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Copyright 1998 by the Genetics Society of America
Meiotic Chromosome Morphology and Behavior in zip1 Mutantsof Saccharomyces cerevisiae
Kuei-Shu Tung† and G. Shirleen Roeder*,†,‡
*Howard Hughes Medical Institute, †Department of Molecular, Cellular and Developmental Biology and ‡Department of Genetics,Yale University, New Haven, Connecticut 06520-8103
ABSTRACTThe yeast Zip1 protein (Zip1p) is a component of the central region of the synaptonemal complex
(SC). Zip1p is predicted to form a dimer consisting of a coiled-coil domain flanked by globular domains.To analyze the organization of Zip1p within the SC, in-frame deletions of ZIP1 were constructed andanalyzed. The results demonstrate that the C terminus but not the N terminus of Zip1p is required forits localization to chromosomes. Deletions in the carboxy half of the predicted coiled-coil region causedecreases in the width of the SC. Based on these results, a model for the organization of Zip1p withinthe SC is proposed. zip1 deletion mutations were also examined for their effects on sporulation, sporeviability, crossing over, and crossover interference. The results demonstrate that the extent of synapsis ispositively correlated with the levels of spore viability, crossing over, and crossover interference. In contrast,the role of Zip1p in synapsis is separable from its role in meiotic cell cycle progression. zip1 mutantsdisplay interval-specific effects on crossing over.
MEIOSIS is essential for diploid organisms to gener- some organisms, full-length axial elements are formedate haploid gametes. Meiotic cells undergo a sin- before the initiation of synapsis (reviewed by von Wett-
gle round of DNA replication followed by a lengthy stein et al. 1984). In wild-type Saccharomyces cerevisiae,prophase and two successive nuclear divisions. During synapsis initiates before axial element formation is com-prophase of meiosis I, homologous chromosomes pair plete (Dresser and Giroux 1988; Alani et al. 1990;and recombine to ensure their proper segregation at Padmore et al. 1991). Throughout this paper, synapsismeiosis I (reviewed by Roeder 1997). is defined as SC formation and is recognized cytologi-
Pairing of homologous chromosomes culminates in cally by the appearance of two parallel lateral elementsthe formation of a proteinaceous scaffold called the separated by a uniform distance.synaptonemal complex (SC), which is morphologically Another prominent and essential feature of meiosisconserved across a wide variety of species (reviewed by is a high level of genetic recombination. Crossing overvon Wettstein et al. 1984). The SC consists of two results in the reciprocal exchange of genetic informa-dense parallel structures called lateral elements, sepa- tion and is essential for proper chromosome segregationrated by a less dense central region. In organisms that at meiosis I. Meiotic recombination and SC formationare particularly favorable for cytological study, two dis- are concurrent events in S. cerevisiae (reviewed by Petes
tinct substructures can be observed within the central et al. 1991; Roeder 1995, 1997; Kleckner 1996). In thisregion (Moses 1968; Wettstein and Sotelo 1971; organism, most or all meiotic recombination is initiatedSchmekel et al. 1993a,b; reviewed by Schmekel and by double-strand breaks (DSBs). DSBs occur early inDaneholt 1995). The central element runs longitudi- prophase before synapsis begins (Padmore et al. 1991).nally along each complex, equidistant between the lat- DSBs are converted to double Holliday junctions arounderal elements; transverse filaments cross the central re- the time of SC formation (Schwacha and Kleckner
gion, lying perpendicular to the longitudinal axis of the 1994). Mature crossover products are detected near theSC. Many transverse filaments span the entire width of end of pachytene, as the SC disassembles (Padmore
the SC from one lateral element to the other, while et al. 1991; Goyon and Lichten 1993; Nag and Petes
others terminate at the central element. The lateral 1993). The SC has been postulated to play a role inelements of the SC are separated by z100 nm through- crossover interference, which decreases the probabil-out the entire length of each pair of homologs. Each
ity of crossing over in regions that have already under-lateral element corresponds to the protein backbone of
gone exchange (Egel 1978; Maguire 1988; King andone pair of sister chromatids and is referred to as an
Mortimer 1990).axial element before it becomes part of mature SC. InIn S. cerevisiae, the ZIP1 gene encodes a component
of the central region of the SC (Sym et al. 1993; Sym
and Roeder 1995). The Zip1 protein (Zip1p) localizesCorresponding author: G. Shirleen Roeder, Department of Molecular,
to synapsed meiotic chromosomes but not to unsyn-Cellular and Developmental Biology, Yale University, P.O. Box 208103,New Haven, CT 06520-8103. E-mail: [email protected] apsed axial elements (Sym et al. 1993). In the zip1 null
Genetics 149: 817–832 ( June, 1998)
818 K.-S. Tung and G. S. Roeder
In each zip1 deletion mutant, both copies of the chromosomalmutant, homologous chromosomes are paired but notZIP1 gene were replaced with the indicated zip1 deletion allelesynapsed, and each pair of full-length axial elements isby the two-step transplacement method (Rothstein 1991).
closely associated at a few sites (Sym et al. 1993; Nag et The zip1 null mutant (MY152) is homozygous for zip1::URA3al. 1995; Rockmill et al. 1995). Based on DNA sequence (Sym and Roeder 1995). Strain MY187 is MY152 carrying zip1-
M1 (zip1-DH2) on a multicopy (2m) plasmid (pMB164; Symanalysis, Zip1p is predicted to contain an a-helical coiledand Roeder 1995).coil and to form a dimer consisting of a long, rod-shaped
Strains used for tetrad analysis are isogenic derivatives ofdomain flanked by globular domains. Mutations that MY261 (Sym and Roeder 1994), which has the followingincrease the length of the Zip1p coiled coil increase the genotype:width of the SC, suggesting that Zip1p is a component
MATa/MATa CENIII::URA3/CENIII::TRP1of transverse filaments (Sym and Roeder 1995). leu2::hisG/leu2::hisG HIS4/his4-B-LEU2The Scp1 protein in rats, mice, and humans, and the lys2/lys2 ho::LYS2/ho::lys2 trp1-H3/trp1-H3 ura3/ura3
homologous Syn1 protein in hamsters are similar toIn all MY261 derivatives, one copy of the ZIP1 gene carriesZip1p in that each contains a long coiled-coil region,
the zip1::LEU2 disruption (Sym et al. 1993); the other copy isand antibodies against these proteins localize specifi- either not changed (for wild type) or replaced with the indi-cally to synapsed regions of meiotic chromosomes (Meu- cated zip1 deletion allele (for deletion mutants) or with the
zip1::LYS2 disruption (for the null mutant; Sym and Roederwissen et al. 1992, 1997; Dobson et al. 1994; Sage et al.1994).1995). Epitope mapping of the hamster Syn1 and rat
In-frame deletions of ZIP1: All zip1 in-frame deletions wereScp1 proteins has shown that the C terminus of each derived from pMB96, which was described previously (Sym etmolecule lies in the lateral element region, and that al. 1993). The zip1-N1 mutation was constructed as follows:the molecules protrude from the lateral elements into First, the 39 fusion site for the deletion was created by removing
a 69-bp NlaIV-NlaIV fragment (nucleotides 420–488) and ligat-the central region of the SC (Dobson et al. 1994; Liu
ing the remaining blunt ends, which generates a BamHI site.et al. 1996; Schmekel et al. 1996). The N termini of Scp1For the 59 fusion site, a BlpI site (position 58) was cut and
molecules from opposite lateral elements may overlap filled in with the Klenow fragment of DNA polymerase I, and(Schmekel et al. 1996). a 12-bp BamHI linker was inserted. Digestion at the 59 and 39
fusion sites with BamHI, followed by ligation, resulted in theThe zip1 null mutation causes meiotic arrest in some,zip1-N1 deletion. The zip1-NM1 mutation was constructed bybut not all, yeast strain backgrounds (Sym et al. 1993;ligation between the blunt ends resulting from digestion at
Sym and Roeder 1994). In zip1 strains that arrest, re- NlaIV (position 486) and HincII (position 726) sites. Thecombination initiates, but no mature recombinants are zip1-NM2 mutation was constructed by cutting at a BlpI siteproduced (Sym et al. 1993). In zip1 strains that sporulate, (position 58), filling in with Klenow fragment, inserting a 12-
bp ClaI linker, cutting at the inserted ClaI site and at a Psp1406Ithe frequency of crossing over is decreased two- to three-site (position 723), and ligating the ends. The zip1-M1 muta-fold, and Holliday junctions persist longer than in wildtion, previously referred to zip1-DH2, was described by Sym
type (Sym and Roeder 1994; Storlazzi et al. 1996). and Roeder (1995). The zip1-M2 mutation was constructedCrossover interference is completely eliminated in zip1 as follows: To create the 59 fusion site, an 8-bp BglII linker
was inserted at an EcoRV site (position 1222). For the 39 fusionstrains, consistent with a role for the SC in mediatingsite, a 12-bp BamHI linker was inserted at a PvuII site (positioninterference (Sym and Roeder 1994).2099). Digestion at the inserted BglII and BamHI sites andTo investigate the structure and function of Zip1p, we ligation of the resulting ends created zip1-M2. The zip1-MC1
have constructed and analyzed a set of in-frame deletion mutation was constructed by cutting at EcoRV (position 1222)and XmnI (position 2393) sites and ligating the resulting bluntmutations affecting different domains of the protein.ends. The zip1-MC2 mutation was constructed by cutting atOur results offer insight into the organization of Zip1pthe BamHI site inserted during construction of zip1-M2, fillingwithin the SC, and they provide information about thein with Klenow fragment, and then ligating to the blunt end
relationship between synapsis and the other functions resulting from digestion at an XmnI site (position 2393). Thethat Zip1p performs. zip1-C1 mutation was created by cutting at a Bsu36I site (posi-
tion 2365), filling in the ends, and ligating to the blunt endresulting from digestion at an HpaI site (position 2469). Thezip1-C2 mutation has a 10-bp XbaI linker (New England Bio-
MATERIALS AND METHODS labs, Beverly, MA) inserted at the HpaI site (position 2469).This insertion results in an in-frame UAG stop codon.
Strains: Yeast manipulations were performed and media A SacI-Sal I fragment containing the zip1-N1 mutation waswere prepared using standard procedures (Sherman et al. subcloned into a YIp vector, pRS306 (Sikorski and Hieter
1986). Cells were grown and induced for meiosis at 308 unless 1989), at SacI-Sal I sites to create pTP86 for integration. Forotherwise indicated. Yeast transformations were carried out the integration of zip1-NM1, zip-NM2, zip1-M1, zip1-M2, andby the lithium acetate method (Ito et al. 1983). Yeast strains zip1-MC2, SacI-XhoI fragments containing the mutations wereused for cytological and sporulation analyses are isogenic de- subcloned into the SacI-XhoI sites of pRS306 to make pTP110,rivatives of BR2495 (Rockmill and Roeder 1990), which has pTP106, pTP62, pTP97, and pTP105, respectively. The BamHI-the following genotype: SphI fragments containing the zip1-MC1 and zip1-C1 deletions
were cloned into the BamHI-SphI sites of YIp5 (Struhl etMATa/MATa leu2-27/leu2-3,112 his4-280/his4-260 al. 1979) to make the integration plasmids pTP5 and pTP6,arg4-8/ARG4 thr1-1/thr1-4 ade2-1/ade2-1 ura3-1/ura3-1 respectively. A SacI-Sal I fragment containing zip1-C2 was sub-
cloned into the SacI-Sal I sites of a YCp vector, pRS316 (Sikor-trp1-1/trp1-289 cyh10/CYH10.
819Mutational Analysis of ZIP1
ski and Hieter 1989), to generate pTP98. Plasmids weretargeted for integration at ZIP1 by cutting with EcoNI (pTP86,pTP110, and pTP106), Bsu36I (pTP62), HpaI (pTP97 andpTP105), or XbaI (pTP5 and pTP6).
Coiled-coil analysis: The predicted amino acid sequence ofZip1p was analyzed using the COILS program (Lupas et al.1991) to calculate the probability that the sequence will adopta coiled-coil conformation. The COILS program used for theanalysis is located at http://ulrec3.unil.ch/software/COILSform.html. Using the myosins, tropomyosins, intermediatefilaments, desmosomal proteins, and kinesins matrix with win-dow 5 21, four stretches of sequence within Zip1p are pre-dicted to adopt a coiled-coil conformation. These are thesegments from residues 180 to 218, from 233 to 321, from395 to 433, and from 463 to 748. Assuming 0.1485 nm perresidue in a coiled-coil conformation (Steinert et al. 1993),these coiled-coil segments are predicted to be 5.79, 13.22,5.79, and 42.47 nm in length. Thus, the total length of thecoiled-coil region of a Zip1p dimer is predicted to be 67.27nm. In the zip1-M2 and zip1-MC1 mutations, a linker regionof 29 amino acids (aa) is included in the deletions; in the Figure 1.—Deletions of Zip1p. Based on the predicted
three-dimensional conformation, Zip1p is divided into threezip1-NM1 mutation, a linker of 14 aa is included. Becauseregions: the N-terminal globular domain (N), the coiled coilthese linkers are very short, it is assumed that the deletion ofin the middle of the protein (M), and the C-terminal globularthe linker does not cause any significant change in the lengthdomain (C). The vertical lines represent the boundaries be-of the Zip1p dimer beyond that caused by the deletion in thetween domains. The portion deleted in each mutant is indi-coiled-coil segments. Also, it is assumed that the 16 aa deletedcated by the gap; the amino acids deleted are indicated onfrom the N-terminal globular domain in zip1-NM1 and the 51the right.aa deleted from the C-terminal globular domain in zip1-MC1
and zip1-MC2 do not significantly affect the length of theZip1p dimer.
Cytology: Meiotic chromosome spreads were prepared ac- domain in the middle of Zip1p, and C for thecording to the method of Dresser and Giroux (1988). All C-terminal globular domain. For example, N1 is con-mutants were examined in multiple, independent spread fined to the N-terminal domain, while NM1 begins inpreparations with similar results. For electron microscopy,
the N-terminal domain and ends in the coiled-coil do-spreads were stained with silver nitrate as described by Dres-
main. Together, the deletion mutations analyzed spanser and Giroux (1988). Immunofluorescence proceduresthe entire length of the ZIP1-coding region.were performed according to Sym et al. (1993). Chromosomal
DNA was visualized by staining with a DNA-binding dye, DAPI. Deletion mutations were analyzed for their effects onThe rabbit anti-Zip1p antibody (Sym et al. 1993) was used chromosome synapsis, Zip1p localization, sporulationat a 1:100 dilution, and Cy3-conjugated secondary antibody efficiency, spore viability, crossing over, and crossoveragainst rabbit IgG (Jackson Laboratories, West Grove, PA) was
interference. To assess spore viability, crossing over, andused at a 1:200 dilution. A Leitz DMRB microscope (Wetzlar,crossover interference, tetrad analysis was performed inGermany) equipped with fluorescence and a PL APO 3100
objective was used to observe antibody-stained preparations. an SK1 strain background in which the zip1 null mutantImages were captured using an Imagepoint CCD camera (Pho- sporulates (Sym and Roeder 1994). Cytological analysestometrics, Tucson, AZ) and processed with the IPLab Spec- and sporulation assays were carried out in a BR2495trum software (Vienna, Virginia).
strain background (Rockmill and Roeder 1990) inwhich the zip1 null mutant undergoes pachytene arrest(Nag et al. 1995; data not shown). For cytological stud-RESULTSies, meiotic chromosomes at various stages of meiotic
In-frame deletions affecting different domains of prophase were surface spread. To assess chromosomeZip1p: According to the predicted amino acid sequence synapsis, spread chromosomes were stained with silverof Zip1p, the internal a-helical coiled-coil domain ex- nitrate and observed in the electron microscope. Totends approximately from aa 180 to aa 748, with three monitor Zip1p localization, spread chromosomes werenon-coiled-coil linker interruptions (aa 219–232, 322– stained with anti-Zip1p antibodies, followed by appro-394, and 434–462). Residues 1–179 comprise the N-ter- priate secondary antibodies. For each mutant, a timeminal globular domain, and aa 749–875 form the C-ter- course analysis of Zip1p localization was performed tominal globular domain. To investigate the relationship monitor the rate of synapsis.between Zip1p structure and function, strains carrying The N-terminal globular domain of Zip1p is not essen-in-frame deletion mutations in different regions of the tial for synapsis: In Zip1-N1p, 80% of the N terminus (aaprotein were constructed and analyzed. Mutations are 21–163) is deleted. The zip1-NM1 deletion (aa 164–243)given letter designations that indicate the domain(s) in includes one end of the N terminus and a segment ofwhich each deletion begins and ends (Figure 1): N for the coiled-coil domain. zip1-NM2 (aa 21–242) is the sum
of zip1-N1 and zip1-NM1.the N-terminal globular domain, M for the coiled-coil
820 K.-S. Tung and G. S. Roeder
Figure 2.—Electron micrographs of meiotic chromosomes from mutants affecting the N- and C-terminal globular domainsof Zip1p. An unsynapsed region in D is indicated by an arrow. Stretches of SC in E and F are indicated by arrowheads. Allspreads were prepared from cells sporulated at 308. Bar, 1 mm.
821Mutational Analysis of ZIP1
Figure 3.—Summary ofphenotypes of zip1 deletionmutants. SC formation:each pair of thick lines indi-cates the lateral elements(axial elements if not syn-apsed) of a pair of homo-logs; short vertical lines rep-resent Zip1p, and the grayvertical lines represent un-stable synapsis in zip1-NM1.Sporulation and spore via-bility data are taken fromTable 3; ts, temperature sen-sitive. Crossing over is thesum of map distances in theMAT-CEN3 and CEN3-HIS4intervals; data are takenfrom Table 4. Symbols forinterference: 1, wild-type in-terference; 6, decreased in-terference; 2, no interfer-ence.
Based on electron microscopy, homologous chromo- from the C terminus of Zip1p. The zip1-C2 deletionbegins at aa 825 and extends to the end of the ZIP1-somes in zip1-N1 undergo full synapsis just as they do
in wild type (Figures 2, A and B, and 3). Fluorescence coding region.In electron microscopic and immunofluorescencemicroscopic analysis demonstrates that the mutant Zip1-
N1p localizes along the lengths of pachytene chromo- analyses, the zip1-MC2 mutant undergoes wild-type SCformation (Figures 2G and 3). Furthermore, Zip1p lo-somes; however, Zip1p staining is not as continuous as
it is in wild type, even at late time points (Figure 4, A calization appears normal with respect to both patternand timing (Figure 4E; Table 1). In the zip1-C1 mutant,and B; Table 1). In most zip1-NM1 nuclei, homologous
chromosomes are fully synapsed (Figures 2C and 3), full-length axial elements are assembled and paired, butno SC is detected in silver-stained preparations (Figuresbut short unsynapsed regions are occasionally observed
(Figure 2D). Zip1p staining appears to be linear, and 2H and 3). The Zip1-C1p does not localize to chromo-somes; instead, it assembles into polycomplexes, whichnuclei displaying linear staining accumulate as cells ar-
rest in pachytene (Figure 4C; Table 1). In the zip1-NM2 are aggregates of Zip1p unassociated with chromosomes(Figure 4F). Polycomplexes are observed when wild-mutant, the extent of synapsis varies among nuclei from
only short stretches of SC to nearly complete synapsis, type Zip1p is overproduced (Sym and Roeder 1995)and in mutants defective in SC formation (Alani et al.but nuclei with fully synapsed chromosomes have not
been detected (Figures 2,E and F, and 3). Zip1p staining 1990; Bishop et al. 1992; Loidl et al. 1994). The zip1-C2mutant is indistinguishable from the zip1 null mutant;is punctate,even at late time points (Figure 4D; Table 1).
Anti-Zip1p antibodies were raised against the C-terminal neither Zip1p staining nor SC formation were observedin spread nuclei of cells carrying this mutation (data264 aa; thus, the less continuous Zip1p staining observed
in the zip1-N1 and zip1-NM2 mutants cannot result from not shown). This result is consistent with a previousreport indicating that the nuclear localization signal fora loss of epitopes recognized by the antibody.
These data suggest that the N terminus of Zip1p is Zip1p is located at the extreme C terminus (Burns etal. 1994), which is deleted in this mutant.not essential for chromosome synapsis. However, this
domain might play a role in stabilizing Zip1p or the These results demonstrate that aa 791–824 are impor-tant for Zip1p localization to chromosomes and thusstructure of the SC.
The C-terminal domain is essential for Zip1p localiza- for synapsis. This region may be essential for the interac-tion of Zip1p with some other component(s) of the SC.tion to chromosomes: The zip1-MC2 deletion removes
the last 48 amino acids of the coiled-coil region and the Deletions in the Zip1p coiled coil can decrease thewidth of the SC: Both the zip1-M2 (aa 409–700) andfirst 51 residues of the C terminus (aa 701–799). In the
zip1-C1 deletion, only 34 aa (791–824) are eliminated zip1-MC1 (aa 409–799) mutants have deletions in the
Figure 4.—Immunolocalization of Zip1p. Spread nuclei from wild-type and zip1-deletion mutants were stained with DAPI andanti-Zip1p antibodies. The two staining patterns are superimposed. For the purpose of better contrast, DAPI staining is shownin red, and anti-Zip1p staining is shown in green. Regions of overlap are yellow, and the intense green-staining bodies in F andL are polycomplexes. Large arrowheads point to the nucleolar region on chromosome XII, which does not undergo synapsisand does not display Zip1p staining. Arrows point to condensed chromosomes that have incomplete Zip1p staining. Sharparrowheads in K and L point to regions of Zip1p staining that are paired, but separate on homologs. The inserts in K and Lare shown at 1.5-fold higher magnification. All spreads were prepared from cells sporulated at 308. Bar, 2 mm.
823Mutational Analysis of ZIP1
TABLE 1
Kinetics of Zip1p localization on chromosomes
Classes of Zip1p staining (number of spreads in 100 nuclei scored)a
Dotty 1 Dotty in Paired butStrain Time (hr) Dotty linear linear array Linear separate No staining
ZIP1 13 22 21 4 36 1715 10 13 1 48 2817 13 21 1 35 30
zip1-N1 13 43 34 2 2115 34 45 3 1817 33 45 3 19
zip1-NM1 13 32 21 2 31 1415 18 5 16 51 1017 9 6 13 63 9
zip1-NM2 13 52 25 2315 26 49 2517 33 53 14
zip1-M1 13 38 18 22 2215 34 7 41 1817 36 1 1 42 20
zip1-M2 13 14 52 23 1115 6 40 49 517 2 23 68 7
zip1-MC1 13 20 48 16 1615 9 34 43 1417 6 25 61 8
zip1-MC2 13 21 26 2 40 1115 9 18 49 2417 20 22 33 25
Zip1p antibody localization in nuclear spreads was scored at three different time points during meiosis ina BR2495 strain background.
a Spreads were classified as follows. Dotty, only dotty Zip1p staining; Dotty 1 linear, both dotty and linearZip1p staining observed in the same spread, or incomplete synapsis (Zip1p staining linear but incomplete oncertain chromosomes); Dotty in linear array, Zip1p staining dotty but in linear order corresponding to DAPIstaining on condensed pachytene chromosomes; Linear, complete and linear Zip1p staining; Paired butseparate, regions of Zip1p staining (linear or dotty) paired but separate on homologs; No staining, no detectableZip1p staining.
carboxyl half of the coiled-coil region (Figure 1). Elec- become fully synapsed (Figures 4, G and H, and 5, Aand B; Table 1).tron microscopic examination demonstrates that ho-
mologous chromosomes are synapsed in both mutants The width of the SC is obviously decreased in thezip1-M2 and zip1-MC1 mutants (Figure 6). To investigate(Figures 3 and 5, A and B). Immunofluorescence analy-
sis indicates Zip1p localization along the lengths of chro- further the relationship between zip1 deletions and SCwidth, the width of the SC in wild-type and all zip1mosomes similar to wild type (Figure 4, G and H). How-
ever, the rate of synapsis in both mutants is slower than deletion mutants that make complete SC was measuredfrom electron micrographs of SCs in which the lateralin wild type (Table 1). In electron microscopic analysis,
chromosomes at certain intermediate stages of synapsis elements could be clearly distinguished. The width ofthe SC in the zip1-N1 and zip1-NM1 mutants is approxi-are frequently detected in the mutants, but they are
rarely observed in wild type. At these intermediate mately the same as in wild type (Table 2). On the otherhand, in the zip1-MC2, zip1-MC1, and zip1-M2 mutants,stages, synapsed chromosomes appear to have short un-
synapsed regions at their ends, and/or unsynapsed axial the width of the SC is decreased by 14, 66, and 52 nm,respectively (Table 2). These data show that the widthelements coexist with fully synapsed chromosomes in
the same nucleus (Figure 5, C and D). Consistent with of the SC can be decreased by deletions in the coiled-coil domain of Zip1p.the electron microscopic observations, incomplete
Zip1p staining on condensed chromosomes (based on Zip1p antibodies stain unsynapsed chromosomes inthe zip1-M1 mutant: The zip1-M1 mutation has a deletionDAPI staining) is detected in both mutants (Figure 4,
I and J). At late time points, the number of nuclei in the N-terminal half of the coiled-coil domain (aa244–511). Previous studies indicated that the zip1-M1at intermediate stages decreases, and all chromosomes
824 K.-S. Tung and G. S. Roeder
Figure 5.—Electron micrographs of meiotic chromosomes from Zip1p coiled-coil mutants. All spreads were prepared fromcells sporulated at 308. Bar, 1 mm.
mutant (formerly referred to as zip1-DH2) makes appar-ently normal SC (Sym and Roeder 1995). However, inthis study, we found that homologous chromosomes arepaired but not synapsed in the zip1-M1 mutant, basedon electron microscopic observations (Figure 5E). Fur-thermore, analysis of Zip1-M1p localization revealedZip1p antibody staining in regions of parallel axial ele-ments that are unsynapsed (Figure 4K). Similar resultswere obtained in an SK1 strain background and in cellscarrying the zip1-M1 allele on a multicopy vector (Fig-ures 4L and 5F). The simplest explanation for the differ-ence between our results and those published previously
Figure 6.—Regions of SC from coiled-coil deletion mutantsis that the SC formed in the zip1-M1 mutant is highlyof Zip1p. Bar, 200 nm.unstable and therefore very sensitive to subtle (and un-known) variations in the spreading procedure.
825Mutational Analysis of ZIP1
TABLE 2
Changes in the width of the SC
Observed change in Predicted changeStrain Width of SC (nm)a SC width (nm) in Zip1p dimer (nm)b
ZIP1 115 6 10zip1-N1 118 6 10 13zip1-NM1 118 6 10 13 27.4zip1-MC2 101 6 10 214 27.1zip1-MC1 49 6 7 266 246.2zip1-M2 63 6 7 252 239.1
a At least 50 intervals of parallel lateral elements were measured in each strain, as described by Sym andRoeder (1995). The distances given are averages with standard deviations.
b Predicted changes in the lengths of Zip1p dimers were calculated as described in materials and methods.
Sporulation is independent of chromosome synapsis: one spore is disomic (Ura1 and Trp1 in the case ofchromosome III).Sporulation was assessed in the same strains used for
cytological analyses. The zip1-MC2 and zip1-N1 strains In the zip1-MC2, zip1-N1, and zip1-NM1 strains, whichmake apparently normal or nearly normal SC, sporesporulate to the same extent as wild type, although spor-
ulation is slightly delayed (Figures 3 and 7A; Table 3). viability is similar to that of wild type (Figure 3; Table3). In the zip1-M2 and zip1-MC1 mutants, which makeSporulation in the zip1-NM1 mutant is temperature sen-
sitive. The sporulation efficiency is 55% at 278 (similar SCs that are significantly narrower than wild-type com-plexes, spore viability is decreased to z90% of wild typeto wild type), but it decreases to 11% at 308 (20% of
the wild-type level; Figures 3 and 7B; Table 3). zip1-NM2 (Figure 3; Table 3). In the zip1-NM2 mutant, whichundergoes incomplete synapsis, spore viability is de-and zip1-C1 fail to sporulate, just like the null mutant
(Figure 3; Table 3). In the mutants just described above, creased even more, but it is still higher than that of thenull mutant (Figure 3; Table 3). In the zip1-M1 mutant,there is a rough correlation between the ability to make
SC and the ability to sporulate. However, other mutants in which Zip1p localizes to unsynapsed chromosomes,spore viability is slightly better than that of the zip1 nullprovide exceptions to this rule. The zip1-M2 and zip1-
MC1 mutants do not sporulate, even though they make mutant (Table 3). Spore viability of zip1-C1, which failsto make any SC, is close to that of the null mutantfully synapsed chromosomes (Figure 3; Table 3). The
fully synapsed chromosomes observed in these mutants (Table 3). These data indicate a correlation betweenSC formation and spore viability (Figure 3).persist even after 42 hr in sporulation medium (data
not shown). Sporulation in the zip1-M1 mutant is sub- The distribution of four-, three-, two-, one-, and zero-spore-viable tetrads and the occurrence of spores diso-stantially delayed but eventually reaches nearly wild-type
levels, even though no stable SC is assembled (Figures mic for chromosome III were also analyzed. In allmutants, the proportion of two- and zero-spore-viable3 and 7A; Table 3). These results demonstrate that the
zip1 defects in chromosome synapsis and sporulation tetrads increases significantly as spore viability decreases(Table 3). Similarly, the frequency of tetrads with a paircan be uncoupled.
Spore viability and chromosome segregation in zip1 of spores disomic for chromosome III increases as sporeviability decreases (Table 3). These results indicate thatmutants: To investigate the effects of zip1 deletion muta-
tions on spore viability, chromosome segregation, and the decrease in spore viability in the deletion mutationsis caused largely by homolog nondisjunction at meiosismeiotic recombination, tetrad analysis was carried out
in an SK1 strain in which the centromereof one chromo- I, as shown previously for the zip1 null mutant (Sym
and Roeder 1994). The exceptions to this rule are thesome III is marked with TRP1 and the centromere ofthe homolog is marked with URA3. Meiosis in wild type zip1-M2 and zip1-MC1 mutants, in which the frequency
of three-spore-viable tetrads is much higher than in wildproduces four viable spores, of which two are Trp1 andthe other two are Ura1. If a pair of homologs segregates type or other mutants (Table 3), suggesting an addi-
tional defect in chromosome segregation. Unexpec-to the same pole during meiosis I (homolog nondisjunc-tion), two of the resulting spores will be disomic for tedly, the increase in three-spore-viable tetrads is not
associated with an increase in the frequency of PSSCthat chromosome (Trp1 Ura1 in the case of chromo-some III), and the other two will be inviable. If one for chromosome III (Table 3; see discussion).
Crossover frequency and distribution: Crossing overchromosome and one chromatid from the homologouschromosome segregate to the same pole at meiosis I was measured in the MAT-CEN3 and CEN3-HIS4 inter-
vals on chromosome III by tetrad analysis (Table 4).[precocious separation of sister chromatids (PSSC)],the result will be a three-spore-viable tetrad in which The map distances determined for the two intervals
826 K.-S. Tung and G. S. Roeder
were added together to obtain an overall measure of In the zip1-MC2, zip1-N1, zip1-NM1, zip1-M2, and zip1-MC1 strains, crossing over is increased or unchangedcrossover frequency in each strain (Figure 3). The zip1-
MC2 strain displays the highest crossover frequency in the MAT-CEN3 interval, but it is decreased in theCEN3-HIS4 interval (Table 4). For all other mutants,(61.7 cM), which is close to the map distance observed
in wild type (58.1 cM). In contrast, the amount of cross- including the zip1 null mutant, the decrease in crossingover in the CEN3-HIS4 interval is much greater than theing over is reduced more than threefold in the zip1 null
(12.7 cM), zip1-M1 (17.8 cM), and zip1-C1 (18.0 cM) decrease in the MAT-CEN3 interval. These data demon-strate that zip1 affects crossing over to different extentsmutants. Crossing over in the other mutants is at inter-in different regions of the genome. The modest increasemediate levels (Table 4; Figure 3). Overall, there is ain crossing over in the MAT-CEN3 interval in the zip1-rough correlation between the frequency of crossingMC2, zip1-N1, and zip1-M2 mutants might be caused byover and the level of spore viability (Figure 3).reduced interference from crossovers in the adjacentCEN3-HIS4 interval, where crossing over is significantlydecreased.
Crossover interference: Crossover interference canbe measured in terms of the frequency of double cross-overs in a given interval. A double crossover involving allfour chromatids results in a nonparental ditype (NPD)tetrad. Therefore, crossover interference can be quanti-tated in terms of the NPD ratio (Snow 1979), which isthe proportion of NPDs observed relative to the propor-tion of NPDs expected in the absence of interference.No interference results in an NPD ratio of 1.0, andabsolute interference results in an NPD ratio of zero.NPD ratios in the zip1 null mutant are z1.0 (Sym andRoeder 1994).
NPD ratios were calculated for the MAT-CEN3 andCEN3-HIS4 intervals (Table 4). In the zip1-MC2, zip1-N1, zip1-M2, and zip1-MC1 strains, both intervals showstatistically significant levels of positive interference.The NPD ratios in these mutants range from 0.278 to0.370 for MAT-CEN3 and from 0.130 to 0.230 for CEN3-HIS4. Interference is slightly decreased in the zip1-NM1mutant and even more in zip1-NM2. The apparent nega-tive interference observed in the CEN3-HIS4 interval inzip1-NM2 (NPD ratio of 3.1) is probably caused by ran-dom variation in recombination frequency resultingfrom limited sample size (Sall and Bengtsson 1989)rather than by a bona fide effect of the zip1-NM2 muta-tion. In the zip1-M1 mutant, NPD ratios for the MAT-CEN3 and CEN3-HIS4 intervals are 1.000 and 1.176, re-spectively, suggesting no interference. In summary, allthe mutants that make stable, full-length SC display ap-proximate wild-type levels of interference, while mu-tants that make little or no stable SC exhibit reducedinterference (Figure 3).
DISCUSSION
The C terminus, but not the N terminus, of Zip1p isessential for localization to chromosomes: Our resultsdemonstrate that the C terminus of Zip1p is importantfor its function. The zip1-C1 mutation, which removes
Figure 7.—Kinetics of spore formation in zip1 mutants. only 34 aa, completely eliminates the ability of Zip1pSpore formation was monitored in wild type, zip1-MC2, zip1- to localize to chromosomes and thus abolishes SC for-N1, and zip1-M1 at 308 (A) and in wild type and zip1-NM1 at
mation. Consistent with the cytological data, the defects278 (B). Cells were examined in the light microscope to assessin sporulation, spore viability, and crossing over causedspore formation at 2-hr intervals from 16–42 hr after transfer
to sporulation medium. by zip1-C1 are similar to those caused by the null muta-
827Mutational Analysis of ZIP1
TABLE 3
Sporulation, spore viability, distribution of tetrad types, and chromosome III missegregationa
Distribution of tetrad types (%)a HomologSporulation Tetrads Spore nondisjunctionb PSSCc
Strain efficiency (%) dissected viability (%) 4-sv 3-sv 2-sv 1-sv 0-sv (%) (%)
ZIP1 60 792 97.9 93.8 4.7 1.1 0.1 0.2 0 (0) 1 (0.1)53d
zip1-MC2 70 240 98.4 95.0 3.8 1.3 0 0 0 (0) 0 (0)zip1-N1 57 648 95.3 87.2 8.5 3.2 0.3 0.8 4 (0.6) 0 (0)zip1-NM1 11 288 96.6 89.9 7.3 2.4 0 0.3 1 (0.3) 0 (0)
55d
zip1-M2 0 768 88.9 70.7 18.5 7.9 1.3 1.6 11 (1.4) 2 (0.3)zip1-MC1 0 840 84.6 63.9 18.5 12.1 2.9 2.6 13 (1.5) 3 (0.4)zip1-NM2 0 768 82.7 69.5 9.8 10.9 1.4 8.3 29 (3.8) 6 (0.8)zip1-M1 49 960 72.9 59.0 5.9 18.0 1.8 15.3 56 (5.8) 6 (0.6)zip1-C1 0 314 54.3 38.5 7.3 19.1 2.9 32.2 10 (3.2) 0 (0)zip1D 0 480 60.2 42.5 5.6 24.8 2.3 24.8 40 (8.3) 3 (0.6)
Sporulation data were analyzed in the BR2495 yeast strain background. Tetrad analysis data were obtained in the SK1 strainbackground. Most zip1 deletion mutants sporulate to the wild-type level in the SK1 strain background (.90% at 308), exceptfor zip1-C1, which sporulates less efficiently (36% at 308). Data were obtained from cells that were grown and sporulated at 308,except for the sporulation efficiencies indicated with d, which were obtained from cells sporulated at 278.
a 4-sv, 3-sv, 2-sv, 1-sv, and 0-sv refer to the frequency of tetrads with four, three, two, one, or zero viable spores, respectively.b The frequency of homolog nondisjunction is the frequency among total tetrads of 2-sv tetrads in which both spores are
disomic for chromosome III.c The frequency of PSSC is the frequency among total tetrads of 3-sv and 2-sv tetrads in which one spore is disomic for
chromosome III.
tion. Of the 34 aa deleted in zip1-C1, the first nine is essential for chromosomal localization, suggestingthat this domain interacts with the lateral elements ofoverlap with the deletions in the zip1-MC1 and zip1-
MC2 mutants, which do not affect Zip1p localization to the SC. The N terminus is neither necessary nor suffi-cient for the localization of Zip1p to chromosomes.chromosomes. Thus, the 25 aa from 800 to 824 are
necessary for Zip1p assembly onto chromosomes. The differential importance of the two Zip1p globulardomains, as well as data on SC width discussed below,In contrast, the N terminus of Zip1p is not critical
for function. The zip1-N1 and zip1-NM1 mutations have lead us to propose a model for the organization ofZip1p within the SC. We assume that Zip1p, like otheronly minor effects on chromosome synapsis. Further-
more, a glutathione S-transferase-ZIP1 fusion gene that characterized coiled-coil proteins, forms dimers inwhich the two monomers are in parallel orientation andlacks the first 20 codons of the ZIP1 open reading frame
complements the zip1 null mutant for SC formation and exact register (reviewed by Steinert and Roop 1988).Then, two dimers form a tetramer in which the twosporulation (data not shown). Together, these mutants
cover the entire N-terminal globular domain of Zip1p, dimers are in antiparallel orientation and partially over-lapping (Figure 8A). It is this tetramer that is the build-yet none has a significant effect on SC formation or
Zip1p localization to chromosomes. Even the zip1-NM2 ing block of the SC and spans the width of the SC fromone lateral element to the other. The C termini aremutant, which is the sum of zip1-N1 and zip1-NM1, makes
a significant amount of SC. Consistent with the cytologi- attached to the lateral elements, and Zip1p dimers fromopposite lateral elements overlap for their N terminical results, spore viability and crossing over in the zip1-
N1 and zip1-NM1 mutants are close to the wild-type and part of the coiled-coil region (Figure 8A). Thisorganization for Zip1p within the SC is similar to thatlevels, and the zip1-NM2 mutant displays much higher
spore viability and crossing over than the null mutant. proposed for the Scp1 and Syn1 proteins, based onhigh-resolution epitope mapping (Dobson et al. 1994;Based on these data, the function of the N-terminal
globular domain of Zip1p remains obscure. The temper- Schmekel et al. 1996). Consistent with our model, epi-tope mapping of Zip1p shows that the C terminus ofature-sensitive sporulation defect of the zip1-NM1 strain
suggests that the mutant protein and/or the SC formed each molecule lies in the lateral element region, andthe N terminus localizes to the central region (H. Dongin the mutant are unstable. The discontinuity of Zip1p
staining in the zip1-N1 and zip1-NM2 mutants might and G. S. Roeder, unpublished results).Zip1p tetramers could be formed before they localizereflect a defect in maintaining Zip1p on chromosomes.
Model for the organization of Zip1p in the SC: Our to chromosomes, or they could be formed in situ withZip1p dimers that have already localized to chromo-results indicate that the N and C termini of Zip1p play
different roles in SC formation. The C terminus of Zip1p somes. The N terminus of Zip1p may contribute to the
828 K.-S. Tung and G. S. Roeder
TA
BL
E4
Tet
rad
anal
ysis
Cla
sses
ofte
trad
sPr
obab
ility
bPr
obab
ility
c
Fold
Exp
ecte
dN
PDSt
rain
Inte
rval
PT
NT
otal
cMde
crea
sea
ZIP1
zip1
DN
PDra
tio
No
inte
rfer
ence
ZIP1
ZIP1
M44
527
16
722
21.3
17.6
0.34
10.
006
H26
343
415
712
36.8
69.8
0.21
5,
0.00
1zi
p1-M
C2
M11
310
23
218
27.5
0.77
0.05
3,
0.00
19.
40.
319
0.03
70.
935
H89
126
421
934
.21.
080.
492
,0.
001
17.4
0.23
00.
001
0.91
4zi
p1-N
1M
298
227
553
024
.20.
880.
234
,0.
001
18.0
0.27
80.
002
0.76
7H
279
243
552
725
.91.
42,
0.00
1,
0.00
121
.70.
230
,0.
001
0.90
3zi
p1-N
M1
M15
696
325
522
.40.
950.
715
,0.
001
6.3
0.47
60.
189
0.69
2H
152
972
251
21.7
1.70
,0.
001
,0.
001
6.6
0.30
30.
073
0.68
7zi
p1-M
2M
284
240
653
026
.00.
820.
048
,0.
001
20.8
0.28
80.
001
0.80
1H
323
200
352
520
.71.
78,
0.00
1,
0.00
113
.20.
227
0.00
50.
936
zip1
-MC
1M
330
182
451
620
.01.
070.
578
,0.
001
10.8
0.37
00.
039
0.91
2H
364
159
152
415
.72.
34,
0.00
10.
001
7.7
0.13
00.
016
0.64
4zi
p1-N
M2
M38
313
73
523
14.8
1.44
0.00
4,
0.00
15.
50.
545
0.28
60.
583
H44
078
552
310
.33.
57,
0.00
10.
139
1.6
3.12
50.
007
,0.
001
zip1
-M1
M46
289
255
39.
12.
34,
0.00
10.
149
2.0
1.00
01.
000
0.31
6H
467
812
550
8.5
4.33
,0.
001
0.44
71.
71.
176
0.81
80.
082
zip1
-C1
M94
221
117
12.0
1.78
0.02
00.
055
0.6
1.66
70.
606
0.31
1H
103
140
117
6.0
6.13
,0.
001
0.78
90.
20
0.65
50.
836
zip1
DM
180
240
204
5.9
3.61
,0.
001
1.00
0d1.
000d
0.16
5d
H17
327
022
06.
85.
41,
0.00
11.
111d
0.79
6d0.
007d
Dat
aw
ere
obta
ined
inan
SK1
stra
inba
ckgr
oun
d.G
enet
ican
dst
atis
tica
lan
alys
esw
ere
perf
orm
edas
desc
ribe
dpr
evio
usly
byC
hu
aan
dR
oed
er
(199
7).N
PDra
tios
and
inte
rfer
ence
data
indi
cate
dw
ith
“d ”ar
efr
omS
ym
and
Ro
ed
er
(199
4).M
,MA
T-C
ENII
I;H
,CEN
III-H
IS4;
P,pa
ren
tald
ityp
e;T
,tet
raty
pe;N
,non
pare
nta
ldit
ype;
cM,c
enti
mor
gan
.a
Th
efo
ldde
crea
sein
map
dist
ance
isre
lati
veto
wild
type
.bPr
obab
ility
refe
rsto
the
likel
ihoo
dth
atth
edi
ffer
ence
incr
ossi
ng
over
betw
een
agi
ven
mut
ant
and
wild
type
orbe
twee
nth
em
utan
tan
dth
ezi
p1D
stra
inis
due
toch
ance
.cT
he
prob
abili
tyof
no
inte
rfer
ence
isth
elik
elih
ood
that
the
diff
eren
cebe
twee
nth
en
umbe
rof
NPD
sob
serv
edan
dth
en
umbe
rex
pect
edin
the
abse
nce
ofin
terf
eren
ceis
due
toch
ance
.T
he
prob
abili
tyof
wild
-type
inte
rfer
ence
indi
cate
sth
elik
elih
ood
that
the
diff
eren
cein
NPD
rati
obe
twee
nw
ild-ty
pean
da
give
nzi
p1m
utan
tis
due
toch
ance
.
829Mutational Analysis of ZIP1
SC is not decreased in the zip1-NM1 mutant, althoughthe mutant Zip1p dimer is predicted to be shorter thanthe wild-type dimer by 7.4 nm. In contrast, a deletionat the C-terminal end of the coiled coil should decreasethe width of the SC by twice the change in length ofthe Zip1p dimer (Figure 8C). In agreement with thisprediction, the decrease in the width of the SC (14 nm)in the zip1-MC2 mutant is twice the decrease in thepredicted length of the dimer (7.1 nm). Also, the differ-ence in the length of the dimer between the zip1-MC1and zip1-M2 mutants is predicted to be 7.1 nm, and thedifference in the width of the SC is 14 nm. Thus, theseFigure 8.—Model for the organization of Zip1p within the
SC. Each Zip1p dimer is represented by a pair of parallel results are consistent with the predictions made by ourhorizontal lines (for the coiled-coil region) flanked by a circle model.(for the N-terminal globular domain) and a square (for the For mutants that have large deletions in the coiled-C-terminal globular domain). The vertical gray lines indicate
coil region of Zip1p, such as zip1-MC1 and zip1-M2, itthe lateral elements of the SC. The relative positions of differ-is difficult to predict their effects on the width of the SCent regions in the coiled-coil domain of Zip1p are indicated
by letters a–f. Figure is not drawn to scale. because of uncertainty regarding the extent of overlapbetween Zip1p dimers. Furthermore, studies of thestructure and assembly of keratin, which shares struc-
stability of the SC by stabilizing the interaction between tural homology to Zip1p, have shown that differentZip1p dimers attached to opposing lateral elements. It is forms of tetramers can be formed depending on thealso possible that there is no direct interaction between alignment of dimers (Aebi et al. 1988; Steinert 1991;Zip1p dimers, and that the dimers are connected by Steinert et al. 1993; reviewed by Stewart 1993). Byother components of the central region of the SC. analogy, it is possible that tetramerization of Zip1p di-
A previous study suggested that a single Zip1p dimer mers in the zip1-MC1 and zip1-M2 mutants uses an alter-spans the width of the SC from one lateral element to native region of overlap.the other (Sym and Roeder 1995). This proposal was Chromosome synapsis and meiotic cell cycle control:based on the observation that duplication or triplication Temporal studies have demonstrated that mature cross-of a restriction fragment encoding part of the Zip1p over products arise near the end of pachytene, as the SCcoiled coil increased the width of the SC by amounts disassembles (Padmore et al. 1991). In zip1-null strains,corresponding to the predicted increases in the length chromosomes fail to synapse, and there is a defect (orof the Zip1p dimer (Sym and Roeder 1995). In the a delay) in the production of crossover products (Sym
previous study, it was assumed that the entire region that et al. 1993; Sym and Roeder 1994; Storlazzi et al.was duplicated (or triplicated) would adopt a coiled-coil 1996). These observations raise the possibility that mei-conformation (Sym and Roeder 1995). However, an otic cells monitor the status of SC formation, preventingimproved program for coiled-coil analysis (see materi- the resolution of recombination intermediates untilals and methods) suggests that nearly 40% of the af- chromosomes are synapsed. Such coordination wouldfected amino acids are within linker regions and, thus, be important if the SC plays a role in regulating theshould not make a significant contribution to the length distribution of crossovers along and among chromo-of the coiled-coil domain. The newly predicted increases somes (reviewed by Egel 1995; Roeder 1997). In thisin the length of Zip1p dimers are much less than the case, crossing over in the absence of SC would lead toobserved increases in the width of the SC. Thus, the a deregulation of crossover distribution. As a result,behavior of the duplication and triplication mutants is some chromosome pairs would fail to recombine andnot consistent with the model that a single Zip1p dimer therefore nondisjoin at meiosis I, generating inviablespans the width of the SC. meiotic products.
The width of the SC can be decreased by deletions In zip1-MC1 and zip1-M2 strains, which make com-in the Zip1p coiled coil: A previous study showed that plete SC that is narrower than wild-type SC, meioticthe width of the SC can be increased by insertions in arrest might be caused by the irregular morphology ofthe coiled coil of Zip1p (Sym and Roeder 1995). Our the complex. This hypothesis is consistent with a previ-results demonstrate that the SC width can also be de- ous study suggesting that a meiosis-specific surveillancecreased by deletions in the coiled-coil region of Zip1p. system monitors the status of recombination complexesAccording to our model, a short deletion at the N-terminal in a specific chromosomal context that includes the SC-end of the Zip1p coiled-coil domain should not cause related proteins Red1p and Mek1p (Xu et al. 1997).a decrease in the width of the SC, though it should Red1p is a component of lateral elements and is essen-shorten the region of overlap between dimers (Figure tial for SC formation (Smith and Roeder 1997), while
Mek1p is a meiosis-specific protein kinase required for8B). Consistent with this prediction, the width of the
830 K.-S. Tung and G. S. Roeder
normal SC morphogenesis (Rockmill and Roeder crossovers is determined at the initiation stage. How-ever, it was recently reported that DSBs are rare in the1991). The defect (or delay) in sporulation caused by
zip1 can be alleviated by a red1 or mek1 mutation (Xu CRY1-PGK1 interval (most of the MAT-CEN3 interval),even though there is a significant level of crossing overet al. 1997; J. M. Bailis and G. S. Roeder, unpublished
results), suggesting that Red1p and Mek1p are essential (Baudat and Nicolas 1997). Several possibilities havebeen suggested to explain this unexpected result (Bau-for the function of the proposed surveillance system.
Our data suggest that the checkpoint machinery re- dat and Nicolas 1997): (1) the genetic distance maybe overestimated; (2) the frequency of DSBs may besponds not only to the absence of SC, but also to aberra-
tions in SC morphology. underestimated, perhaps because the breaks do not oc-cur at discrete sites (hotspots); and (3) some crossoversThe observation that zip1-M1 cells sporulate indicates
that the irregular structure of chromosomes in this mu- might be initiated by lesions other than DSBs. If possibil-ity (2) or (3) is the case, the interval-dependent effectstant is not recognized as abnormal by the surveillance
system. One explanation is that an interaction between on crossing over observed in the zip1 mutants might beDSB related. Perhaps Zip1p (or the SC) affects onlyZip1p and some other component(s) of meiotic chro-
mosomes is required to produce the signal that activates recombination events that are initiated by DSBs at re-combination hotspots.the recombination machinery (or blocks the inhibition
of this machinery). According to this model, the rele- zip1-M2 and zip1-MC1 mutants increase the frequencyof three-spore-viable tetrads: The proportion of three-vant protein-protein interaction occurs in the zip1-M1
mutant, but not in the zip1-MC1 and zip1-M2 mutants. spore-viable tetrads in the zip1-M2 and zip1-MC1 mutantsis two- to fivefold higher than in wild-type and otherChromosome synapsis and crossing over: The de-
crease in crossing over in the zip1-null mutant could be zip1 mutants (Table 3), suggesting that the frequencyof PSSC is increased in these mutants. However, thecaused by the absence of SC, which in turn is caused
by the absence of Zip1p. Alternatively, Zip1p may play frequency of PSSC for chromosome III in zip1-M2 andzip1-MC1 is not correspondingly higher (Table 3). It isa role in meiotic recombination that is independent of
its role in SC formation, as proposed by Storlazzi et possible that the increase in three-spore-viable tetradsin these mutants is caused by chromosome missegrega-al. (1996). According to this model, it might be possible
to isolate zip1 mutants that are proficient in crossing tion at meiosis II or by chromosome loss, but not byPSSC at meiosis I. Alternatively, the increase in three-over but defective in synapsis.
The results of this study demonstrate a correlation spore-viable tetrads may be caused by an increase inPSSC that is not detectable in our assay. Chromosomebetween the extent of synapsis and the level of crossing
over. If zip1 deletion mutations are ranked with respect III is one of the smallest chromosomes in yeast, raisingthe possibility that the defect that causes PSSC in theseto the extent of chromosome synapsis and with respect
to the amount of crossing over, there is an excellent mutants is more pronounced on large chromosomes,whose missegregation was not scored. The zip1-M2 andcorrespondence between the two rank orders (Figure
3). The simplest interpretation of these results is that zip1-MC1 mutants both make SC that is significantlynarrower than wild-type SC (Figure 6). It is possible thatthe SC promotes crossing over, perhaps by providing
a context that favors the resolution of recombination the aberrant SC presents a difficulty for SC disassembly,which perturbs subsequent chromosome segregation.intermediates in the direction of crossing over. We have
been unsuccessful in identifying any mutations that af- Bigger chromosomes might be affected more becauseof the greater length of SC that must be disassembled.fect synapsis but not crossing over. Nevertheless, our
results do not exclude the possibility that Zip1p affects Chromosome synapsis and crossover interference:Most models for crossover interference suggest thatrecombination independent of its function in SC poly-
merization. the SC is involved in the transmission of an inhibitorysignal along the chromosome from one crossover siteInterval-dependent effects on crossing over: In all of
the zip1 mutants examined, crossing over in the CEN3- to nearby potential sites of crossing over (Egel 1978;Maguire 1988; King and Mortimer 1990; Sym andHIS4 interval is decreased more than in the MAT-CEN3
interval (Table 4). These results suggest that crossovers Roeder 1994; for review, see Jones 1984 and Roeder
1995, 1997). The observation that chromosomes fail toin the CEN3-HIS4 interval are more sensitive to varia-tions in SC morphology or abundance. synapse and crossover interference is completely elimi-
nated in zip1 null mutants has provided molecular evi-In yeast, most or all meiotic recombination is initiatedby DSBs, and meiotic recombination hotspots are pref- dence for a functional relationship between the SC and
interference (Sym and Roeder 1994).erential sites for DSB formation (Wu and Lichten 1994;Lichten and Goldman 1995; Roeder 1995). It has In this study of zip1 deletion mutations, we found that
all of the mutants that make stable, full-length SC displaybeen shown that the distribution of DSBs is similar tothe distribution of meiotic crossovers in the CEN3-LEU2 crossover interference. Even in zip1-M2 and zip1-MC1
strains, which decrease the width of the SC and reduceregion contained within the CEN3-HIS4 interval (Wu
and Lichten 1994), suggesting that the distribution of crossing over, interference is unaffected. In the zip1-
831Mutational Analysis of ZIP1
in Controlling Events in Meiosis, edited by C. W. Evans and H. G.NM1 mutant, interference appears to be decreased (al-Dickinson. The Company of Biologists Ltd., Cambridge.
though the difference between wild type and the mutantKing, J. S., and R. K. Mortimer, 1990 A polymerization model of
is not statistically significant). However, short stretches chiasma interference and corresponding computer simulation.Genetics 126: 1127–1138.of unsynapsed chromosomes have been observed in
Kleckner, N., 1996 Meiosis: how could it work? Proc. Natl. Acad.spread chromosomes of zip1-NM1, raising the possibility Sci. USA 93: 8167–8174.that any decrease in interference is caused by the occa- Lichten, M., and A. S. H. Goldman, 1995 Meiotic recombination
hotspots. Annu. Rev. Genet. 29: 423–444.sional failure in synapsis or in maintaining SC structure.Liu, J.-G., L. Yuan, E. Brundell, B. Bjorkroth, B. Daneholt et al.,The zip1-NM2 mutant makes considerably less SC than 1996 Localization of the N-terminus of SCP1 to the central
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