Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.     

Genetic and Morphological Study of Aggregation in the Cellular Slime Mold POLYSPHONDYLIUM VIOLACEUM

A system for genetic analysis in the cellular slime mold P. violaceum has been developed. Two growth-temperature-sensitive mutants were isolated in a haploid strain and used to select rare diploid heterozygotes arising by spontaneous fusion of the haploid cells. A recessive mutations to cycloheximide resistance in one strain enables selection of segregants, which often appear to be aneuploid.—Aggregation-defective (ag^- ) mutants having a wide range of phenotypes were isolated in both temperature-sensitive strains after nitrosoguanidine treatment, and complementation tests were performed between pairs of these mutants. Of 380 diploids isolated, 32 showed defective aggregation and were considered to contain 2 noncomplementing ag^- mutations. Among noncomplementing mutants interallelic complementation is common. Noncomplementing mutants fall into 4 complementation groups, and those within each complementation group are phenotypically similar. Statistical analysis of the results suggests that the number of complementation units involved in aggregation is about 50.     

Genetic analysis of tag mutants of Myxococcus xanthus provides evidence for two developmental aggregation systems

Temperature-dependent aggregation mutants (tag) of the myxobacterium Myxococcus xanthus aggregated into mounds and developed into fruiting bodies normally at 28 degrees C; however, they failed to form mounds at 34 degrees C. The timing of sporulation was unaffected by the mutations, and normal numbers of spores were produced at both permissive and restrictive temperatures. This class of mutations was originally identified through screening of ethyl methanesulfonate (EMS)-generated mutations. Subsequent work identified a linked insertion of transposon Tn5, which was used to map the EMS-generated mutations to four loci. In this paper, we describe the cloning of the tag loci and the use of transposon mutagenesis to further analyze the tag loci. Nine tag complementation groups spanning 8.5 kilobase pairs of DNA were identified through mapping of 28 independent Tn5 insertions. All insertion and deletion mutants had the same phenotype as the EMS mutants: they were temperature sensitive for mound formation. This result suggests that M. xanthus has at least two sets of genes for developmental aggregation. The tag genes constitute one set of these genes; they are required for normal development at 34 degrees C but are not required for normal development at 28 degrees C.     

Mechanism of conjugation and recombination in bacteria XVI

Summary Protein synthesis by ribosomes from several cryptopleurine-resistant yeast mutants is also resistant to emetine and tubulosine. These mutants can be classified into two different types: Class I mutants which display high levels of resistance to emetine and tubulosine and Class II mutants that are only weakly resistant to tubulosine and are slightly more sensitive to emetine than those of Class I. Apparently all mutants have similar levels of resistance to cryptopleurine. The distinct phenotypes of Class I and Class II strains are expressed through their 40S ribosomal subunit. Genetic analysis has shown that the mutations to cryptopleurine resistance are allelic and that in a particular case (strain CRY6) the pleiotropic phenotype is a result of the expression of the cryl locus. It is suggested that Class I and Class II mutants arise from two independent mutational events within the cryl allele. in heterozygous (+/cryl) diploids both the sensitive and the resistant genes are expressed as shown by studies of the action of cryptopleurine on polyphenylalanine-synthesizing system derived from each parental sensitive and resistant haploid strain and heterozygous diploid strains. The apparent dominance of sensitivity over resistance which may be observed in vivo in heterozygous (+/cryl) diploids has been explained in terms of the mode of action of the inhibitors.     

Genetic analysis of thr mutations in Salmonella typhimurium

Previous workers divided threonine-requiring (Thr(-)) strains of Salmonella into three phenotypes with mutations in four complementation groups. The mutations were deemed to define four genes in the order thrD-C-A-B at minute zero on the Salmonella linkage map. In the present study 12 of these mutants were reexamined together with eight new Thr(-) strains. The three phenotypes were: homoserine-requiring (Hom(-)); Thr(-), feeders of Hom(-) strains; Thr(-), nonfeeders. Exact correlation between these phenotypic groups and three complementation groups was confirmed by abortive transduction. No evidence was found for intergenic complementation between mutations in Hom(-) strains. It is proposed that thr mutations define three genes rather than four and that these be renamed thrA (Hom(-)), thrB (Thr(-) feeders), and thrC (Thr(-) nonfeeders) to correspond with the sequence of reactions in threonine biosynthesis. Double mutant trpRthr strains were used in reciprocal three-point transduction tests to establish the order of thr mutation sites. Although revisions were made in the classification or location of several mutations, there was an overall correlation of complementation group, phenotype, and map position. The present data provide a basis for further correlation of threonine genes and biosynthetic enzymes, and analysis of cross regulation in aspartate amino acid biosynthesis in Salmonella.     

A new gene (madI) involved in the phototropic response of Phycomyces

Summary Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.     

P-Element Mutations Affecting Embryonic Peripheral Nervous System Development in Drosophila Melanogaster

The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study the molecular mechanisms governing neural development. To identify genes controlling PNS development, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the P-element insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS.     

Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.     

The hydrophobin HCf-1 of Cladosporium fulvum is required for efficient water-mediated dispersal of conidia

Six hydrophobin genes (HCf-1 to -6) have thus far been identified in the tomato pathogen Cladosporium fulvum. HCf-1 to -4 are Class I hydrophobins and HCf-5 and -6 are Class II hydrophobins. In this paper we describe the isolation of deletion mutants that lack HCf-1, HCf-2, or both these genes. Global down-regulation of the expression of Class I hydrophobins is achieved by homology-dependent gene silencing. Analysis of the mutant strains shows that HCf-1 confers hydrophilic character to the conidia and this facilitates the dissemination of conidia on the surface of water droplets. Other Class I hydrophobins, such as HCf-3 or HCf-4, may be involved in the development and germination of conidia.     

Physical and genetic map of a Rhizobium meliloti nodulation gene region and nucleotide sequence of nodC.

Infection of alfalfa by the soil bacterium Rhizobium meliloti proceeds by deformation of root hairs and bacterial invasion of host tissue by way of an infection thread. We studied an 8.7-kilobase (kb) segment of the R. meliloti megaplasmid, which contains genes required for infection. Site-directed Tn5 mutagenesis was used to examine this fragment for nodulation genes. A total of 81 R. meliloti strains with mapped Tn5 insertions in the 8.7-kb fragment were evaluated for nodulation phenotype on alfalfa plants; 39 of the insertions defined a 3.5-kb segment containing nodulation functions. Of these 39 mutants, 37 were completely nodulation deficient (Nod-), and 2 at the extreme nif-distal end were leaky Nod-. Complementation analysis was performed by inoculating plants with strains carrying a genomic Tn5 at one location and a plasmid-borne Tn5 at another location in the 3.5-kb nodulation segment. Mutations near the right border of the fragment behaved as two distinct complementation groups. The segment in which these mutations are located was analyzed by DNA sequencing. Several open reading frames were found in this region, but the one most likely to function is 1,206 bases long, reading from left to right (nif distal to proximal) and spanning both mutation groups. The genetic behavior of this segment may be due either to the gene product having two functional domains or to a recombinational hot spot between the apparent complementation groups.     

Seventeen Complementation Groups of Mutations Decreasing Meiotic Recombination in Schizosaccharomyces Pombe

We have analyzed 43 recessive mutations reducing meiotic intragenic recombination in Schizosaccharomyces pombe. These mutations were isolated by a screen for reduced plasmid-by-chromosome recombination at the ade6 locus. Sixteen of the mutations define 10 new complementation groups, bringing to 17 the number of genes identified to be involved in meiotic recombination. The mutations were grouped into three discrete classes depending on the severity of the recombination deficiency in crosses involving the ade6-M26 recombination hotspot. Class I mutations caused at least a 1000-fold reduction in M26-stimulated intragenic recombination at the ade6 locus. Class II mutations reduced M26-stimulated recombination approximately 100-fold. Class III mutations caused a 3-10-fold reduction in either M26-stimulated or non-hotspot recombination. We obtained multiple alleles of class I and class II mutations, suggesting that we may be nearing saturation for mutations of this type. As a first step toward mapping, we used mitotic segregation to assign fourteen of the rec genes to chromosomes. Mutations in the six rec genes tested also caused a decrease in intragenic recombination at the ura4 locus; five of these mutations also reduced intergenic recombination between the pro2 and arg3 genes. These results indicate that these multiple rec gene products are required for high level meiotic recombination throughout the S. pombe genome.     

Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae

Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morphology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na(+)/H(+) exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.     

Genes Required for Vacuolar Acidity in Saccharomyces Cerevisiae

Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein. Thirty nine vacuolar pH (Vph-) mutants were identified. Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes. The remaining mutants defined eight complementation groups of vph mutations. No alleles of the VAT2 or TFP1 genes (known to encode subunits of the vacuolar H(+)-ATPase) were identified in the Vph- screen. Strains bearing mutations in any of six of the VPH genes failed to grow on medium buffered at neutral pH; otherwise, none of the vph mutations caused notable growth inhibition on standard yeast media. Expression of the vacuolar protease, carboxypeptidase Y, was defective in strains bearing vph4 mutations but was apparently normal in strains bearing any of the other vph mutations. Defects in vacuolar morphology at the light microscope level were evident in all Vph- mutants. Strains that contained representative mutant alleles of the 17 previously described PEP genes were assayed for vacuolar pH; mutations in seven of the PEP genes (including PEP3, PEP5, PEP6 and PEP7) caused loss of vacuolar acidity.     

Mutations in Escherichia coli that effect sensitivity to oxygen

Fifteen oxygen-sensitive (Oxys) mutants of Escherichia coli were isolated after exposure to UV light. The mutants did not form macroscopic colonies when plated aerobically. They did form macroscopic colonies anaerobically. Oxygen, introduced during log phase, inhibited the growth of liquid cultures. The degree of inhibition was used to separate the mutants into three classes. Class I mutants did not grow after exposure to oxygen. Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent. Class III mutants formed filaments in response to oxygen. Genetic experiments indicated that the mutations map to six different chromosomal regions. The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent. Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min. P1 transduction data indicated that mutations in three of these five mutants, Oxys-6, Oxys-14, and Oxys-17, mapped to 8.4 min. The correlation of low enzyme levels and mapping data suggests that a single gene may regulate several enzymes in response to oxygen. The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity. The class II and III mutants had enzyme activities similar to those of the wild-type parent. Our results demonstrate that mutations in at least six genes can be expressed as oxygen sensitivity. Some of these genes may be involved in respiration or cell division or may regulate the expression of several enzymes.     

Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities. This mutant carries a mutation located near minute 58 in the genome. Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system. A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12. Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated. These mutants were divided into three groups. Class I mutants were restored to the wild-type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1. Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2. Class III mutants were unaffected by both pRW1 and growth with NiCl2. The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity. Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al. (1985) J. Bacteriol., 162, 353-360). None of the three classes of mutants possess mutations in hydrogenase structural genes.     

A Female Sterile Screen of the Drosophila Melanogaster X Chromosome Using Hybrid Dysgenesis: Identification and Characterization of Egg Morphology Mutants

We have conducted a hybrid dysgenic screen of the X chromosome for mutations affecting female fertility, with particular attention to those causing abnormal egg and eggshell morphology. In a screen of 4017 dysgenic strains, 398 mutants derived from 168 different germ lines were isolated and assigned to eight classes according to their diverse phenotypes. One interesting class consists of mutants that block oogenesis at specific stages. Our analysis has focused on mutations affecting eggshell formation, including mutants that lay morphologically abnormal sterile eggs as well as those that lay no eggs but exhibit blocks in the late stages of oogenesis. A subset of 48 mutants was assorted into 30 allelic groups by inter se complementation and genetically localized by interval mapping. Two multiallele complementation groups, de1 (7 alleles) and ne1 (8 alleles), were identified as well as five two-allele complementation groups. A search for alleles among mutants generated in other female sterile screens was unsuccessful, pointing to the distinctive nature of the dysgenic mutant collection. The single case of allelism determined in this study was one with a lethal allele of the Broad-Complex, l(1)npr, suggesting a possible involvement of ecdysone in choriogenesis. A subset of 18 dysgenic strains was analyzed for P element hybridization and 16 of these were found to have hybridization signals in the appropriate cytogenetic interval. By examining these signals in two or more alleles of the same complementation group, we have been able to tentatively localize two mutations. Light and electron microscopy of the eggshell in 43 different strains has revealed a variety of effects. The respiratory appendages were defective in 27 of these mutants. Effects on the ultrastructure of the main body of the endochorion were not strongly correlated with the appendage defects, and could be classified as minor (14 mutants) or major (16 mutants). Although 13 mutants showed no ultrastructural chorion defects, six of these had defective respiratory appendages.     

The Hydrophobin HCf-1 of Cladosporium fulvum Is Required for Efficient Water-Mediated Dispersal of Conidia

Six hydrophobin genes (HCf-1 to -6) have thus far been identified in the tomato pathogen Cladosporium fulvum. HCf-1 to -4 are Class I hydrophobins and HCf-5 and -6 are Class II hydrophobins. In this paper we describe the isolation of deletion mutants that lack HCf-1, HCf-2, or both these genes. Global down-regulation of the expression of Class I hydrophobins is achieved by homology-dependent gene silencing. Analysis of the mutant strains shows that HCf-1 confers hydrophilic character to the conidia and this facilitates the dissemination of conidia on the surface of water droplets. Other Class I hydrophobins, such as HCf-3 or HCf-4, may be involved in the development and germination of conidia.     

Identification and characterization of TnphoA mutants of Salmonella that are unable to pass through a polarized MDCK epithelial cell monolayer

Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.