sequence. Th e lack of functional differentiation between a and j3, show n by classic studies, suggested that one locus might be a duplication of the other (Raper 1966). Althoug h we A mating-type specificity can be determined no w show that there are mor e than two genes, the same by four genes argumen t could apply because of th e obvious similarities in transcript sizes. If th e genes have a commo n evolution Th e introduction of a compatible A factor into a haploid w e could expect to detect some sequence similarity and hos t by transformation has the same effect as a compat­ accordingly have looked for cross-hybridization. Five of ible mating; it elicits >1-regulated clamp cell develop­ th e A42 genes have been cloned separately as Sail frag­ men t (Mutasa et al. 1990; May et al. 1991). Each of the ment s (pUKl-5; Fig. 4); a sixth Sail fragment cloned in A4 2 genes was tested separately to see whether it was pUK6 contains the overlapping p4 and 135 genes. Each of sufficient to activate A-regulated development in five th e plasmids was used separately as hybridization probe different A-factor backgrounds, A42 (control), A3, AS, 570 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Coprinus A mating-type gene complexes A4 2 a p pab-1 ade-8 —a 0.07 map units 0.5 1.3 (50 kb) Homologous Hole \ p-Comple x (i1-1 (3 2-1 p3-1 p 4-1 p 5-1 7 kb 2.1 kb 2.5 kb 2.2 kb 2.5 kb 1.1 kb Figure 3. Correlation of the genetic map, based on recombination data of Day (1960), and the physical map derived from this study. a2-l, 131-1, ^2-1, and p4-l (stippled boxes) are the i4-factor specificity genes that have alleles with unique sequence and that promote A-regulated sexual development when introduced by transformation into a host cell with a different allele of the gene. /33-1 (open box) is a nonspecificity gene with unique allele sequences, all and ^5-1 [solid boxes) are nonspecificity genes that have common sequences in all A factors tested (Fig. 6; Table 1). Sizes of transcripts and direction of transcription (Fig. 2) are indicated below the genes. A6, and A43. N o genetic data were available to indicate whethe r any of these factors share the same a or /3 gene —' CM r^ r o m m S complex; Day's (1963) analysis showed only that ^ 5 and A6 have different a and (3 alleles. T o select transformants, a cotransformation system — pBluescript KS' based on tryptophan auxotrophy was used. Cotransfor­ matio n occurs with 30-60% efficiency in C. cinereus (Mellon et al. 1987; Casselton and de la Fuente Herce [33 (J2 1989), bu t not all transforming DNA is expressed (Mel­ lon and Casselton 1988). Trp'^ transformants were screened for clamp cell development. Where a negative result was recorded, 50—100 transformants were checked. The results of these tests are summarized in (3 4 + [3 5 a 2 a 1 Table 1. Four genes, a2, pi, 132, and (34, individually elic­ ited A-regulated clamp cell development when intro­ duced into two hosts having either the A5 factor or the A3 factor. The obvious morphologic change caused by introductio n of these four A42 genes into an A 5 host is illustrate d in Figure 5. a2-l did not promote clamp cell developmen t in an A 6 host, but did in th e A43 host. )84-i failed to induce clamp cell development in both the A 6 and the A43 hosts. As expected, none of the A42 genes elicited clamp cell development when introduced into th e A42 host. The al-1, p3-l, and (35-1 gene s caused no •pBluescript KS' detectabl e phenotypic change in any of the hosts. Be­ cause w e kno w the precise location of the 3 ' ends of the transcript s of each gene, we are confident that the frag­ ment s used for transformation (Sail fragments of 4.2, 3.55, and 4.8 kb) are large enough to contain the entire Figure 4. Cross-hybridization analysis of A42 factor genes. [A] Plasmids pUKl-6 were cut with Sail to release the subcloned coding sequence together with —500 bp of 5'-flanking genes, and each plasmid was then used individually as probe to sequence. Thi s transformation analysis has allowed u s to the same Southern filter, pi, p2, etc., refer to the respective distinguis h at least two functionally distinct classes of genes present in each lane and to the corresponding autoradio- genes i n th e A factor. Th e genes that promote clamp cell graph obtained using the cloned gene as hybridization probe. [B] developmen t clearly determine A-factor specificity, and pUK6 cut with Sail and EcoRl to release j84 and j85 on separate we now refer to these as the specificity genes. fragments and corresponding hybridization data. GENES & DEVELOPMENT 571 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Kiies et al. Table 1. Identification of the four A42-factOT specificity th e A factors we have tested share th e sam e a or /3 allele s genes that promote A-regulated clamp cell development by (Day 1963), but our hybridization and transformation DNA-mediated transformation into hosts with different data indicate that AS and A4 2 share the same a specific­ A factors ity. Thes e studies demonstrat e that these tw o factors can also share alleles of a gene withi n the )3 comple x and still a Comple x /3-Complex hav e a different overall specificity. AM gene al-1 a2-l ^1-1 ^2- 1 fi3-l fi4-l 135-1 Ther e are tw o genes, one in each cluster, that appear to be the same in all A factors, al and j85. As far as we can Host A factor detect, there are no differences in the level of cross-hy­ A 3 - + + - + - bridizatio n between al genes in the different DNAs. For + + + AS - + - - /35, the corresponding gene in the AS factor was barely + + A6 - ~ - ~ - + + detectable, suggesting that there is some sequence vari­ A4 3 - + - ~ - AM - ~ ~ ~ - ~ - atio n among its alleles. We have, however, distinguished tw o classes of genes by this analysis: unique sequence Clamp cell development identifies a specificity gene ( + ); (~) genes [a2, /3i, /32, /33, and /34) and common sequence failure of the specificity gene to promote clamp cells in this genes [al and j85). host resulting from the fact that the host A factor has the same allele of the gene. (-) The gene did not promote clamp cell Th e 7-kb region that separates the a and /3 gen e com­ development in any host. plexes (see Fig. 2) was shown to be homologous in all strain s (Fig. 6), as expected by the reciprocal recombina­ tio n that occurs between a and jS (Day 1960). We have called this region the homologous hole of th e A factor. It Most A genes have alleles with unique sequence An unexpected feature of the fSl and /32 genes described previously was the lack of cross-hybridization between cloned genes and their alleles in other A factors (Mutasa et al. 1990). Thi s analysis has no w been extended to look for similarity between alleles of all seven genes and has allowed us to identify shared alleles in different A fac­ tors. This is of special interest to the specificity genes. Genomi c DNAs of five strains having the A factors 1 0|j.m A42 (control), A3, A5, A6, and A43 were digested with Hindlll and probed separately with each of the A41 genes, as show n in Figure 6. T o avoid any possible flank­ ing sequence homology, cloned cDNAs were used as probes. If we assume that all A factors have the same a 2-1 [5 1-1 genes, we can conclude that five of these genes, a2, (31, ^2, j33, and /34, have alleles with unique sequence. a2-l failed to hybridize to a corresponding A gene in A3 , AS, and A43 DN A but hybridized strongly to A6 DNA. Sig­ nificantly, a2-l failed to induce clamp cell development in an A6 host (Table 1), therefore, we conclude that AAl and A6 share the a2-l allele. Similarly, /34-2 did not cross-hybridize to A3 and AS DNA but did hybridize strongly to A6 and A43 DNA. /84-2 failed to induce clam p cell development in an A6 and an A43 host, indi­ cating that A42, A6, and A43 share the fi4-l allele. It is p2-J (5 4-1 interestin g to note that there is a restriction polymor­ phis m withi n the /34-J gene. Th e j8i-i allele showed faint hybridizatio n to corresponding alleles in all other factors tested, but homology was restricted to the 3 ' ends of the genes. The strong hybridization illustrated in Figure 6 wa s only detected when the probe was a short 0.3-kb EcoKl-Pstl fragment from the 3 ' end of pl-1 cDNA. ^2-1 failed to hybridize to a corresponding /32 gene in any other factor, and )33-l hybridized weakly to the allele in A4 3 (the result of some homology at the 3 ' end; data not Figure 5. A-regulated clamp cell development elicited in an A5 shown). host cell by transformation with A42 specificity genes. The un- From classic genetics we can expect different A factors transformed A5 monokaryon has only simple septa [top]. Trans- to share alleles of genes at either the a or the j8 locus . It formants with a2-l, pi-1, ^2-1, and /34-i [below] all produce is no t know n from recombination studies whethe r any of unfused clamp cells at each septum. GENES &. DEVELOPMENT Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Coprinus A mating-type gene complexes a 2 Homologous Hole 3' P 1 o j CO C\J CO T t -^ CO ur> CO Ti- -^ CO i n CO < < < < < < < < < < 7.1 - 4.3 - 3.0 - 1.9 - P4 P5 CM CO CM CO •<t -^ CD LO CO • * Tt CD i n CO < < < < < < < < < < 3.0- 0.8 - Figure 6. Genomic analysis of five different A factors. cDNAs corresponding to each of the ^442 genes were used to probe Hindlll-cut genomic DNA (5 fig) of monokaryons having the A factors indicated for each lane on the autoradiographs illustrated. Size markers (in kb) indicate the Hindlll fragments of the A42 factor. The respective hybridization pattern obtained for each probe is indicated by the gene designation above the autoradiograph. The results of using two different probes are illustrated for pi, (/3I and 3' j3i) to show the common 3' sequence in all of its alleles. The noncoding sequence between the a and /3 complexes was investigated using two genomic Hindlll fragments of 4.3 and 1.9 kb cloned in pAMT6 and pAMT7. The two Hindlll-BamHI fragments of pAMT7 (see physical map in Fig. 2) gave the same hybridization pattern as the whole plasmid (panel 3, top) confirming a length of —7.0 kb of homologous DNA constituting the homologous hole. is homologous because all A factors share this DN A se­ determine s compatibility in mating by regulating a de­ quenc e and a hole , from the point of view that we cannot velopmenta l sequence that leads to fruit body develop­ ment , meiosis, and sporulation. We have identified detect any transcripts from it. seven genes separated by 7 k b of noncoding homologous DN A sequence into two subcomplexes that correspond The A-factor specificity gene proteins contain a homeo to the a and j8 loci identified by classic recombination domain-related motif analysis. Th e organization of these genes is summarized Althoug h the complete sequences of the four A42 spec­ in Figure 3 . We have used sequence similarity and func­ ificity genes are no t yet available, preliminary data show tion as criteria for characterizing the A genes, and this tha t all encode proteins containing a putative DNA- has enabled us to distinguish the three classes repre­ binding domain, a home o domain-related motif (Scott et sented by th e different boxes in Figure 3 . Four genes, one al. 1989). The sequences of these domains have been a and three (3 (stippled boxes), determine A-factor spec­ compared with those present in the al and al mating- ificity. Th e alleles of each gene have uniqu e sequences. It type proteins of Saccharomyces cerevisiae (Fig. 7). In the is only whe n the same allele is presen t in tw o different A (x2-l and j82-2 proteins, the home o domain is more sim­ factors that we can detect cross-hybridization (Fig. 6). ilar to that of al , and in the pi-1 and ^-1 proteins it is Transformatio n data suggest that it requires only one of mor e similar to that of a2. thes e genes to be mad e heteroallelic in a cell to promote A-regulated sexual development (i.e., a2-l, not present Discussion i n A3 , A5 , or A43 , triggers clamp cell developmen t when introduce d into host cells with any one of these A fac­ Th e genes we have described constitute an A mating- tors). A third gene with unique sequence alleles (open typ e factor of th e mushroo m C. cinereus, a complex that GENES & DEVELOPMENT 573 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Kiies et al. RQIEVWFQNH R R A R K E LEKYFEYNAY P C. cinereus a2-l LEKYFEYNAY P R Q I E V W F Q N H R R A R K E C. cinereus fi2-l LEj Q VlFjR RKQSLNSK E KlE I T P L [QJV R |V W F| I [N. Ri S K K m S. cerevisiae al WLKDNWYNPYP S SRKDIDAWFIDARRRIGW N C. cinereus [il-l S R K D I D A W F I D A RITI R I G W N C. cinereus (H-l WLKDNWYNPYP S " L IS U 11 T1 1 I> VVJF A K|_NJI E[N_ S R|I | Q l | K N|_WJ V S N R R R R K E K T S. cerevisiae <i2 P Y * * * Figure 7. Comparison of homeo domain-related motifs of A41 specificity gene proteins of C. cinereus and those of the al and al mating-type proteins of 5. cerevisiae (Shepherd et al. 1984). The most invariant amino acids in the homeo domain, WF-N-R, which are found in the recognition helix (Scott et al. 1989), are marked by asterisks (*). box; Fig. 3) an d two genes that are shared in all A factors alleles lack any detectable homology, but heteroallelism tested (solid boxes; Fig. 3) have been detected only by of this gene alone is insufficient to promot e y4-regulated sexual development. With so man y specificity genes and thei r transcripts; their functions are unknown. their apparent redundancy, it is possible that some of their alleles are nonfunctional. We do not know, as yet, Function of the h-factor genes th e function of the other two genes. It is tempting to speculate that they may have a role in post-translational A compatible mating requires that the A factors of the activation, as we have been unable to detect any devel­ tw o mating cells have different specificities. We have opmenta l regulation of A-gene transcription. show n by DNA-mediated transformation that there are four genes in the AAl factor that determine this speci­ ficity, al, 131, /32, and /34. Preliminary sequence data Determination of h-factor specificity sho w that these encode putative DNA-binding proteins, each with a home o domain-related motif (Fig. 7), which Thi s study shows that it requires only one of the four suggests that they are transcription factors. The role of A-factor specificity genes to be made heteroallelic for fungal mating-type proteins as transcription factors has A-regulated sexual development to proceed. We con­ been elegantly demonstrated in S. cerevisiae (for review, clude that nonself recognition and developmental regu­ see Herskowitz 1989), and increasing evidence suggests lation are jointly governed by the ability of a cell to syn­ tha t this is also true in other species. Th e three polypep­ thesize polypeptides coded by heteroalleles of these tides coded by the S. cerevisiae al, al , and al genes all genes. It is the n logical to suggest that heteroallelic poly­ bind DN A sequences upstream of regulated genes to ac­ peptides can associate to form novel regulatory proteins tivat e or repress a- and a-specific haploid cell functions no t present in unmate d cells. Thi s idea is similar to mod­ or as an al-a 2 dimer to repress haploid functions in a els proposed by Schulz et al. (1990) to explain the recog­ diploid cell (see Herskowitz 1989). It is particularly rel­ nitio n function of the h gene of the hemibasidiomycete evan t that a l and al polypeptides have a home o domain- smu t fungus U. maydis. A precedent for the interaction related motif (Shepherd et al. 1984; Scott et al. 1989). A of different mating-type polypeptides is provided by S. similar motif is found in the matl-Pl mating-type pro­ cerevisiae, where the al-a2 dimer is a new regulatory tei n of Schizosaccharomyces pombe (Kelly et al. 1988) protei n that can only be formed in mated cells (see Her­ and in the multiallelic b gene polypeptides of Ustilago skowit z 1989; Dranginis 1990) . It may be significant maydis (Schulz et al. 1990). Preliminary sequence data tha t a l and a 2 have dissimilar home o domain s (Shepherd for Aa gene alleles of S. commune show that these al­ et al. 1984). Two of the homeo domains in the C. ci­ leles also encode proteins with homeo domain-related nereus AAl mating-type proteins {a2-l and j82-i) are motifs (Ullrich et al. 1991). mor e similar to that of al , and two [pi-1 and (34-1) are mor e similar to that of al (Fig. 7) . Thi s could suggest Transcrip t analysis has enabled us to identify three tha t the interaction that triggers sexual development in othe r genes associated with the specificity genes. The C. cinereus is between nonallelic homeo domain pro­ tight clusters tha t these genes form strongly suggest that teins . Our transformation experiments do not rule out the y are part of the A-factor complex, al and )85 are the thi s possibility because of th e large numbe r of specificity flanking genes and have a DN A sequence that appears to genes present in each factor. be at least partly homologous in the different A factors examined, but there is n o homologou s flanking sequence Th e problem of polypeptide recognition in basidio- betwee n these genes and the adjacent specificity genes mycete s is more complex than in S. cerevisiae because a2 and j84. j84 and j85 are transcribed in opposite direc­ ther e are multipl e alleles, no t just two alleles at th e mat­ tion s with overlapping 3 ' ends, and several long tran­ ing-type locus. In U. maydis there are —25 alleles of the scripts of al, only identified by cDN A cloning (U. Kues, b gene, which controls both mating compatibility and unpubl.), overlap the 5' end of the a2 gene. Th e jSS gene formation of the pathogenic dikaryon (Rowell and is particularl y puzzling: As wit h the specificity genes, its DeVay 1954; Puhalla 1968). Thes e alleles are largely ho- 57 4 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Coprinus A mating-type gene complexes mologou s in sequence, and alleles encode polypeptides revealed one specificity gene at the a locus and two genes in addition to (i4-l at the /3 locu s (U. Kiies et al., wit h only a variable amino-terminal domain (Kronstad unpubl.). As expected from the hybridization data (Fig. and Leong 1990; Schulz et al. 1990). In both hymeno- 6), all of these alleles are different from those present in mycete s studied, C. cinereus shown here and S. com­ A42 . Because bot h sets of specificity genes trigger A-reg- mune (Giasson et al. 1989), alleles of the A mating-type ulate d development in an A3 host, we can conclude that genes do not cross-hybridize, indicating very different at least two of the (3 specificity genes are multiallelic. nucleotid e sequences. This does not preclude some sim­ Thi s study shows that the A mating-type factor is far ilarity at the protein level, as shown by preliminary se­ mor e complex than predicted by classic genetics. With quenc e data for alleles of the same mating-type gene of S. four different genes, all contributin g a uniqu e specificity, commune (Ullrich et al. 1991). Allelic genes are different our analysis provides an insight into how such large enoug h not to cross-hybridize, but their proteins may number s of different A factors have developed in nature. still have conserved regions necessary for polypeptide recognition. This leads to an important problem, high­ lighted in our study. With four specificity genes, appar­ Materials and methods entl y identical in function, how are polypeptides brought together by mating distinguished from those already C. cinereus strains include AAlBAl: JV6 wild type; LN118 ade-2 presen t in the unmate d cell? It seems likely that some of trp-1.1,1.6; A6B6: H9 wild type; LT2 trp-1.1,1.6; A5B6: H5 wild th e specificity genes are redundant. It will require us to type; FA2222 trp-1.1,1.6; A3S1: C692 ade-3; 218 trp-l.1,1.6; A43fi43: OK130 ade-8; ATS ade-8 trp-1.1,1.6. Media and meth­ generate null mutants to demonstrate this, a difficult ods for culturing C. cinereus have been described by Lewis tas k in C. cinereus because homologous gene replace­ (1961), with modifications summarized by Mutasa et al. (1990). men t by transforming DNA is extremely rare (Binningcr The transformation procedure was based on that of Binninger et etal . 1991). al. (1987), as modified by Casselton and de la Fuente Herce (1989). Plasmid pCclOOl, containing the C. cinereus trp-1 gene (Binninger et al. 1987), and pDBl, containing the C. cinereus Multiple A-factor specificities trp-3 gene (D.M. Burrows, T.J. Elliott, and L.A. Casselton, un­ Day (1963) identified 31 A factors in a sample of 33 in­ publ.), were used for cotransformation experiments with trp dependen t isolates of C. cinereus. From this analysis it is auxotrophic C. cinereus strains. Photographs of clamp cells were taken with a Zeiss photomicroscope using phase contrast. predicted that >I60 A factors exist (Raper 1966). We have shown that four genes determine specificity and tha t different A factors can share their alleles. If all allele DNA manipulations combination s arc possible, only four alleles of each gene are required to generate 256 A factors. It is unlikely, Genomic DNAs of C. cinereus strains were prepared either by the method of Wu et al. (1983) or by the small-scale method of however, that these genes are easily reassociated in na­ Zolan and Pukkila (1986). Routine cloning and plasmid ampli­ ture . Separation of the a2 gene into a separate subcom- fication was in Escherichia coli strains XL-1 Blue \recA\, lac , plex allows this to be rccombined with all specificity endAl, gyM96, thi, hsdRll, supEAA, relAl, {¥' proAB, lacl'^, genes in the j3 complex , but the organization within the lacZ^Ml5, TnlO)l (Stratagene) or DH5a (F , endAl, hsdRll /3 comple x suggests that its genes arc rarely rccombined. (r^ , m^ * ), .sup£44, thi , recAl, gyM96, reMl, ^^QMacTMlS] No t only do alleles of th e genes lack sequence similarity, (BRL) using standard DNA techniques (Sambrook et al. 1989). bu t flanking sequences are also nonhomologous (Mutasa et al. 1990). Reciprocal recombination is possible only if a region of homology is presented by a shared p allele. Plasmids constructs with C. cinereus DNA Th e only shared alleles detected in this study were /34-i pECl, pEC12, and pEC16 are recombinant clones from a cosmid and ^5-1. Because of their positions at the border of the library constructed in Lorist2 and contain parts or the whole complex, recombination within these genes would not sequence of the A42 mating-type factor (Mutasa et al. 1990). recombin e the other specificity genes to generate new A Gel-purified fragments or restriction digests of these cosmid factors. clones were subcloned into the polylinker of either pBlue- script(KS-) or pUC13 (pBG-series) and used for restriction and Because of the close linkage of a and j3, th e only esti­ hybridization analysis to map the A42 a and /3 loci and to com­ mate s of allele numbers at each locus are from a small plete the restriction map given earlier (Mutasa et al. 1990). survey of 10 A factors studied by Day (1963), whic h iden­ (Compare Fig. 2 to localize subcloned fragments especially men­ tified four a and five to seven p alleles. Unfortunately, tioned for transformation, hybridization, and sequencing exper­ only tw o of th e characterized factors (A5 and A6) are still iments.) available. Day's studies lead us to expect at least four pBluescript(KS-) subclones of genomic fragments used for Co­ alleles of the a2 specificity gene. A molecular approach prinus transformations pAMTl contains ^1-1 on a 3.0-kb wil l be required to determine whether the three (3 spec­ Hindlll fragment, and pESMl contains 132-1 on a l.S-BamHl- ificity genes are also multiallelic. The A43 factor of C. Sall fragment (Mutasa et al. 1990). pUKl, pUK2, and pUK4- cinereus used in this study has been cloned by May et al. pUK6 have genes al-1, a2-l, ^2-1, (33-1, and )35-2 on Sail frag­ (1991). Th e investigators provide evidence for three spec­ ments of lengths 4.2, 2.1, 3.4, 3.55, and 4.8 kb, respectively. ificity genes in this factor. We have shown here tha t A43 pUK6 contains /35-2, together with an inactive truncated copy of and A42 share the allele of one of these [(34-1) (Fig. 6). )34-l on a 4.8-kb Sail fragment. pESM2 contains the complete Better resolution of AA3 by transformation analysis has /34-J gene on a 4.0-kb BamHl fragment. GENES & DEVELOPMENT 575 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Kiies et al. Subclones used for Noithein analyses pAMTl, pUKl , pESM2, Gel-isolated DNA fragments or plasmid clones for Southern and the following plasmid subclones were used for Northern blots were labeled with [a-'^^P]CTP using a nick-translatio n kit analyses. pUK7 contains a 2.9-kb £coRI-HindIII fragment con­ (BRL). High-activity hybridization probes for RNA blots were taining mos t of th e a2-l gene. pAMT 6 and pAMT 7 contain 4.0- generated by labeling double-stranded DNA (dsDNA) or single- and 1.9-kb Hindlll fragments, respectively, which identify the stranded DNA (ssDNA) with [a-^^PjCTP (>3000 Ci/mmole, noncodin g sequence between the a and /3 genes. pUK3 has a Amersham ) using a random primer kit (Boehringer). dsDNA 3.5-kb Sail fragment containing the fil-l gene. A 0.5-kb Hindlll probes used for Norther n analyses, indicated in Figure 2 bu t not and a 0.4-kb HindIII-£coRI fragment with the 5' end of gene described above, were gel-purified restriction fragments of the /32-i was subcloned after partial restriction of pCE12 to give cosmids or pUC13 and pBluescript(KS - ) subclones. To deter­ pBGl . pBG2 contains a 2.9-kb EcoRl-HindlU fragment with the min e transcript directions, single-stranded DNA s were prepared 3 ' end of gene 132-1, an d pBG3 contains a 2.5-kb Hindlll-EcoRl from pBluescript(KS-) subclones using the helper phage fragment with most of the /33-I gene sequence. pESM3 is a M13K07 (Sambrook et al. 1989), and labeled strands were gen­ subclone of pBG2 containing a 1.2-kb Pstl-Sall fragment. erated using the Klenow fragment of £. coli DNA polymerase I pESM4, pESM5, pESM6, and pUK8 carry a 1.3-kb BamHl- and the M13 reverse primer (Ley et al. 1982; Sambrook et al. Hindlll fragment, a 1.0-kb Hindlll fragment, a 1.7-kb Hindlll- 1989). BamHl fragment, and a 0.7-kb HindIII-£coRI fragment, respec­ tively, each with parts of the /34-i gene sequence. pUK9 has a Construction of a cDNA library 1.0-kb £coRI-B<3mHI fragment carrying part of /35-1. pLAC l was isolated from a plasmid genomic library of C. A XgtlO cDNA library was contructed with mRNA isolated cinereus strain H9 constructed by Pukkila et al. (1984), using from wild-type strain JV6 and the cDNA synthesis system plus th e A42 a2-l gene as hybridization probe. pLACl contains the and cDNA cloning system-X.gtlO from Amersham (RPN 1256 tw o genes of the A6 a locus . Because A6 and ^442 have the same and RPN 1257) using the recommended £. coli strain NM514 alleles of these two genes, fragments from pLACl subcloned {hsdRSl4 (r^ m,, ) argH, galE, galX, StrA, lycB7* (Hfl^)] as int o pBluescript(KS - ) could be used as gene probes in Northern host. The library was screened with pBluescript(KS-) sub­ hybridizations . pHH5 and pHH7 contain the 5' and 3 ' ends of clones. Screening of 10"^ t o lO'" plaque s was needed to detect one th e al-1 gene cloned on 2.4-kb and 1.1-kb £coRI fragments, copy of each gene. respectively. pUKlO contains part of the a2-l gene on a 2.0-kb Clal-BamHl fragment. Sequencing Subclones used for Southern analysis pUKl-pUK6 were used All sequencing was performed with dsDNA of pBlue- to detect homology between different ^4 2 genes. cDNA s for all script(KS - ) or pUC13 subclones, "'''S-labeled ATP, the T7 poly­ A-gene transcripts were subcloned from \gtlO into pBluc- merase kit of Pharmacia, and either the M13 universal or re­ script(KS- ) with either the £coRI or the BamHl sites of the verse primers. Home o domain motifs (Fig. 7) were deduced from \gtl O adapter. These were used to detect homoallclic genes in partial sequence data of cDN A clones. Transcription directions genomic DNA digests. The length of the homologous hole was of all genes were confirmed by sequence analysis. cDN A 3 ' ends defined using gcl-elutcd fragments of pAMT6 and pAMT7. were located within specific genomic fragments identified by th e transcript mapping analysis. The results of these analyses RNA isolation can be summarized with respect to the gene, the plasmid con­ struct, sequence, and relative position on the restriction map Strain JV6 was grown in liquid minimal medium, and total show n in Figure 2: al-1, pHH7, TCAGAATCCA'TTCTC- RNA was prepared using the guanidinium thiocyanate-CsCl ACGGG , 0.25 kb upstream of the BamHl site; a2-l, step gradient procedure (Glisin et al. 1974; Sambrook et al. pAMT6A£coRI, TTGCATTTCA'AGCACATTTC , 0.12 kb up­ 1989), wit h the modification suggested by Chirgwi n et al. (1979) strea m of the £coRI site; (31-1, pAMTl, ATCCAGCATA*C- of resuspending the pellet in 7.5 M guanidiniu m hydrochloride TATCGACCA , 0.3 kb upstream of the Hindlll site; p2-l, after the ultracentrifugation step. Standard extractions were pESMl , CCGATCGCTA*GTTGCAATAC , 0.2 kb downstream from 10 gram s wet weight mycelium ground in 10 m l of extrac­ of the Pstl site; fi3-l, pBG3, TCTGCCTCCA-GAGTGGCAAA, tion buffer with a polytron homogenizer (Kinematica PTlO/35). 0.5 kb upstream of the £coRI site; (M-1 and /35-2, pUK8 mRN A was selected using oligo(dT) cellulose (Pharmacia) as C AAATAC AA G *' TA G AC AAGC ATTTGGGTTCCTTT C A * ^- described by Sambrook et al. (1989). For Northern blotting, ATCCTCGTAG , 0.05 kb from the £coRI site. Th e asterisk in mRN A (5 jJLg) was denatured at 55°C for 15 min with 17.5% each case indicates the polyadenylation site. *' marks the po­ formaldehyde and 50% formamide and electrophoresed on a 1 % sition of the pA site of the 135-1 gene transcript in the opposite agarose gel containing 17.5% formaldehyde. An RNA ladder of DN A strand; *^ indicate s the end of the ^4-1 transcript. 0.24—9.5 kb (BRL) was used to determine transcript sizes. Acknowledgments Nucleic acid blots and hybridizations Gels prepared for Southern or Northern analyses were blotted We thank Flora Banuett for her critical reading of the manu­ ont o Hybond- N membranes (Amersham) in 20 x SSC (3 M NaCl , script and her valuable discussion, and Georgiana May for the 0.3 M sodiu m citrate). For Southern blots, hybridizations were cloned A43 factor. This work was supported by Science and performed overnight at 65°C as described by Mello n et al. (1987). Engineering Research Council grants GR/E90175 and GR/ Hybridization s of Norther n blots and washes were performed as F78781 to L.A.C. and studentship to A.M.T., and a Gatsb y Char­ recommende d for Hybond-N by the manufacturers. For autora­ itable Foundation Sainsbury studentship to W.V.J.R. diography, filters were exposed to Fuji X-ray film. Filters for Th e publication costs of this article were defrayed in part by bot h Southern and Northern analyses were stripped (see man­ paymen t of page charges. This article mus t therefore be hereby ufacturers ' instructions) and reused to allow direct size compar­ marke d "advertisement" in accordance with 18 USC section isons. 1734 solely to indicate this fact. GENES & DEVELOPMENT 576 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press Copiinus A mating-type gene complexes Pukkila , P.J., B.M. Yashar, and D.M. Binninger. 1984. Analysis References of meiotic development in Copiinus cineieus. Controlling Binninger, D.M., C. Skrzynia, P.J. Pukkila, and L.A. Casselton. event s in meiosis. Symp. Soc. Exp. Biol. 38: 177-194. 1987. DN A mediated transformation of the basidiomycete Raper, C.A. 1983. Control s for development and differentiation Copiinus cinereus. EMBO /. 6: 835-840. of th e dikaryon in Basdiomycetes. In Secondary metabolism Binninger, D.M., L. LeChevanton, C. Skrzynia, CD . Shubkin, and diffeientiation in fungi (ed. J.W. Bennet t and A. Ciegler), and P.}. Pukkila. 1991. 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Papazian, H.P. 1951. Th e incompatibility factors and a related gene in Schizophyllum commune. Genetics 36: 441-459. Puhalla, J.E. 1968. Compatibilit y reactions on solid mediu m and interstrai n inhibition in Ustilago maydis. Genetics 60: 461 - GENES & DEVELOPMENT 577 Downloaded from genesdev.cshlp.org on October 25, 2021 - Published by Cold Spring Harbor Laboratory Press The combination of dissimilar alleles of the A alpha and A beta gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus. U Kües, W V Richardson, A M Tymon, et al. Genes Dev. 1992, 6: Access the most recent version at doi:10.1101/gad.6.4.568 This article cites 30 articles, 8 of which can be accessed free at: References http://genesdev.cshlp.org/content/6/4/568.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. 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