Genetic characterization of Tetrahymena thermophila mutants unable to secrete capsules

Under appropriate conditions, Alcian Blue-induced exocytosis of Tetrahymena mucocysts leads to formation of a capsule that surrounds the cell. This phenomenon is an example of regulated secretion, a mechanism of fundamental significance in eukaryotic cells. In order to dissect genetically the mechanism of mucocyst biogenesis and regulated exocytosis, mutants unable to form capsules (Caps–) were isolated. In this paper we report a genetic characterization of Caps– mutants in this collection. The mutations in mutants SB255 and SB281 behave as single recessive Men-delian mutations. The mutation in SB251 is restricted to the macronucleus, and could not be further characterized by the genetic methods we used. Complementation tests suggest the existence of at least 2 genes, named exoA and exoB; additional mutant loci are likely to be included in the mutant collection. Deletion mapping using nulli-somic strains showed that exoA and exoB are located on the left arm of chromosome 4. The exo-3 mutation, which behaves as recessive and complements with exoA1 in SB255 and exoB2 in SB281, maps to chromosome 3. These Caps– mutants may be useful for the elucidation of the developmental pathway of mucocyst biogenesis and the control of regulated secretion in eukaryotic cells. © 1992 Wiley-Liss, Inc.     

Conjugation Rescue of Exocytosis Mutants in Tetrahymena thermophila Indicates the Presence of Functional Intermediates in the Regulated Secretory Pathway

ABSTRACT. Tetrahymena thermophila possesses a regulated secretory pathway in which mucin proteins are stored in dense-core granules, called mucocysts. Exocytosis-defective mutants exist that fail to secrete mucin in response to secretagogues. Four of the mutants (SB281, SB283, SB285 and SB715) appear to be blocked at different steps of the regulated secretory pathway. SB281 and SB285 accumulate mucin proteins in heterogeneous cytoplasmic organelles which have not yet been identified; SB283 makes mucocyst-like structures but they contain no immunologically identifiable 80-kDa or 50-kDa mucin proteins; and SB715 has more than normal amounts of immature and undocked mucocysts. The organelles that accumulate in exocytosis-defective mutants could be either normal intermediates in the biosynthetic pathway or aberrant structures that form as a result of the mutations. We have used conjugation rescue to analyze steps in the biogenesis of exocytosis-competent mucocysts and to identify functional intermediates. The cytoplasmic organelles that accumulate in SB281 appear to be unidentified biosynthetic intermediates, and the defect is in a cytosolic protein essential for mucocyst maturation. The organelles which accumulate in the other mutants are likely biosynthetic, but their mutations are in proteins which are labile or not free to diffuse into the mutant conjugant.     

Maturation of dense core granules in wild type and mutant Tetrahymena thermophila.

Tetrahymena thermophila, a ciliated protozoan, has a well-developed pathway of regulated secretion from dense core granules called mucocysts. Since exocytosis-defective mutants are available, steps in the biogenesis of dense core granules and their fusion with the plasma membrane may be resolved genetically. To describe the steps in biochemical terms, we have generated antisera against mucocyst content proteins. One antiserum is directed against a calcium binding protein, p40, that is released on stimulation of exocytosis. p40 is shown to associate with an insoluble matrix in mature mucocysts. In addition, the antiserum recognizes a larger protein, p60, that is soluble, is not found in mature mucocysts and is not released on stimulation. Pulse-chase experiments support a precursor-product relationship between p60 and p40. Using these proteins as markers, two mutant Tetrahymena strains defective in exocytosis have been shown to accumulate the putative precursor p60 in organelles that can be distinguished from one another and from wild type mucocysts on the basis of density. The kinetics of appearance of insoluble p40 and the mutant phenotypes suggest a model of mucocyst maturation in which sorting precedes matrix condensation.     

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules.

Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.     

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.     

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

Mutations in Genes Encoding Essential Mitotic Functions in DROSOPHILA MELANOGASTER

Temperature-sensitive mutations at 15 loci that affect the fidelity of mitotic chromosome behavior have been isolated in Drosophila melanogaster. These mitotic mutants were detected in a collection of 168 EMS-induced X-linked temperature-sensitive (ts) lethal and semilethal mutants. Our screen for mutations with mitotic effects was based upon the reasoning that under semirestrictive conditions such mutations could cause an elevated frequency of mitotic chromosome misbehavior and that such events would be detectable with somatic cell genetic techniques. Males hemizygous for each ts lethal and heterozygous for the recessive autosomal cell marker mwh were reared under semirestrictive conditions, and the wings of those individuals surviving to adulthood were examined for an increased frequency of mwh clones. Those mutations producing elevated levels of chromosome instability during growth of the wing imaginal disc were also examined for their effects on chromosome behavior in the cell lineages producing the abdominal cuticle. Fifteen mutations affect chromosome behavior in both wing and abdominal cells and thus identify loci generally required for the fidelity of mitotic chromosome transmission. Mapping and complementation tests show that these mutations represent 15 loci. One mutant is an allele of a locus (mus-101) previously identified by mutagen-sensitive mutants and a second mutant is an allele of the lethal locus zw 10.--The 15 mutants were also examined cytologically for their effects on chromosomes in larval neuroblasts. Taken together, the results of our cytological and genetical studies show that these mutants identify loci with wild-type functions necessary for either maintenance of chromosome integrity or regular disjunction of chromosomes or chromosome condensation. Thus, these mutations define a broad spectrum of genes required for the normal execution of the mitotic chromosome cycle.     

Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit.

Suppressors of the blue fluorescence phenotype of the Arabidopsis trp1-100 mutant can be used to identify mutations in genes involved in plant tryptophan biosynthesis. Two recessive suppressor mutations define a new gene, TRP4. The trp4 mutant and the trp1-100 mutant are morphologically normal and grow without tryptophan, whereas the trp4; trp1-100 double mutant requires tryptophan for growth. The trp4; trp1-100 double mutant does not segregate at expected frequencies in genetic crosses because of a female-specific defect in transmission of the double mutant genotype, suggesting a role for the tryptophan pathway in female gametophyte development. Genetic and biochemical evidence shows that trp4 mutants are defective in a gene encoding the beta subunit of anthranilate synthase (AS). Arabidopsis AS beta subunit genes were isolated by complementation of an Escherichia coli anthranilate synthase mutation. The trp4 mutation cosegregates with one of the genes, ASB1, located on chromosome 1. Sequence analysis of the ASB1 gene from trp4-1 and trp4-2 plants revealed different single base pair substitutions relative to the wild type. Anthranilate synthase alpha and beta subunit genes are regulated coordinately in response to bacterial pathogen infiltration.     

Absence of complementation in non-allelic mutants of Dictyostelium discoideum with defects in the transition from growth to development

In Dictyostelium discoideum, growth and development are mutually exclusive and the transition between the two phases of the life cycle is regulated by the environment. This regulation is disturbed in HBW3, a chemically induced mutant with an unknown molecular defect. The mutant develops rapidly and expresses developmental markers during growth. Here we show that HBW3 fails to complement another mutant which has a similar phenotype: a targeted knock-out of the gdt1 gene. We further show that both mutations are recessive and that both are located on chromosome III, suggesting that the two mutations might be allelic. Molecular analysis, however, demonstrates that the gdt1 gene is not mutated in HBW3. Thus, although a wild-type copy of each gene is present in diploid cell lines, the defects due to the recessive mutations synergize to produce a detectable phenotype. The phenotypic similarities and differences between the two mutants are discussed.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt^+/−→aprt^−/−) was 1.3 × 10⁻⁵/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt^−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.     

Recessive hyperekplexia mutations of the glycine receptor α1 subunit affect cell surface integration and stability

The human neurological disorder hyperekplexia is frequently caused by recessive and dominant mutations of the glycine receptor α1 subunit gene, GLRA1 . Dominant forms are mostly attributed to amino acid substitutions within the ion pore or adjacent loops, resulting in altered channel properties. Here, the biogenesis of glycine receptor α1 subunit mutants underlying recessive forms of hyperekplexia was analyzed following recombinant expression in HEK293 cells. The α1 mutant S231R resulted in a decrease of surface integrated protein, consistent with reduced maximal current values. Decreased maximal currents shown for the recessive α1 mutant I244N were associated with protein instability, rather than decreased surface integration. The recessive mutants R252H and R392H encode exchanges of arginine residues delineating the intracellular faces of transmembrane domains. After expression, the mutant R252H was virtually absent from the cell surface, consistent with non-functionality and the importance of the positive charge for membrane integration. Surface expression of R392H was highly reduced, resulting in residual chloride conductance. Independent of the site of the mutation within the α1 polypeptide, metabolic radiolabelling and pulse chase studies revealed a shorter half-life of the full-length α1 protein for all recessive mutants as compared to the wild-type. Treatment with the proteasome blocker, lactacystin, significantly increased the accumulation of α1 mutants in intracellular membranes. These observations indicated that the recessive α1 mutants are recognized by the endoplasmatic reticulum control system, and degraded via the proteasome pathway. Thus, the lack of glycinergic inhibition associated with recessive hyperekplexia may be attributed to sequestration of mutant subunits within the endoplasmatic reticulum quality control system.     

Identification of a gene, included in control of root system development in Arabidopsis thaliana

Genetic and molecular genetic analysis of a lethal root mutant of Arabidopsis thaliana was carried out. The mutant was obtained from a collection created earlier by means of insertion mutagenesis. The mutation was found to be recessive. It was caused by an insertion of the T region of vector pLD3 used for transformation of germinating seeds when creating the collection of insertion mutants. A 118-bp DNA fragment flanking the left border of the insertion was isolated using the TAIL PCR technique, and its nucleotide sequence was determined. Computer analysis of this DNA region demonstrated that it was located in exon 32 of the YUP8HI2R.44 gene in chromosome 1.     

Genetic analysis of a cluster of genes required for synthesis of the calcofluor-binding exopolysaccharide of Rhizobium meliloti.

Rhizobium meliloti produces an acidic, Calcofluor-binding exopolysaccharide which plays a role in nodulation of alfalfa plants by this bacterium. We constructed and mapped 102 transposon insertions in a 48-kilobase (kb) region previously shown to contain several exo genes. Mutations affecting production of the Calcofluor-binding exopolysaccharide were clustered in a 22-kb region and fell into 12 complementation groups. Strains carrying mutations in seven of the complementation groups (exoA, exoB, exoF, exoL, exoM, exoP, and exoQ) produced no Calcofluor-binding exopolysaccharide and induced non-nitrogen-fixing nodules on alfalfa. Mutants in an eighth complementation group, exoH (Leigh et al., Cell 51:579-587, 1987), produce an altered exopolysaccharide and also induce the formation of non-nitrogen-fixing nodules. Mutants in the remaining four complementation groups produced less Calcofluor-binding material than the wild type. Mutants carrying mutations in two of these complementation groups (exoK and exoN) formed apparently normal, nitrogen-fixing nodules, while mutants in the other two groups (exoG and exoJ) formed normal nodules less efficiently than the wild type.     

Identification of the gene whose mutation leads to hypocotyl tropism in Arabidopsis thaliana

Genetic and molecular analysis of a mutant of Arabidopsis thaliana with bended hypocotyl from a previously obtained collection of insertion mutants is presented. The examined mutation was shown to be recessive and based on a single insertion of pLD3 vector T-region into the A. thaliana genome. Computer-aided analysis of a DNA region adjacent to the left border of the insertion revealed a putative site of T-DNA insertion, the At1g15760 gene from 609-bp chromosome 1 represented by a single exon.     

Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.     

A novel dnaC mutation that suppresses priB rep mutant phenotypes in Escherichia coli K-12

The loading of a replisome in prokaryotic and eukaryotic cells at an origin of DNA replication and during replication restart is a highly ordered and regulated process. During replication restart in Escherichia coli, the PriA, PriB, PriC, DnaT and Rep proteins form multiple pathways that bind to repaired replication forks. These complexes are then recognized by DnaC as sites to load DnaB, the replicative helicase. Several dnaC mutations have been isolated that suppress phenotypes of some replication restart mutants. A new dnaC mutation (dnaC824) is reported here that efficiently suppresses priB rep mutant phenotypes. Furthermore, it is shown that dnaC824 will suppress phenotypes of priB priA300, rep priA300 and priB priC strains. Unlike other dnaC suppressors, it can only weakly suppress the absence of priA. Others have reported a different type of dnaC mutation, dnaC1331, is able to mimic priB mutant phenotypes. This is supported herein by showing that like dnaC1331, a priB mutation is synthetically lethal with a dam mutation and this can be rescued by a mutH mutation. Furthermore, priB dam lethality can also be suppressed by dnaC824. Like a priB mutation, a dnaC1331 mutation causes a priA2::kan-like phenotype when combined with priA300. Lastly, we show that dnaC824 is dominant to wild type and that dnaC1331 is recessive to wild type. Several models are discussed for the action of these mutant dnaC proteins in replication restart.     

A new mouse insertional mutation that causes sensorineural deafness and vestibular defects.

This article describes a new recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice. Histologic analysis revealed virtually complete loss of the cochlear neuroepithelium (the organ of Corti) in adult mutant mice. In association with the neuroepithelial changes, there is a dramatic reduction of the cochlear nerve supply. Adult mutants also show morphological defects of the vestibular apparatus, including degeneration of the saccular neuroepithelium and occasional malformation of utricular otoconia. Audiometric evaluations demonstrated that the mice displaying the circling phenotype are completely deaf. Molecular analysis of this mutant line revealed that the transgenic insertion occurred without creating a large deletion of the host DNA sequences. The mutant locus was mapped to a region on mouse chromosome 10, where other spontaneous, recessive mutations causing deafness in mice have been mapped.     

Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. II. Homozygous Effect of Polygenic Mutations

Polygenic mutations affecting viability were accumulated on the second chromosome of Drosophila melanogaster by treating flies with EMS in successive generations. The treated chromosomes were later made homozygous and tested for their effects on viability by comparison of the frequency of such homozygotes with that of other genotypes in the same culture. The treated wild-type chromosomes were kept heterozygous in Pm/+ males by mating individual males in successive generations to Cy/Pm females. The number of generations of accumulation was 1 to 30 generations, depending on the concentration of EMS. A similar experiment for spontaneous polygenic mutations was also conducted by accumulating mutations for 40 generations. The lower limit of the spontaneous mutation rate of viability polygenes is estimated to be 0.06 per second chromosome per generation, which is about 12 times as high as the spontaneous recessive lethal mutation rate, 0.005. EMS-induced polygenic mutations increase linearly with the number of treated generations and with the concentration of EMS. The minimum mutation rate of viability polygenes is about 0.017 per 10(-4)m, which is only slightly larger than the lethal rate of 0.013 per 10(-4) m. The maximum estimate of the viability reduction of a single mutant is about 6 to 10 percent of the normal viability. The data are consistent with a constant average effect per mutant at all concentrations, but this is about three times as high as that for spontaneous mutants. It is obvious that one can obtain only a lower limit for the mutation rate, since some mutants may have effects so near to zero that they cannot be detected. The possibility of measuring something other than the lower limit is discussed. The ratio of the load due to detrimental mutants to that caused by lethals, the D/L ratio, is about 0.2 to 0.3 for EMS-induced mutants, as compared to about 0.5 for spontaneous mutants. This is to be expected if EMS treatment produces a large fraction of small deletions and other chromosome rearrangements which are more likely to be lethal.