The molecular mechanism of protoplasmic incompatibility and its relationship to the formation of protoperithecia in Podospora anserina.

In Podospora anserina, protoplasmic incompatibility due to interactions between non-allelic genes was suppressed by the effect of mutations in two modifier genes, mod-I and mod-2. It is shown that mod-I and mod-2 are involved in the production of three specific proteins, a phenoloxidase and two previously identified proteases (Bégueret & Bernet 1973 a) which are associated with the phenomenon of protoplasmic disintegration. These enzymes, whose messengers are normallly latent during vegetative growth, appear at this stage of the life cycle only as a consequence of incompatible gene interactions. The mode-I and mod-2 genes and each of the five incompatibility loci involved in non-allelic incompatibility systems also participate in the formation of the protoperithecia. This pleiotropic effect suggests that protoplasmic incompatibility is a deviation in the normal physiological processes of protoperithecial formation.     

Genetics analysis of mouse mutations Abnormal feet and tail and rough coat, which cause developmental abnormalities and alopecia

Mutations in the mouse Brachyury (T) gene are characterized by a dominant reduction of tail length and recessive lethality. Two quantitative trait loci, Brachyury-modifier 1 and 2 (Brm1 and Brm2) are defined by alleles that enhance the short-tail Brachyury phenotype. Here we report on a genetic analysis of a visible dominant mutation Abnormal feet and tail (Aft) located in the vicinity of Brm1. Affected animals display kinky tails and syndactyly in the hindlimbs, both likely resulting from a defect in apoptosis. We observed an unusual genetic incompatibility between Aft and certain genetic backgrounds. We show that Aft and T are likely to interact genetically, since some double heterozygotes are tailless. In addition to the tail and hindlimb phenotypes, Aft-bearing mutants display characteristic late-onset skin lesions. We therefore tested for allelism between Aft and a closely linked recessive mutation rough coat (rc) and found that these two mutations are likely nonallelic. Our results provide a valuable resource for the study of mammalian skin development and contribute to the genetic analysis of Brachyury function.     

Protoplasmic Incompatibility in PODOSPORA ANSERINA: a Possible Function for Incompatibility Genes

The suppression of protoplasmic incompatibility resulting from nonallelic gene interactions has been obtained by the coupled effect of mutations in the modA and modB genes (Bernet 1971). Due to their female sterility, modA modB strains provide an experimental tool to determine whether or not the mod and incompatibility loci are involved in a function other than protoplasmic incompatibility. Present results show that modA modB female sterility is a nonautonomous trait since heterokaryotic mycelia that include a modA modB nucleus and a female fertile nucleus (wild-type, modA or modB) produce modA modB protoperithecia, which are also formed by culture on medium supplemented with specific amino acids. Using modA modB strains, which are sterile at 32 degrees and fertile at 26 degrees , we have shown that the mod genes have no specific sequential timing. Indeed, the mod mutations may prevent the achievement of the female sexual cycle at any developmental stage from before early differentiation of protoperithecia until ascospore maturation. Employing different modA and modB mutations, we have shown that protoperithecia in modA modB cultures are generally distributed in female fertile rings; this result indicates that protoperithecia occur only in mycelial areas that have a restricted range of age at the time that modA modB thalli complete growth. Furthermore, nonsense mutations of incompatibility genes suppress the modA modB female fertile rings or restrict their width, suggesting that incompatibility loci, like the mod loci, are involved in protoperithecium formation. Taken together, these results lead to the postulate that mod and incompatibility genes do not determine, sensu stricto, protoperithecial function, as previously supposed (Boucherie and Bernet 1974), but may be involved in the homeostatic control of stationary cell functions essential for the complete development of the female sexual cycle.     

Conditional impairment of cell division and altered lethality in hipA mutants of Escherichia coli K-12.

Mutations in hipA, a gene of Escherichia coli K-12, greatly reduce the lethality of selective inhibition of peptidoglycan synthesis. These mutations have also been found to reduce the lethality that accompanies either selective inhibition of DNA synthesis or heat shock of strains defective in htpR. In addition, the mutant alleles of hipA are responsible for a reversible cold-sensitive block in cell division and synthesis of macromolecules, particularly peptidoglycan. Recombination between the chromosome of hipA mutants and plasmids containing noncomplementing fragments of hipA+ revealed that the mutations responsible for both cold sensitivity and reduced lethality were probably identical and, in any case, lay within the first 360 base pairs of the coding region of hipA, probably within the first 50 base pairs. We suggest that the pleiotropic effects of mutations in hipA reflect the involvement of this gene in cell division.     

Biosynthesis of hepatitis B virus e antigen: directed mutagenesis of the putative aspartyl protease site.

The C gene products of all mammalian hepadnaviruses contain a region with sequence similarities to the catalytic center of the aspartyl proteases. This region could have the capacity to cleave precore proteins, leading to the synthesis of e antigen. By site-directed mutagenesis on a plasmid containing the hepatitis B virus C gene, we have replaced either the Asp residue of the putative aspartyl protease catalytic center or an Asp residue located 3 amino acids upstream. Transient expression of the mutated hepatitis B virus C gene in human and mouse cells showed that none of these mutations prevented the secretion of an accurately processed HBe antigen. Thus, we demonstrated that the aspartyl protease responsible for e antigen precursor processing is not C gene encoded but is more likely to be a cellular enzyme. From these results, we suggest a model for the mechanism of e antigen synthesis.     

Rules of nonallelic noncomplementation at the synapse in Caenorhabditis elegans

Nonallelic noncomplementation occurs when recessive mutations in two different loci fail to complement one another, in other words, the double heterozygote exhibits a phenotype. We observed that mutations in the genes encoding the physically interacting synaptic proteins UNC-13 and syntaxin/UNC-64 failed to complement one another in the nematode Caenorhabditis elegans. Noncomplementation was not observed between null alleles of these genes and thus this genetic interaction does not occur with a simple decrease in dosage at the two loci. However, noncomplementation was observed if at least one gene encoded a partially functional gene product. Thus, this genetic interaction requires a poisonous gene product to sensitize the genetic background. Nonallelic noncomplementation was not limited to interacting proteins: Although the strongest effects were observed between loci encoding gene products that bind to one another, interactions were also observed between proteins that do not directly interact but are members of the same complex. We also observed noncomplementation between genes that function at distant points in the same pathway, implying that physical interactions are not required for nonallelic noncomplementation. Finally, we observed that mutations in genes that function in different processes such as neurotransmitter synthesis or synaptic development complement one another. Thus, this genetic interaction is specific for genes acting in the same pathway, that is, for genes acting in synaptic vesicle trafficking.     

Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes

Rearrangements of our genome can be responsible for inherited as well as sporadic traits. The analyses of chromosome breakpoints in the proximal short arm of Chromosome 17 (17p) reveal nonallelic homologous recombination (NAHR) as a major mechanism for recurrent rearrangements whereas nonhomologous end-joining (NHEJ) can be responsible for many of the nonrecurrent rearrangements. Genome architectural features consisting of low-copy repeats (LCRs), or segmental duplications, can stimulate and mediate NAHR, and there are hotspots for the crossovers within the LCRs. Rearrangements introduce variation into our genome for selection to act upon and as such serve an evolutionary function analogous to base pair changes. Genomic rearrangements may cause Mendelian diseases, produce complex traits such as behaviors, or represent benign polymorphic changes. The mechanisms by which rearrangements convey phenotypes are diverse and include gene dosage, gene interruption, generation of a fusion gene, position effects, unmasking of recessive coding region mutations (single nucleotide polymorphisms, SNPs, in coding DNA) or other functional SNPs, and perhaps by effects on transvection.     

Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome

Dubin-Johnson syndrome (DJS) is an autosomal recessive disease characterized by conjugated hyperbilirubinemia. Previous studies of the defects in the human canalicular multispecific organic anion transporter gene (MRP2/cMOAT) in patients with DJS have suggested that the gene defects are responsible for DJS. In this study, we determined the exon/intron structure of the human MRP2/cMOAT gene and further characterized mutations in patients with DJS. The human MRP2/cMOAT gene contains 32 exons, and it has a structure that is highly conserved with that of another ATP-binding-cassette gene, that for a multidrug resistance-associated protein. We then identified three mutations, including two novel ones. All mutations identified to date are in the cytoplasmic domain, which includes the two ATP-binding cassettes and the linker region, or adjacent putative transmembrane domain. Our results confirm that MRP2/cMOAT is the gene responsible for DJS. The finding that mutations are concentrated in the first ATP-binding-cassette domain strongly suggests that a disruption of this region is a critical route to loss of function.     

The Isolation of Mutagen-Sensitive Nuv Mutants of Aspergillus Nidulans and Their Effects on Mitotic Recombination

More than 200 mutants of Aspergillus nidulans were isolated as hypersensitive to the monofunctional alkylating agent MNNG and/or UV-irradiation (designated nuv mutants). Of these, 23 were selected for further characterization. All were markedly hypersensitive to both MNNG and the quasi-UV-mimetic mutagen 4-NQO. The hypersensitive phenotype of each mutant was shown to result from mutation of a single gene. The nuv mutants exhibited a diverse range of growth responses on solid media containing various concentrations of MNNG or 4-NQO. This suggested that they represented many nonallelic mutations. Analysis to determine the dominance/recessiveness of the nuv mutations with respect to hypersensitivity revealed that most were fully recessive, although several appeared to be semidominant. A novel system to assay homologous mitotic recombination using simple plating tests was developed. The system was exploited to determine the effects of the nuv mutations on mitotic recombination. Of the 23 mutations tested, 10 caused a hypo-recombination phenotype and three a hyper-recombination phenotype, while 10 appeared to have no effect on recombination. The hypo-rec effect of one of the mutations, nuv-117, appeared to be semidominant. Transcomplementation analysis between seven of the nuv mutations defined at least six nonallelic loci.     

Genetic interactions between the Escherichia coli umuDC gene products and the beta processivity clamp of the replicative DNA polymerase

The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA-single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD' (which enables TLS). In addition, even modest overexpression of the umuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD(2)C. We have previously reported that overexpression of the epsilon proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of epsilon with UmuD(2)C is important for the DNA damage checkpoint control function of the umuDC gene products. Here, we report that overexpression of the beta processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of the umuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD'C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant beta proteins possibly deficient for physical interactions with the umuDC gene products, we selected for novel dnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD'C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of beta or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaN missense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eight dnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of the umuDC gene products, suggesting that the interactions between UmuD' and beta are a subset of those between UmuD and beta. Taken together, these findings suggest that interaction of beta with UmuD(2)C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD(2)C with the epsilon and the beta subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.     

Superkiller mutations in Saccharomyces cerevisiae suppress exclusion of M2 double-stranded RNA by L-A-HN and confer cold sensitivity in the presence of M and L-A-HN.

In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30 degrees C but not at 20 degrees C. Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes. The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mks1 and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes. Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30 degrees C and prevent the cells from growing at 8 degrees C. Elimination of M double-stranded RNA from a cold-sensitive ski- strain results in the loss of cold sensitivity. ski- [KIL-sd1] strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- [KIL-k1] strains and are only slightly cold sensitive. The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication. We propose that the elevated levels of M in ski- strains divert the host LTS5 product away from the host and to the M replication process. We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI+ products.     

Hybrid breakdown caused by substitution of the X chromosome between two mouse subspecies

Hybrid breakdown is a type of reproductive failure that appears after the F2 generation of crosses between different species or subspecies. It is caused by incompatibility between interacting genes. Genetic analysis of hybrid breakdown, particularly in higher animals, has been hampered by its complex nature (i.e., it involves more than two genes, and the phenotype is recessive). We studied hybrid breakdown using a new consomic strain, C57BL/6J-X(MSM), in which the X chromosome of C57BL/6J (derived mostly from Mus musculus domesticus) is substituted by the X chromosome of the MSM/Ms strain (M. m. molossinus). Males of this consomic strain are sterile, whereas F1 hybrids between C57BL/6J and MSM/Ms are completely fertile. The C57BL/6J-X(MSM) males showed reduced testis weight with variable defects in spermatogenesis and abnormal sperm head morphology. We conducted quantitative trait locus (QTL) analysis for these traits to map the X-linked genetic factors responsible for the sterility. This analysis successfully detected at least three distinct loci for the sperm head morphology and one for the testis weight. This study revealed that incompatibility of interactions of X-linked gene(s) with autosomal and/or Y-linked gene(s) causes the hybrid breakdown between the genetically distant C57BL/6J and MSM/Ms strains.     

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.     

Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina

In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi-6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi-7. We have used a GFP-IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi-7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.     

Cluster of mrdA and mrdB genes responsible for the rod shape and mecillinam sensitivity of Escherichia coli.

Two closely linked genes, mrdA and mrdB, located at ca. 14.2 min on the Escherichia coli chromosomal linkage map, seen to be responsible for the normal rod shape and mecillinam sensitivity of E. coli. The product of mrdA was concluded to be penicillin-binding protein 2, because mrdA mutations caused formation of thermosensitive penicillin-binding protein 2. The product of the mrdB gene is unknown. At 42 degrees, C, mutation in either of these genes caused formation of spherical cells and mecillinam resistance. Both mutations was recessive, and complementation, as detected in +-/-+ meroheterodiploids having the wild-type phenotype, provided strong evidence that the two mutations are in different complementation groups. P1 transduction suggested that the most plausible gene order is leuS-mrdA-mrdB-lip. The rodA mutation reported previously seems to be similar to the mrdB murations, but the identities of the two have not yet been proven.     

Role for poliovirus protease 2A in cap independent translation.

Viral protein synthesis in poliovirus infected cells was found to be influenced by mutations in part of the viral 5'-non-coding region (NCR) in a temperature dependent manner. At elevated temperatures these mutations resulted in virus titre reductions that allowed selection of revertant viruses. Some revertants were found to have retained the 5'-NCR mutations but had compensating mutations in the 2A protease gene that were responsible for the suppression of the temperature sensitive phenotypes. The mutations in 2A enhanced viral protein synthesis at a stage when cap dependent translation was already abolished, suggesting that the virally encoded protein 2A is directly involved in the process of cap independent translation in addition to its role in abolishing cap dependent translation.