DNA ligase gene disruptions can depress viral growth and replication in poxvirus-infected cells.

Poxvirus-encoded DNA ligases are assumed to play a role in viral DNA replication; however mutational inactivation of vaccinia ligase has not been reported to affect viral growth rates in culture. This communication re-examines this surprising aspect of poxviral biology using both Shope fibroma virus (SFV) and vaccinia virus. SFV and vaccinia ligase deficiencies create essentially identical phenotypes. In particular, ligase-deficient SFV strains are mildly UV sensitive and etoposide resistant, phenotypes previously shown to characterize ligase-deficient vaccinia strains. Moreover, we find that ligase mutations can inhibit the growth of both SFV and vaccinia virus in vitro. The poor growth observed in the absence of a viral ligase is correlated with a two- to tenfold reduction in viral and extragenomic DNA synthesis. This phenotype is host dependent. No differences in viral growth or DNA yield were seen when vaccinia strains were cultured on rabbit (SIRC) cells, but ligase deficiencies reduced growth and DNA yields when vaccinia was plated on BSC-40 cells or SFV on SIRC cells. Despite these replicative defects, mutational inactivation of SFV ligase produced no detectable increase in the number of viral DNA breaks and had no effect on virus-catalyzed extragenomic DNA recombination or UV repair. We conclude that poxviral ligases do play a role in viral DNA replication, but the replicative defect is obscured in some cell lines.

An etoposide-induced block in vaccinia virus telomere resolution is dependent on the virus-encoded DNA ligase.

Etoposide, an inhibitor of the breakage-reunion reaction associated with cellular type II DNA topoisomerases, was shown to inhibit plaque formation of vaccinia virus. This drug had a major effect on the segregation of newly replicated DNA concatemers. Gene expression and the initiation and elongation phases of viral DNA replication were essentially unaffected. Pulsed-field gel electrophoresis of viral DNA replicated in the presence of etoposide revealed two major classes of DNA: the mature monomeric linear genome and DNA that failed to enter the gel (the relative proportions depending on the concentrations of drug). Restriction enzyme analysis showed a severe defect in telomere resolution. In addition, slowly migrating restriction fragments were suggestive of a general recombination defect. We have isolated several etoposide-resistant mutants and used marker rescue and DNA sequencing to localize the resistance-causing mutation to the amino terminus of the viral DNA ligase gene. Inactivation of the DNA ligase also resulted in an etoposide-resistant phenotype, but to a lesser extent. The telomere resolution and segregation defects were corrected both in the drug-resistant mutants and in the DNA ligase knockout mutants. Reinsertion of wild-type or mutant DNA ligase in the viral thymidine kinase locus confirmed the role of the viral DNA ligase in conferring sensitivity not only to etoposide but also to another topoisomerase II inhibitor, 4'-(9-acridinylamino) methanesulphon-m-anisidide (mAMSA). The data suggest that the nonessential DNA ligase is involved in telomere resolution, possibly as part of a general recombinase.

The vaccinia virus-encoded uracil DNA glycosylase has an essential role in viral DNA replication.

The vaccinia virus conditional-lethal temperature-sensitive (ts) mutant ts4149 is, at the nonpermissive temperature, severely impaired in its ability to replicate its DNA genome. Compared to wild type, the amount of replication is suppressed by several orders of magnitude, and the little DNA that is replicated is not converted to mature linear genomes. We have demonstrated that this "DNA-" phenotype is not the result of a failure to produce early proteins. In agreement with the DNA- phenotype, intermediate and late gene expression were not detected. Marker rescue and DNA sequencing located the mutation in ts4149 to open reading frame D4. This gene has recently been shown to encode a 25-kDa protein with uracil DNA glycosylase activity (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden (1993), J. Virol. 67, 2503-2512). We speculate on the function of this "essential" viral repair enzyme and its role(s) in viral DNA replication.

Identification of a temperature-sensitive mutant of vaccinia virus defective in late but not intermediate gene expression.

The vaccinia virus conditional-lethal temperature-sensitive (ts) mutant tsC63 is defective in the synthesis of some but not all postreplicative proteins. Synthesis of the temporal "intermediate" class of proteins was unaffected, whereas "late" proteins were absent at the nonpermissive temperature. At the DNA level, DNA synthesis was unaffected, but telomere resolution was severely inhibited. In order to identify the defective gene responsible for this ts defect, we performed marker rescue and DNA sequencing experiments. We localized the lesion to open reading frame (ORF) A1L, which has recently been identified as one of the three intermediate genes required for the transcription of late genes (J.G. Keck, C.J. Baldick, Jr., and B. Moss, (1990). Cell 61, 801-809). S1 nuclease analysis of viral mRNA demonstrated that the ts defect in late protein synthesis was caused by a defect in the transcription of stable mRNA and therefore provides evidence for a role of the A1L gene product during in vivo transcriptional activation of late genes or stabilization of late RNA. Furthermore, the kinetics of early protein synthesis in tsC63-infected cells suggests that, in addition to its role in trans-activation of late genes, intermediate gene expression mediates suppression of early protein synthesis. The telomere resolution defect of this mutant is presumably a secondary consequence of the defect in late gene expression.

A temperature-sensitive lesion in the small subunit of the vaccinia virus-encoded mRNA capping enzyme causes a defect in viral telomere resolution.

Using pulsed-field gel electrophoresis, we demonstrated that the temperature-sensitive (ts) conditional lethal mutant ts9383 is, at the nonpermissive temperature, defective in the resolution of concatemeric replicative intermediate DNA to linear 185-kb monomeric DNA genomes. The resolution defect was shown to be the result of a partial failure of the mutant virus to convert the replicated form of the viral telomere to hairpin termini. In contrast to other mutants of this phenotype, pulse-labeling of viral proteins at various times postinfection revealed no obvious difference in the quantity or temporal appearance of members of the late class of polypeptides. Using the marker rescue technique, we localized the ts lesion in ts9383 to an approximately 1-kb region within the HindIII D fragment. Both the ts phenotype and the resolution defect were shown to be caused by a single-base C----T point mutation resulting in the conversion of the amino acid proline to serine in codon 23 of open reading frame D12. This gene encodes a 33-kDa polypeptide which is known to be the small subunit of the virus-encoded mRNA capping enzyme (E. G. Niles, G. J. Lee-Chen, S. Shuman, B. Moss, and S. S. Broyles, Virology 172:513-522, 1989). The data are consistent with a role for this capping enzyme subunit during poxviral telomere resolution.

Identification of temperature-sensitive mutants of vaccinia virus that are defective in conversion of concatemeric replicative intermediates to the mature linear DNA genome.

Pulsed-field gel electrophoresis was used to screen temperature-sensitive mutants of vaccinia virus for the ability to convert replicated viral DNA into mature linear 185-kilobase hairpin-terminated genomes. Of 30 mutually noncomplementing mutants tested, 5 displayed a temperature-sensitive defect in the resolution of the telomere fusion configuration within concatemeric replicative intermediates, resulting in a failure to convert such intermediates to the linear monomeric genome. Adjacent genomic units in the concatemeric arrays generated in these mutants were arranged in both tandem and inverted orientations. The observation that four of the five mutants had a severe general defect in the synthesis of the late class of viral proteins suggests that at least one late protein is directly required to resolve the telomere fusion intermediate to hairpin termini. The identification of such telomere resolution proteins should be facilitated by genetic and molecular characterization of resolution-defective mutants, such as C63, in which late protein synthesis is not severely affected.

Tumorigenic poxviruses: fine analysis of the recombination junctions in malignant rabbit fibroma virus, a recombinant between Shope fibroma virus and myxoma virus.

Malignant rabbit fibroma virus (MRV) has been shown to be a lethal tumorigenic poxvirus of rabbits derived from a recombination event between Shope fibroma virus (SFV), which induces benign fibromas in rabbits, and myxoma virus, the agent of myxomatosis. We have cloned and sequenced all of the MRV recombination junctions, which are located near the left and right terminal inverted repeat (TIR) regions, and present a composite map of the MRV genome with respect to the relevant gene products. The two junctions closet to the MRV termini, at identical positions at the left and right ends, are at nucleotide 5272 and result in an in-frame fusion protein (ORF T-5) in which the N-terminal 232 aa are derived from an SFV sequence linked to a C-terminus derived from myxoma. At the left MRV TIR the recombination junction distal from the terminus maps to nucleotide 9946 but leaves the adjacent gene virtually unchanged from its SFV homolog. At the right terminus, the relevant junction sequences from MRV and myxoma could not be cloned in wild-type Escherichia coli but were maintained stably in a recA recBC sbcB host. The SFV/myxoma junction at this location maps 5' to a growth factor gene (SFGF) which is related to those encoding epidermal growth factor and transforming growth factor-alpha. As a result, the myxoma growth factor gene has been deleted in MRV and replaced in toto by the SFV gene. The recombination junction upstream from the SFGF gene creates an in-frame fusion in ORF T11-R in which the N-terminal amino acids are derived from myxoma and the remainder from SFV. In summary, MRV has received the following ORFs from SFV: at the left terminus T5 (fusion), T6, T7, and T8; at the right terminus, T5 (fusion), T6, T7, T8, T9-R, SFGF, and T11-R (fusion).

Tumorigenic poxviruses: genomic organization and DNA sequence of the telomeric region of the Shope fibroma virus genome.

Shope fibroma virus (SFV), a tumorigenic poxvirus, has a 160-kb linear double-stranded DNA genome and possesses terminal inverted repeats (TIRs) of 12.4 kb. The DNA sequence of the terminal 5.5 kb of the viral genome is presented and together with previously published sequences completes the entire sequence of the SFV TIR. The terminal 400-bp region contains no major open reading frames (ORFs) but does possess five related imperfect palindromes. The remaining 5.1 kb of the sequence contains seven tightly clustered and tandemly oriented ORFs, four larger than 100 amino acids in length (T1, T2, T4, and T5) and three smaller ORFs (T3A, T3B, and T3C). All are transcribed toward the viral hairpin and almost all possess the consensus sequence TTTTTNT near their 3' ends which has been implicated for the transcription termination of vaccinia virus early genes. Searches of the published DNA database revealed no sequences with significant homology with this region of the SFV genome but when the protein database was searched with the translation products of ORFs T1-T5 it was found that the N-terminus of the putative T4 polypeptide is closely related to the signal sequence of the hemagglutinin precursor from influenza A virus, suggesting that the T4 polypeptide may be secreted from SFV-infected cells. Examination of other SFV ORFs shows that T1 and T2 also possess signal-like hydrophobic amino acid stretches close to their N-termini. The protein database search also revealed that the putative T2 protein has significant homology to the insulin family of polypeptides. In terms of sequence repetitions, seven tandemly repeated copies of the hexanucleotide ATTGTT and three flanking regions of dyad symmetry were detected, all in ORF T3C. A search for palindromic sequences also revealed two clusters, one in ORF T3A/B and a second in ORF T2. ORF T2 harbors five short sequence domains, each of which consists of a 6-bp short palindrome and a 10- to 18-bp larger palindrome. The significance of these palindromic domains in this ORF is unclear but the coincidence of the end of one larger palindrome with the end of the translated protein sequence that has homology with the B chain of insulin suggests that the palindromes may divide the T2 protein into several functional units. The salient organizational features of the complete SFV TIR are also discussed in light of what is known about other poxviral TIRs.

Efficient resolution of replicated poxvirus telomeres to native hairpin structures requires two inverted symmetrical copies of a core target DNA sequence.

The terminal hairpin sequences of the linear double-stranded DNA genome of the leporipoxvirus Shope fibroma virus (SFV) has been cloned in Saccharomyces cerevisiae and in recombination-deficient Escherichia coli as a palindromic insert within circular plasmid vectors. This sequence configuration is equivalent to the inverted repeat structure detected as a telomeric replicative intermediate during poxvirus replication in vivo. Previously, it has been shown that when circular plasmids containing this palindromic insert were transfected into SFV-infected cells, efficient replication and resolution generated linear minichromosomes with bona fide viral hairpin termini (A. M. DeLange, M. Reddy, D. Scraba, C. Upton, and G. McFadden, J. Virol. 59:249-259, 1986). To localize the minimal target DNA sequence required for efficient resolution, a series of staggered unidirectional deletions were constructed at both ends of the inverted repeat. Analyses of the resolution efficiencies of the various clones indicate that up to 240 base pairs (bp) centered at the symmetry axis were required for maximal resolution to minichromosomes. To investigate the role of the AT-rich central axis sequences, which in SFV include 8 nonpalindromic bp, a unique AflII site at the symmetry axis was exploited. Bidirectional deletions extending from this AflII site and insertions of synthetic oligonucleotides into one of the deletion derivatives were constructed and tested in vivo. The efficiency with which these plasmids resolved to linear minichromosomes with hairpin termini has enabled us to define the minimal target DNA sequence as two inverted copies of an identical DNA sequence between 58 and 76 bp in length. The nonpalindromic nucleotides, which, after resolution, constitute the extrahelical residues characteristic of native poxviral telomeres, were not required for resolution. The close resemblance of the SFV core target sequence to the analogous region from the orthopoxvirus vaccinia virus is consistent with a conserved mechanism for poxviral telomere resolution.

Replication and resolution of cloned poxvirus telomeres in vivo generates linear minichromosomes with intact viral hairpin termini.

The covalently closed terminal hairpins of the linear duplex-DNA genomes of the orthopoxvirus vaccinia and the leporipoxvirus Shope fibroma virus (SFV) have been cloned as imperfect palindromes within circular plasmids in yeast cells and recombination-deficient Escherichia coli. The viral telomeres inserted within these recombinant plasmids are equivalent to the inverted-repeat structures detected as telomeric replicative intermediates during poxvirus replication in vivo. Although the telomeres of vaccinia and SFV show little sequence homology, the termini from both viral genomes exist as AT-rich terminal hairpins with extrahelical bases and alternate "flip-flop" configurations. Using an in vivo replication assay in which circular plasmid DNA was transfected into poxvirus-infected cells, we demonstrated the efficient replication and resolution of the cloned imperfect palindromes to bona fide hairpin termini. The resulting linear minichromosomes, which were readily purified from transfected cells, were shown by restriction enzyme mapping and by electron microscopy to have intact covalently closed hairpin termini at both ends. In addition, staggered unidirectional deletion derivatives of both the cloned vaccinia and SFV telomeric palindromes localized an approximately 200-base-pair DNA region in which the sequence organization was highly conserved and which was necessary for the resolution event. These data suggest a conserved mechanism of the resolution of poxvirus telomeres.

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (ii) amplification of intracellular plasmid DNA; and (iii) density shift analysis in the presence of BrdUrd. Replication of plasmid DNA was observed in the cytoplasm of cells infected with the tumorigenic leporipoxviruses Shope fibroma virus (SFV) and myxoma, and less extensively with the orthopoxvirus vaccinia, but not in uninfected cells. Unexpectedly, all input plasmids tested, including pBR322, pUC13, polyoma, PM2 phi X174 replicative form (RF), and M13 RF, replicated with equal efficiency in SFV-infected cells, indicating that no specific replication origin sequence is required. The transfected plasmid DNA was replicated concomitantly with the infecting poxviral DNA and by 24 hr post-transfection, it resided predominantly in high molecular weight Dpn I-resistant head-to-tail tandem repeats. The failure to detect unreplicated Dpn I-sensitive plasmid concatemers early in replication together with the absence of significant levels of integrated plasmid sequences in the poxviral genome suggest that replication of the transfected plasmid DNA is not the consequence of nonhomologous recombination of concatemeric plasmid DNA into the poxvirus genome, but rather of an autonomous process that is dependent on trans-acting replication factors produced during virus infection, and that does not require a specific origin sequence on the substrate plasmid DNA.

Tumorigenic poxviruses: construction of the composite physical map of the Shope fibroma virus genome.

The sites for the restriction enzymes BamHI, Bg/I, HindIII, PstI, PvuII, and SstI on the linear DNA genome of Shope fibroma virus, a tumorigenic poxvirus of rabbits, have been determined by digestions of the cloned BamHI and HindIII restriction fragments and by hybridization of 32P-labeled cloned fragments to Southern blots of Shope fibroma virus DNA cleaved partially or completely with the various enzymes. The linear genome is shown to be 160 kilobases in length and to possess terminal inverted repeat sequences of between 12.2 and 12.5 kilobases extending inwards from the cross-linked DNA telomeres. The fine map of the Shope fibroma virus terminal inverted repeats has been constructed and shown to be distinctly different from that of members of the orthopoxvirus group, such as vaccinia, by the absence of detectable tandemly repeated sequences near the termini and by the lack of detectable sequence homology with vaccinia termini.

Cloning of the vaccinia virus telomere in a yeast plasmid vector.

The vaccinia virus DNA telomere, which contains a covalently closed hairpin structure, has been cloned in a yeast plasmid vector. Restriction mapping indicates that the cloned vaccinia telomere is maintained in yeast not in its native hairpin configuration but as an inverted repeat structure, within a circular plasmid, with the sequences of the viral hairpin now at the axis of symmetry of an imperfect palindrome. As such, the cloned telomere resembles the telomeric replicative intermediate observed during vaccinia virus DNA replication. Small deletions and duplications in the viral inverted repeats of different clones suggest a model in which the observed circular plasmids were generated in yeast by the replication of hybrid linear DNA molecules consisting of the linearized yeast vector flanked by two hairpin-containing vaccinia termini.

Physical characterization and molecular cloning of the Shope fibroma virus DNA genome.

DNA from several independent strains of Shope fibroma virus, a tumorogenic leporipoxvirus of rabbits, was isolated and analyzed by restriction endonuclease digestion and Southern blotting. The restriction profiles indicated a high degree of sequence conservation among the isolates but blotting under standard stringencies revealed no detectable cross homology with a member of the orthopoxvirus group, vaccinia. The genome of the fibroma virus was calculated to be in excess of 160 kilobases and shown to possess two features analogous to the orthopoxvirus group: (1) the terminal restriction fragments possess covalently closed hairpin structures; and (2) the terminal sequences are present as inverted repeats of greater than 10 kilobases. The terminal 3.6 kilobase BamHI restriction fragment was cloned in pBR322 after removal of the hairpin structure with mung bean single strand-specific endonuclease and addition of BamHI linkers. SFV sequences within this terminal region were shown, using 32P SFV cloned terminal probe, to have none of the sequence heterogeneity characteristic of vaccinia DNA termini. The remaining 20 internal SFV BamHI restriction fragments were propagated in bacterial plasmids either as intact fragments, or after secondary digestion with HindIII, and together constitute the complete cloned SFV sequence library.

Characterization of MMS-sensitive mutants of Neurospora crassa.

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer assistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increased (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histidine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 X) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to the number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.

The mutation SK(ad-3A) cancels the dominance of ad-3A+ over ad-3A in the ascus of Neurospora.

A newly induced mutant of Neurospora, when crossed with an ad-3A mutant, produces asci with four viable black and four inviable white ascospores. The survivors always contain the new mutant allele, never ad-3A. The new allele, which is called SK(ad-3A) (for spore killer of ad-3A), is located at or very near the ad-3A locus.--In crosses homozygous for ad-3A, each ascus contains only inviable white ascospores. This defect in ascospore maturation is complemented by the wild-type allele, ad-3A+ (crosses heterozygous for ad-3A and ad-3A+ produce mainly viable ascospores), but it is not complemented by the new SK(ad-3A) allele (all ad-3A ascospores from crosses heterozygous for SK(ad-3A) and ad-3A are white and inviable). In crosses homozygous for SK(ad-3A) or heterozygous for SK(ad-3A) and ad-3A+, each ascus contains only viable black ascospores. SK(ad-3A) does not require adenine for growth, and forced heterokaryons between SK(ad-3A) and ad-3A grow at wild-type rates and produce conidia of both genotypes with approximately equal frequency. Thus, the action of SK(ad-3A) is apparently restricted to ascospore formation. Possible mechanisms of the action of this new allele are discussed.

The isolation of mms- and histidine-sensitive mutants in Neurospora crassa.

A simple method of replica plating has been used to isolate mutants of Neurospora crassa that have increased sensitivity to methyl methanesulfonate (MMS) and/or to histidine. Twelve mutants with increased sensitivity to MMS and one mutant with increased sensitivity to histidine showed Mendelian segregation of the mutant phenotypes. Three mutants were mapped to loci not previously associated with MMS sensitivity. Two other were allelic to the UV- and MMS-sensitive mutant, mei-3. Survival curves indicate that conidia (mutant or wild-type) survive on much higher concentrations of MMS at 25 degrees than at 37 degrees. In contrast, mycelial growth is more resistant to MMS at 37 degrees. The possibility of qualitatively different repair processes at these two temperatures is discussed.

Meiosis in Neurospora crassa. I. The isolation of recessive mutants defective in the production of viable ascospores.

A scheme has been devised for efficient isolation of recessive meiotic mutants of Neurospora crassa. These mutants were detected by their reduced fertility or by the abortion of ascospores. Their isolation involved the selection and screening of the strains arising from ascospores disomic (n + 1) for linkage group I (LG I), which bears the mating-type locus. These strains are self-fertile heterokaryons that contain two types of haploid nuclei of opposite mating types (A + a). Selfings of these strains are homozygous for genes on all linkage groups except LGI and therefore allow the expression of recessive mutants with an altered sexual cycle. Using this selection procedure, three classes of mutants were detected. In one class, mutants had an early block in perithecial development (class I), and in another mutants had altered perithecia, but apparently unaltered fertility (class III). No recessive mutants were observed and all mutants tested (eight of class I and two of class III) were expressed only when used as the maternal parent. A third mutant class displayed normal production of perithecia, but defective formation of asci (class IIA), or black ascospores (class IIB). Four of 13 class IIA mutants were analyzed, and two of them [asc(DL131) and asc (DL400)] were definitely recessive analysis of 10 of 13 class IIB mutants disclosed six recessive, mutually complementing mutants: ase(DL95), asc(DL243), asc(DL711), asc(DL879), asc(DL917m) and asc(DL961). Mutants asc(DL95), asc(DL243) and the previously studied mei-1 mutant (Smith 1975) complemented one another in crosses, but did not recombine. These may be alleles of the same gene, or they may comprise a gene cluster.

Meiosis in Neurospora crassa. II. Genetic and cytological characterization of three meiotic mutants.

Three recessive meiotic mutants, asc(DL95), asc(DL243) and asc(DL879), were detected by the abortion of many of their ascospores and were analyzed using both cytological and genetic methods. Even though asc(DL95), asc (DL243) and the previously studied meiotic mutant, mei-1 (Smith 1975; Lu and Galeazzi (1978), complement one another in crosses, they apparently do not recombine (DeLange and Griffiths (1980). Thus, they may represent alleles of the same gene or comprise a gene cluster. Ascospore abortion in these mutants is caused by abnormal disjunction of meiotic chromosomes. In crosses homozygous for asc(DL95), asc(DL879) or mei-1, both pairing of homologs and meiotic recombination frequencies are reduced. In each case, this primary defect is followed by the formation of univalents at metaphase I and their irregular segregation. The mutant asc(DL243) has a defect in ascus formation, and later in disjunction during the second meiotic and post-meiotic divisions. The first-acting defect before or during karyogamy results in the abortion of most cells. Some cells manage to proceed past this block. During the second meiotic division, most chromosomes of the few resulting asci are attached to only one of the two spindle-pole bodies. Disjunction at the post-meiotic division is also highly irregular. This mutant appears to be defective in the attachment of one spindle-pole body to a set of chromosomes. The defect may involve either a centromere-associated product or a spindle-pole body.