physical mapping of meiotic crossover events in a 200-kb ......physical mapping of crossovers 1-...

Copyright 0 1993 by the Genetics Society of America

Physical Mapping of Meiotic Crossover Events in a 200-kb Region of Neurospora crassa Linkage Group I

Mario R. Mautino, Sergio D. Haedo and Albert0 L. Rosa'

Departamento de Quimica Biolbgica (CIQUIBIC-CONICET), Facultad de Ciencias Quimicas, Universidad Nacional de Cbrdoba, 5016-Cbrdoba, Argentina

Manuscript received March 8, 1993 Accepted for publication April 29, 1993

ABSTRACT We propose a general restriction fragment length polymorphism-based strategy to analyze the

distribution of meiotic crossover events throughout specific genetic intervals. We have isolated 64 recombinant chromosomes carrying independent meiotic crossover events in the genetic interval eth- 1-un-2 on linkage group I of Neurospora crassa. Thirty-eight crossover events were physically mapped with reference to a 200-kb region cloned by chromosome walking, using N . crassa X and cosmid libraries. Crossovers were homogeneously distributed at intervals of 5.0 f 2.3 kb along the entire cloned interval. The ratio of physical to genetic distance appears to be higher in the region than in the overall N . crassa genome, suggesting that recombinational activity is less in large chromosomes than in small ones. The present work provides a method for defining the centromeric-telomeric orientation of single cloned DNA fragments. Their physical distance can also be estimated with respect to linked loci, provided that crossover events are distributed homogeneously in the interval. This strategy overcomes typical difficulties in defining the position and direction of chromosome walking steps on conventional linkage maps.

-I ,-

C URRENT progress in genome mapping projects is unraveling the hidden arrangement of lower

and higher eukaryotic chromosomes (OLIVER et al. 1992; NIH/CEPH 1992). The fungus Neurospora crassa is a suitable organism for studying patterns of genome organization and function (PERKINS 1992a). Cytological aspects of the small N . crassa chromosomes have been studied (PERKINS and BARRY 1977), and particular chromosomal domains such as telom- eres and the nucleolus organizer (NOR) are currently being explored (BUTLER and METZENBERG 1989; SCHECHTMAN 1990; BUTLER 1992). Detailed classical and restriction fragment length polymorphism (RFLP)/rapid amplified polymorphic DNA (RAPD) maps are available (PERKINS 1992b; METZENBERG and GROTELUE~CHEN 1992). The genetics of Neurospora has been developed for more than 60 years. The organism is an abundant resource of mapped mutants (-700 loci defined) and chromosomal rearrangements stocks (-300) (PERKINS 1992a,b; PERKINS and BARRY 1977), and well developed genetic methodologies (DAVIS and DE SERRES 1970) making it a powerful model for examining chromosome function and organization.

We are studying the N . crassa centromeric locus sn (snowflake; MITCHELL 1959; ROSA, ALVAREZ and MAL- DONADO 1990; HAEDO et al. 1992; TEMPORINI and ROSA 1993; ALVAREZ et al. 1993). To clone sn, we

' To whom correspondence should be addressed.

Genetics 134: 1077-1083 (August, 1993)

have initiated a convergent chromosome walk toward the centromeric region (CENZ) of linkage group I (LGI), starting from cloned DNA fragments which flank CENZ. A general strategy, based on the use of RFLP markers (BOTSTEIN et al. 1980) and classical genetic analyses, has been developed to physically map crossover events throughout specific genetic intervals and to define the centromere-distal and proximal ends of single cloned DNA fragments. The method allows the physical distance of these fragments to be estimated with respect to linked loci, when crossovers are homogeneously distributed in the region. We describe here the use of this approach to study the correspondence between genetic and physical maps on a 200-kb cloned region around the N . crassa locus arg-3, which is closely linked to sn and CENZ.

MATERIALS AND METHODS

Strains, culture conditions and crosses: N . crassa mutants eth-1 (FGSC 121 2/1220), csp-1 (FGSC 2554), mei-3 (FGSC 2764), un-2 (FGSC 1956), arg-3 (MEP35) (FGSC 3844), T(I+ZZ)39311; to1 trp-4 (FGSC 2976), and wild type strains 74-OR23-1A (Oak Ridge; FGSC 987) and 25a (Lin- degren; FGSC 353), were obtained from the Fungal Ge- netics Stock Center. Standard N. crassa methodologies were used (DAVIS and DE SERRES 1970). Ethionine (Sigma) was added to sterile media at 50 Mg/ml. Partially duplicated strains were isolated among the progeny of the cross Normal Sequence X T(I+IZ)39311; to1 trp-4 (PERKINS 1986). The genetic background at the CENI region in eth-1 strains was defined as Oak Ridge, while T(I+II)39311; to1 trp-4 and un- 2 strains were defined as Lindegren, by RFLP analysis. The

1078 M. R. Mautino, S. D. Haedo and A. L. Rosa

overall genome in these strains is largely Oak Ridge (Fungal Genetics Stock Center 1992). Oak Ridge (FGSC 987) and Lindegren (FGSC 353) wild-type genetic backgrounds are highly polymorphic for RFLPs (HAEDO, MAUTINO and ROSA 1992; S. D. HAEDO and A. L. ROSA, unpublished results). N. crassa libraries and screening procedures: Colony

replicas of the 74-OR23-1A ordered genomic cosmid libraries pSV50 (3072 independent clones; VOLLMER and YAN- OFSKY 1986) and pMOcosX (4800 independent clones; con- structed by M. SACHS and M. ORBACH, and distributed by FGSC) were made on Hybond-N (Amersham) nylon mem- branes (8 X 12 cm) at a density of 384 colonies/filter. These libraries were stored in multiwell plates at -70” (VOLLMER and YANOFSKY 1986). A 74-OR23-1A hJ1 library (ORBACH, PORRO and YANOFSKY 1986) was plated on Escherichia coli K802 and processed and hybridized as indicated (ALLDAY and JONES 1987; SAMBROOK, FRITSCH and MANIATIS 1989). Oligolabeled DNA probes (FEINBERG and VOGEISTEIN 1983) and riboprobes (LITTLE and JACKSON 1989) were obtained by using Klenow and T3/T7 RNApol enzymes from Promega. [a-’*P]dATP (3000 Ci/mmol) and [‘Y-’~P] UTP (800 Ci/mmol) were from NEN-Du Pont.

DNA manipulations and protoplast transformation: Total N . crassa DNA was purified and subjected to Southern blot analysis as described (HAEDO et al. 1992). Cosmid and h phage DNA was purified following published protocols (SAMBROOK, FRITSCH and MANIATIS 1989). Protoplasts were prepared and transformed as indicated (VOLLMER and YAN- OFSKY 1986). Benomyl (I .O rg/ml; pSV50 clones) or hygromycin (250 Pg/ml; pMOcosX clones) were included in the bottom media to select transformant cells (VOLLMER and YANOFSKY 1986). In some experiments, protoplasts were co-transformed with pMOcosX clones and the plasmid pBT6, selecting colonies resistant to benomyl (ORBACH, PORRO and YANOFSKY 1986). About 80% of these Bml’ transformants were Hyg‘. Novozyme 234 and benomyl (95%) were from Novo BioLabs (Denmark), and Du Pont (United States), respectively. Hygromycin B was from Cal- biochem (398 units/mg solution).

RESULTS

Analysis of cosmid clones linked to CENI: CENZ is included between the proximal breakpoints of the insertional translocations T(Z+ZZ)3931 I and T(Z+ VZI;Z;V;VZZ)ARI73, which involve part of the left and right arms of LGI, respectively (Figure 1A) (PERKINS and BARRY 1977). Cosmid clones 28: 12D and 26: 1 1 B, complementing the mutation arg-3 (Figure 1A) have been isolated from the pSV50 N . crassa library (VOLL- MER and YANOFSKY 1986). We confirmed the linkage of the DNA inserts contained in 28: 12D and 26: 1 1 B to CENZ by RFLP segregation analysis (not shown). Two additional experiments confirmed linkage to this region. First: EcoRI, EcoRV and XbaI RFLPs detected in genomic DNAs of parental strains 74-OR23-1A (Oak Ridge) and T(I-dZj3931 I (Lindegren) (see MA- TERIALS AND METHODS section), by using either 28:lZD or 26:llB as probes, were heterozygous in partially duplicated Dp(Z+ZZ)39311 progeny strains isolated from the cross 74-OR23-1A X T(I+IZ)39311. Thus, these clones map to the left of the centromeric breakpoint of T(Z+ZZ)39311, within the chromosomal fragment duplicated in Dp(Z-+ZZ)3931 I strains. Sec-

ond, 28:12D, used as a probe, detected aberrant EcoRI and EcoRV hybridizing bands in genomic Southern blots of the arg-3 mutant MEP35. This mutant has been genetically characterized as a translocation event inseparable from arg-3 (PERKINS et al. 1982). Thus, 28: 12D overlaps the arg-3 linked breakpoint occurring in MEP35 (data not shown).

Cosmids 28: 12D and 26: 1 1 B were physically characterized by digestion with the restriction enzymes EcoRI and Not1 (Figure 1B). In order to determine the centromeric and telomeric ends of the 50-kb region defined by 28: 12D and 26: 1 lB, the cosmids were used in two sorts of experiments: (i) as probes in genomic Southern blots of the strains 25a-Lindegren and T(Z+ZZ)3931 I (see MATERIALS AND METHODS), in which identical EcoRI, KpnI and Not1 hybridizing band patterns were found between Normal Sequence and Translocated strains, indicating that the cloned region did not span the T(Z-+ZZ)3931 I breakpoint; and (ii) in protoplast transformation experiments of mutants csp-2, mei-3 and eth-1, all closely linked to arg-3 (Figure 1A). Among these mutants, putative products for eth-1 (S-adenosylmethionine synthetase) (METZ- ENBERG, KAPPY and PARSON 1964; KERR and FLAVIN 1970; JACOBSON, CHEN and METZENBERG 1977) and mei-3 (RecA-like protein) (CHENG et al. 1993) have been defined. Recessiveness of the csp-1, mei-3 and eth-1 phenotypes (PERKINS et al. 1982) was confirmed by using forced primary heterokaryons carrying vari- able gene doses (nuclear input ratios 10: 1, 1: 1 and 1: 10) of the wild type and mutant genes (DAVIS and DE SERRES 1970; TEMPORINI and ROSA 1993) (not shown). Cosmids 28:12D and 26: 11B failed to com- plement the phenotype of any of these mutants.

Thus, from these experiments it was not possible to orient the small “arg-3” cosmid contig (50 kb) on the chromosome.

Chromosomal orientation of 261 1B by mapping crossover events in the arg-3 region: We defined the chromosomal orientation of the cloned region around arg-3 by mapping the right and left ends of the DNA insert contained in cosmid 26: 1 1B to regional crossover events. The cross eth-1 a X un-2 A was designed (see MATERIALS AND METHODS section) to isolate a unique type of recombinant progeny, wild type in this study, carrying segregating RFLP types. These RFLPs will be Lindegren to the left and Oak Ridge to the right of each crossover point (Figure 2). The eth-l- linked marker mt (mating type), on the left arm of LGI (Figure 2), was scored among the recombinant progeny to discard strains in which an additional, external crossover event had taken place.

Sixty-four wild-type recombinant individuals were isolated as ascospores germinating in minimal medium at 39“ (eth-I and un-2 strains did not growth at 39”) (HOULAHAN, BEADLE and CALHOUN 1949; METZEN-

Physical Mapping of Crossovers

T393 11

1- CEN’ L TAR173

arg- 7 eth- 1 arg-3 csp-1 (sn os-4) un-2

1079

A

- 50 kb

B

E N N E ’ E E N N E E N E

1 1 E 8 E 26:i 1 B

28:i 2D

- 5 kpb

FIGURE 1.-Genetic and physical map of the region surrounding the locus arg-3 on the N . crassa LGI. (A) Linkage map. Positions of relevant loci and translocation breakpoints are indicated. Genetic linkages are from published data (PERKINS et al. 1982; PERKINS 1992b) and from our laboratory (A. L. ROSA and M. R. MAUTINO, unpublished). Physical size of CENI is unknown and this uncertainty is represented as an empty space between square brackets. The loci sn and os-4 map between the proximal breakpoints of translocations 39311 and AR1,73; their position with respect to CENI is not known (PERKINS et al. 1982; TEMPORINI and ROSA 1993). The locus mei-3 (not drawn) is probably located right of eth-1 and arg-3 (1%; PERKINS et al. 1982). Bars of 50 and 200 kb spanning the locus arg-3 represent pSV50 (28:12D)-26:1lB) and pMOcosX (Cl-C13) cosmid contigs, respectively. For details see text and Figure 4. (B) EcoRI (E) and Not1 (N) restriction map (“Oak Ridge”) of the 50-kb genomic region covered by insert DNAs of cosmids 26: 1 1B and 28: 12D. Restriction fragments I IE and 8 E were used to define the chromosome orientation of 26: 1 1 B. The arg-3 locus should be included in the region where cosmids overlap. A “Lindegren” EcoRI site (arrowhead) defining RFLP I1 (see Figure 3) is shown.

BERG, KAPPY and PARSON 1964; JACOBSON, CHEN and METZENBERC 1977). All of them were Eths (ethionine sensitive), grew as wild type at 39”, and were of mating type A . Physical mapping of crossover events in these recombinant chromosomes was performed by using different DNA probes on Southern blots of genomic DNA (Figure 2; see below). Initial DNA probes in- clude the Z ZE and 8E EcoRI DNA fragments mapping at the left and right ends of 26: 1 lB, respectively (Figure 1B). Figure 3A shows the segregation of an EcoRI RFLP detected with the probe 8E (RFLP 111) in the parental strains eth-Z and un-2, among the 64 wild type progeny. As can be seen, chromosomes Z of 15 progeny (8, 11, 17, 19, 23, 24, 29, 30, 31, 37, 43, 45, 56, 60 and 62) originated by crossing over to the left of the RFLP 111, while the remaining strains resulted from crossover events to the right (Figure

3B). A similar analysis with an EcoRI RFLP detected with the probe ZZE (RFLP 11) showed that 11 chromosomes I of the 64 have originated from crossovers left of this marker. Thus, RFLP I1 is closer than RFLP I11 to eth-I. Taken together, the results indicate the order: eth-I-RFLP 11-RFLP 111-un-2 (Figure 3B).

The experiments show that four crossover events had taken place in the 21.3-kb interval between RFLPs I1 and I11 (Figures 3B and 4A). Assuming that the distribution of the 64 crossover events isolated in this study is homogeneous, the eth-Z mutation is predicted to be 60 kb left of the RFLP 11. Thus, to clone eth-I, a chromosome walk should be initiated in the direction arg-3 + eth-2, using Z ZE as the probe (Figure 3B).

Molecular cloning of a 200-kb region surrounding the arg-3 locus: To study a larger chromosomal re-

1080 M. R. Mautino, S . D. Haedo and A. L. Rosa

a eth-7 un-2 +

1 1 2 3 4 5

" _ " " " " - - - - - - -.

_ " I I

a b

FIGURE 2.--Isolation of recombinant chromosomes carrying marked crossover events in the interval eth-1-un-2. A cross is made between efh-1 a and un-2 A strains which possess Oak Ridge and Lindegren genetic backgrounds, respectively (filled and dashed lines), which are highly polymorphic for restriction sites ( S . HAEDO and A. L. ROSA, unpublished results). A unique type of recombinant progeny (wild type in the scheme) is selected. Position of the crossover point in each recombinant chromosome ( I , 2 , 3, 4, 5 ) is marked by flanking, recombinant RFLP types and could be physically mapped in Southern blot analyses by using flanking probes (a and b) .

gion in the interval eth-2-un-2, we performed nine chromosomal walking steps on a 4800-clone ordered N . crussu cosmid library ("pMOcosX"; see MATERIALS AND METHODS). A total of 13 cosmid clones were isolated and characterized at both sides of urg-3 (Fig- ure 4B). A physical map of the cloned region, which covers about 200 kb around urg-3, was obtained by single and double digest restriction analyses of the cosmid clones. In addition, we confirmed the entire map, and its colinearity with the chromosome, by Southern blot analyses (not shown). EcoRI, EcoRV and Not1 maps are shown in Figure 4A. Studies of heterozygous RFLPs and hybridizing band patterns in genomic Southern blots of the strains Dp(Z+ZZ)39322 and T(Z+ZZ)39322, using the entire cosmid C13 as a probe (Figure 4B), indicated that the entire contig is left of the T(Z+ZZ)39322 proximal breakpoint.

Mapping of regional crossover events in the 64 recombinant chromosomes Z isolated from the cross eth-2 X un-2, was performed in different experiments by using the cosmids C2, C12 and C13 (Figure 4, B and C). Linkage of EcoRI RFLPs I , 11, IV and V detected with these probes (Figure 4C), to the 64 crossover events, was performed as indicated above and in Figures 2 and 3. Results of these experiments are presented in Figure 4C. The final map included 38 crossover events positioned in the 200-kb cloned

A 0 L 1 2 3 d 5 6 7 8 9 1 0 1 1 1 2 I 3 1 4 1 5 1 6

0 L 17 IO 1 9 20 21 22 23 24 25 26 27 20 29 30 31 32

0 L 33 34 35 36 37 30 30 40 I1 4 2 43 44 I 5 4 6 I 7 I8

0 L I 9 50 51 52 53 54 55 56 57 50 59 60 61 62 63 64

B 11 4 49

I I I l l - eth- 7 arg-3 un-2

t t 1 l E 8E -

FIGURE 3.-Chromosomal orientation of the cosmid 26: 1 1 B by regional crossover linkage mapping. (A) Southern blot analysis of genomic DNAs from 64 wild-type individuals (1-64) randomly isolated among the progeny of the cross diagrammed in Figure 2. The probe used was the EcoRI restriction fragment 8E (see Figure 1B) which detects a Oak Ridge/Lindegren RFLP (111) with the alternative forms of 10.0 kb (eth-1 strain) (0, Oak Ridge) and 13.5 kb (un-2 strain) (L, Lindegren). (B) Linkage relationships and distribution of 64 crossover events in the interval el-1-un-2 with reference to the RFLP markers I1 (detected with probe I l E ) and 111 (detected with probe BE). I l E and 8E probes correspond to the EcoRI end restriction fragments of the cosmid 26: 1 1 B (Figure 1 B).

region and 26 crossovers mapped between RFLP V and un-2.

The experiments support the postulated orientation of cosmid 26:€ 1B (Figure 3B), and show that the distribution of crossovers, between successive EcoRI RFLPs, was about one recombinational event occurring each 5.0 f 2.3 kb, in the set of chromosomes analyzed. No great changes in crossover distribution

1081

6

C

10 kb

26:11 B

28 120 L1 - c1

c3 c2

c4 C8

c9 c5 C6

- c10 La - - c11

+- c7 - c12

C13

6 5 4 14 9 26 ”L__i”,L[ j“

I 1 1 1 1 1 I

I I [ ’ 1 eth- 1 arg-3 un-2

T T 11E- 8E -

c2 c12

C13

FIGURE 4.-Physical positioning of meiotic crossover events on a 200-kb restriction map around the locus arg-3. (A) EcoRI, EcoRV and Not1 restriction maps (“Oak Ridge”) of the 200-kb region cloned by bidirectional chromosome walking around the locus arg-3. EcoRI sites (arrowheads) defining RFLPs I, 111, IV and V are “Oak Ridge.” The EcoRI site defining RFLP I1 is “Lindegren” (see Figure 1B). (B) Relative position of the insert DNAs present in pSV50 (26:l lB, 28:12D), pMOcosX (Cl-C13), and XJ1 (Ll,L2) clones, characterized in the 200-kb cloned region. (C) Distribution of the 64 crossover events in the interval eth-l-un-2. Number of crossover events taking place between eth-l and RFLPs I to V is indicated in the upper row. The physical distance between the right end of clone C13 and un-2, and the physical distribution of the 26 crossovers occurring between RFLP V and un-2 is unknown. This uncertainty is indicated by an empty space between square brackets. Position of EcoRI RFLPs (I-V) and DNA probes (C2, 1 lE, 8E, C12, C13) used to map the position of crossovers is also indicated.

were observed along the cloned region, close or distal to the centromere. If it is assumed that there is no crossover interference in the centromeric region, un- 2 would be -320 kb from eth-1 and the right end of the cloned region about - 130 kb from un-2, with the centromere of chromosome Z included in this chromosomal interval. However, as crossover suppression is a well described phenomenon in centromere regions of several fungal species (NAKASEKO et al. 1986; YOSH- IKAWA and ISONO 1990), the distance between the right end of the cloned region and CENZ may be much longer than estimated above (see DISCUSSION).

Molecular cloning of the eth-l gene: The results presented above suggested that eth-I should be at approximately 30 k 14 kb left of RFLP I, on either clone C 5 , L2 or C7 (Figure 4C). We prepared protoplasts from the eth-1 mutant and transformed them, in separate experiments, with the cosmids C 4 , C5 and C7 (Figure 4C; see MATERIALS AND METHODS). Trans- formed strains were scored for reversion of the ther- mosensitive phenotype associated with the eth-l mu-

tation (METZENBERG, KAPPY and PARSON 1964; JA- COBSON, CHEN and METZENBERG 1977). Only C7 transformed eth-1 to a vigorously growing phenotype at 39”. Additional experiments (M. R. MAUTINO and A. L . ROSA, unpublished data) indicate that eth-1 has been cloned. The experiments show that both the chromosomal direction and distance between the left end of the “arg-3” contig (Figure 1B) and the eth-I gene were as predicted from the regional crossover linkage analysis.

DISCUSSION

We have developed a general strategy to determine the distribution of crossover events in small genetic intervals defined by successive RFLPs. The approach combines previously characterized loci, anonymous RFLPs and classical genetic analysis, and allows the chromosomal orientation of single cloned DNA fragments to be established by mapping the ends of a clone to regional crossover events. In chromosome walking protocols, a specific direction can be accu-

1082 M. R. Mautino, S. D. Haedo and A. L. Rosa

rately defined and the physical distance to a target locus can also be estimated. To make this estimation, an homogeneous distribution of crossover events in the region must be assumed. It is necessary to note that this assumption would give wrong estimates of physical distances for larger chromosomal intervals, where suppression or enhancement of recombinational activity can take place (OLIVER et al. 1992). This strategy makes it possible to avoid bidirectional walking steps typically used to find the desired orientation of DNA clones on the chromosome (MCCLUNG, Fox and DUNLAP 1989; CABIBBO et al. 1991). The approach was successfully applied to define the orientation of a small genomic DNA fragment (38 kb). The physical distance between the distal end of this fragment and eth-1 was estimated from the number of crossover events occurring between two EcoRI RFLPs defined by the ends of the fragment. eth-1 was local- ized at the expected molecular distance, and molecular cloning of eth-1 was achieved by chromosome walking and confirmed by protoplast transformation and phenotypic rescue of the eth-1 mutant.

The strategy described here would be of general and direct application in any haploid organism. It could also be used in diploid organisms where appro- priate methods are available for selecting regional crossover events between linked loci. When the available pair of mutants are of identical genetic backgrounds, a first cross would be required to isolate a double mutant. The double mutant would then be crossed against the “exotic” parent. A continuum set of RFLP-marked crossover events, distributed throughout the region, could be isolated among any of the two alternative types of single mutant progeny from this cross. To check the progress of chromosome walking steps in large molecular cloning projects, different sets of RFLP-marked chromosomes, spanning several contiguous genetic intervals, can be used as landmarks of chromosomal position. To find the chromosomal orientation of cosmid or YAC clones carrying phage RNA polymerase promoters (e.g., T3/T7), no restriction map is necessary to define the ends of the DNA start clone, and riboprobes can be used to map regional crossover events. We are using this general approach to analyze the relationship between physical and genetic maps along the entire CENI region (M. R. MAUTINO, s. D. HAEDO and A. L. ROSA, unpublished).

Physical mapping of 38 crossover events in the 200- kb cloned region of LGI suggests that these events are distributed in a nearly homogeneous fashion in- cluding those in the cloned region more closely linked to the centromere. Other workers have found marked suppression of recombination across the N . crassa CENVII, where 0.02 map unit in the interval qa-2- met-7 corresponds to 440kb (1 map unit, -22,000 kb;

M. CENTOLA and J. CARBON, personal communica- tion). These results suggest that our cloned region on LGI is still far from CENI and from the region of strong centromeric crossover suppression. Although existing data on recombination values in the vicinity of CENI may be of limited value to establish physical:genetic ratios, due to the local action of rec genes (CATCHESIDE and CORCORAN 1973) and to the putative regional influence of the centromere, values observed for the intervals his-2-nuc-1 (0.37 map unit, -23 kb; METZENBERG and CHIA 19’79; KANG and METZENBERG 1990), un-2-his-2 (1 map unit, >80 kb; S. D. HAEDO, M. R. MAUTINO and A. L. ROSA, unpublished results), arg-3-csp-1 (1 map unit, >lo0 kb; V. ALVAREZ, M. R. MAUTINO and A. L. ROSA, unpublished results), and eth-1-arg-3 (1 map unit, -80 kb; this work), are consistently 2-3 times higher than ratios observed in a small chromosome such as LGVII (see MCCLUNG, Fox and DUNLAP 1989; PAIETTA 1989; CABIBBO et al. 1991). This observation could be supported by the KABACK’S (KABACK, STEENSMA and DE JONGE 1989; KABACK et al. 1992) rule: if at least one meiotic crossover event in each pair of homologous chromosomes is required to ensure ap- propriate pairing and segregation (BAKER et al. 1976), physical distances between loci in a large chromosome should be larger than expected from the ratio of physical and genetic maps in the overall genome.

We would like to thank to members of the Neurospora group at Cbrdoba and M. E. ALVAREZ, C. ARGARA~A, C. LANDA and H. J. F. MACCIONI for comments and suggestions about the manuscript. We are grateful to M. CENTOLA and J. CARBON for sharing with us unpublished data about CENVII, and to M. ORBACH, M. SACHS, S. VOLLMER and C. YANOFSKY for making the N. crassa libraries, vectors and plasmid pBT6 used in this work freely available at the Fungal Genetics Stock Center (FGSC). Libraries and mutant strains were provided by C. WILSON and P. HUBBARD from the FGSC. We thank to Novo BioLabs (via Crispex S.R.L.) and S. R. FOOR (Du Pont) for gifts of Novozym 234 and benomyl, respectively. This work was supported by grants from Fundacion Antorchas (Argen- tina), Conicet (Consejo Nacional de Investigaciones Cientificas y Tkcnicas), Conicor (Consejo de Investigaciones Cientificas y Tkc- nicas de Cbrdoba), and Third World Academy of Sciences (Trieste, Italy).

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Communicating editor: R. H. DAVIS

physical mapping of meiotic crossover events in a 200-kb ......physical mapping of crossovers 1-...

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