Mitochondrial DNA and suppressiveness of petite mutants in Saccharomyces cerevisiae

Ethidium bromide is known to be a powerful mutagen for the induction of cytoplasmically inherited petite mutations in yeast. The effect of ethidium bromide on the degree of suppressiveness of the induced mutants as a function of exposure time is described. The mitochondrial DNA of 20 ethidium bromide-induced petite mutants has been studied to determine its absence or presence and its buoyant density. Ten mutants, in which we were not able to detect any mitochondrial DNA, were neutral petites. The 10 remaining mutants with mitochondrial DNA simultaneously showed a measurable degree of suppressiveness. It was not possible to correlate the buoyant density of the mutant mitochondrial DNA with the degree of suppressiveness.This study was supported in part by USPHS grant GM 10017. G.M. received a Fulbright Travel Grant.     

Isolation or group B streptococci defective for the type antigen Ia by transposon-mediated mutagenesis

Transposon-mediated mutability has been used to isolate the isogenic strains of the group B serotype Ia streptococci with the mutations in the genes coding for the production of capsular antigen. The transconjugants have lost the ability to bind type Ia antiserum as demonstrated by immunoblotting technique. The loss of type-specific antigen by the strains has resulted in a dramatic decrease in virulence for mice while the absorbtion indexes of transconjugants increased 2-3 fold. The mutant clones deficient in capsule expression had the increased buoyant density in the percoll gradient as compared with the parent strains of group B. The stable mutants impaired in ability to produce the polysaccharide capsule on the cell surface were obtained as a result of site-specific insertion of transposon Tn916 into the genome of group B serotype I streptococcus.     

Mutations affecting the initiation of plasmodial development in Physarum polycephalum

In the heterothallic myxomycete Physarum polycephalum, uninucleate amoebae normally differentiate into syncytial plasmodia following heterotypic mating. In order to study the genetic control of this developmental process, mutations affecting the amoebal-plasmodial transition have been sought. Numerous mutants characterized by self-fertility have been isolated. The use of alkylating mutagens increases the mutant frequency over the spontaneous level but does not alter the mutant spectrum. Three spontaneous and 14 induced mutants have been analyzed genetically. In each, the mutation appears to be linked to the mating type locus. In three randomly selected mutants, the nuclear DNA content is the same in amoebae and plasmodia, indicating that amoebal syngamy does not precede plasmodium development in these strains. These results indicate that a highly specific type of mutational event, occurring close to or within the mating type locus, can abolish the requirement for syngamy normally associated with plasmodial differentiation. These mutations help define a genomic region regulating the switch from amoebal to plasmodial growth.     

A genetic and biochemical analysis of petite mutations in yeast

A series of spontaneous cytoplasmic petite mutants was isolated from a grande strain of Saccharomyces cerevisiae doubly marked with the cytoplasmically inherited determinants to erythromycin and oligomycin resistance. The petites were characterized with regard to the genetic stability of these antibiotic resistance markers and to their degree of suppressivity. No relation was found between the genetic instability of a petite mutant and the degree of suppressivity exhibited by that mutant. Three petites of 19.4%, 57.4% and 90.4% suppressivity were selected and their mitochondrial DNA characterized with regard to molecular weight, buoyant density in analytical cesium chloride density gradients, and the percentage of the total cellular DNA represented by the mitochondrial DNA. From these results it appears that the molecular weight of the mitochondrial DNA of the petite strains examined is the same as that shown by the parental grande strain, regardless of the degree of suppressivity exhibited.     

Linker insertion mutations in the adenovirus preterminal protein that affect DNA replication activity in vivo and in vitro.

Eighteen linker insertion mutants with mutations in the adenovirus precursor to terminal protein (pTP), which were originally constructed and tested in virions by Freimuth and Ginsberg (Proc. Natl. Acad. Sci. USA 83:7816-7820, 1986), were transferred to expression plasmids for assay of the various functions of the isolated pTP. Function was measured by the ability of individual pTP mutant proteins to participate in the initiation of replication from an adenovirus DNA end, by their activity in assays of DNA elongation, and by the intracellular distribution of pTP demonstrated by indirect immunofluorescence. Ten of the 11 mutants that were active in virion formation were also functional in DNA replication reactions in extracts, while 1 had reduced function. Four mutants with mutations that were lethal to virus production were also inactive in DNA replication reactions. These four mutations are probably located at sites required for the function of pTP in DNA synthesis. Three pTP mutants with mutations that were lethal or partially defective with respect to virion formation were active in reactions requiring pTP for initiation and elongation in extracts. All three of these mutant pTPs targeted normally to the nucleus, suggesting a defect after this step in replication. Since pTP has been reported to bind the nuclear matrix, these pTP mutants may have mutations that define sites necessary for binding to this structure. Several mutants with mutations that lie outside the putative nuclear targeting region were aberrantly localized, suggesting either that additional domains are important in nuclear localization or that there are alterations in protein structure that affect nuclear transport for some pTP mutants.     

Purification on Renografin Density Gradients of Chlamydia trachomatis Grown in the Yolk Sac of Eggs

Chlamydia trachomatis grown in the yolk sac of embryonated eggs was purified by centrifugation on continuous isopycnic Renografin density gradients. A band of chlamydial particles with a buoyant density of 1.20 contained 70% of the starting particles, and electron microscopy revealed the virtual absence of contaminating egg material. Centrifugation on Renografin gradients caused only a moderate decrease in infectivity. For large-scale purification, infected yolk sac was centrifuged through Renografin solutions, resulting in greater than 60% recovery of starting chlamydial particles, but less than 1% recovery of the dry weight and protein.     

Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha.

Bacteriophage P4 DNA replication depends upon the phage-encoded alpha protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [alpha cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp alpha that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of alpha protein in vitro. The alpha cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp alpha domain that binds specifically to DNA and that gp(alpha)cr mutations impair this interaction. We hypothesize that gp alpha-Cnr interaction is essential for the control of P4 DNA replication.     

Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli.

The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age. In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis. The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells. Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam. The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate. In this range, however, the average cell volume was also constant. Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age). A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.     

Characterization of mutations of the bacteriophage P1 mod gene encoding the recognition subunit of the EcoP1 restriction and modification system.

This study characterized several mutations of the bacteriophage P1 mod gene. This gene codes for the subunit of the EcoP1 restriction enzyme that is responsible for DNA sequence recognition and for modification methylation. We cloned the mutant mod genes into expression vectors and purified the mutant proteins to near homogeneity. Two of the mutant mod genes studied were the c2 clear-plaque mutants described by Scott (Virology 41:66-71, 1970). These mutant proteins can recognize EcoP1 sites in DNA and direct restriction but are unable to modify DNA. Methylation assays as well as S-adenosylmethionine (SAM) binding studies showed that the c2 mutants are methylation deficient because they do not bind SAM, and we conclude that the mutations destroy the SAM-binding site. Both of the c2 mutations lie within a region of the EcoP1 mod gene that is not conserved when compared with the mod gene of the related EcoP15 system. EcoP15 and EcoP1 recognize different DNA sequences, and we believe that this region of the protein may code for the DNA-binding site of the enzyme. The other mutants characterized were made by site-directed mutagenesis at codon 240. Evidence is presented that one of them, Ser-240----Pro, simultaneously lost the capacity to bind SAM and may also have changed its DNA sequence specificity.     

Sex-reversing mutations affect the architecture of SRY-DNA complexes.

The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination.     

Wet and dry bacterial spore densities determined by buoyant sedimentation

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

Isolation and properties of genetically defined strains of the methylotrophic yeast Hansenula polymorpha CBS4732

Genetically defined strains of the yeast Hansenula polymorpha were constructed from a clone of H. polymorpha CBS4732 with very low mating and sporulation abilities. Mating, spore viability, and the percentage of four-spore-containing asci were increased to a level at which tetrad analysis was possible. Auxotrophic mutations in 30 genes were isolated and used to construct strains with multiple markers for mapping studies, transformation with plasmid DNA, and mutant screening. Various other types of mutants were isolated and characterized, among them mutants that displayed an altered morphology, methanol-utilization deficient mutants and strains impaired in the biosynthesis of alcohol oxidase and catalase. Also, the mutability of H. polymorpha CBS4732 vs H. polymorpha NCYC495 was compared. The data revealed clear differences in frequencies of appearance and mutational spectra of some mutants isolated. Many of the mutants isolated had good mating abilities, and diploids resulting from their crossing displayed high sporulation frequencies and high spore viability. Most of the markers used revealed normal Mendelian segregation during meiosis.The frequency of tetratype spore formation was lower than in Saccharomyces cerevisiae suggesting a lower frequency of recombination during the second meiotic division. The properties of genetically defined strains of H. polymorpha CBS4732 as well as their advantages for genetics and molecular studies are discussed.     

Wet and dry bacterial spore densities determined by buoyant sedimentation.

The wet densities of various types of dormant bacterial spores and reference particles were determined by centrifugal buoyant sedimentation in density gradient solutions of three commercial media of high chemical density. With Metrizamide or Renografin, the wet density values for the spores and permeable Sephadex beads were higher than those obtained by a reference direct mass method, and some spore populations were separated into several density bands. With Percoll, all of the wet density values were about the same as those obtained by the direct mass method, and only single density bands resulted. The differences were due to the partial permeation of Metrizamide and Renografin, but not Percoll, into the spores and the permeable Sephadex beads. Consequently, the wet density of the entire spore was accurately represented only by the values obtained with the Percoll gradient and the direct mass method. The dry densities of the spores and particles were determined by gravity buoyant sedimentation in a gradient of two organic solvents, one of high and the other of low chemical density. All of the dry density values obtained by this method were about the same as those obtained by the direct mass method.     

Mutations at the smo genetic locus affect the shape of diverse cell types in the rice blast fungus

Teflon film surfaces are highly conducive to the formation of infection structures (appressoria) in the plant pathogenic fungus, Magnaporthe grisea. We have utilized Teflon films to screen and select for mutants of M. grisea that are defective in appressorium formation. This approach and several others yielded a group of 14 mutants with a similar phenotype. All the mutant strains make abnormally shaped conidia and appressoria. When two mutant strains are crossed, abnormally shaped asci are formed. Ascus shape is normal when a mutant strain is crossed with a wild-type strain. Despite dramatic alterations in cell shape these strains otherwise grow, form conidia, undergo meiosis, and infect plants normally. This mutant phenotype, which we have termed Smo(-), for abnormal spore morphology, segregates in simple Mendelian fashion in crosses with wild-type strains. Some ascospore lethality is associated with smo mutations. In genetic crosses between mutants, smo mutations fail to recombine and do not demonstrate complementation of the abnormal ascus shape phenotype. We conclude that the smo mutations are alleles of a single genetic locus and are recessive with regard to the the ascus shape defect. Mutations at the SMO locus also permit germinating M. grisea conidia to differentiate appressoria on surfaces that are not normally conducive to infection structure formation. A number of spontaneous smo mutations have been recovered. The frequent occurrence of this mutation suggests that the SMO locus may be highly mutable.     

Mutations that alter initiation codon discrimination by Escherichia coli initiation factor IF3.

This work describes the isolation of mutations in infC, the structural gene for IF3, using different genetic screens. Among 21 mutants characterised, seven were shown to produce stable variant IF3 proteins unable to fully complement a strain carrying a chromosomal deletion of the infC gene. The mutants were also shown to be unable to normally discriminate against several non-canonical initiation codons such as AUU and ACG. The two mutants with the strongest complementation or discrimination defects carry changes in the C-terminal domain of IF3, which is responsible for the binding of the factor to the 30 S ribosomal subunit. We show that the first mutant has an expected decreased but the second an unexpected increased capacity to bind the 30 S subunit. The in vivo defects of the second mutant are explained by its capacity to bind unspecifically to other targets, as shown by its increased affinity for the 50 S subunit, which is normally not recognised by the factor. Interestingly, this mutant corresponds to a change of an acidic residue that might play a negative discriminatory role in preventing interactions with non-cognate RNAs, as has been reported for acidic residues of aminoacyl-tRNA synthetases shown to be involved in tRNA recognition.     

Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor.

When Saccharomyces cerevisiae a cells bind alpha-factor pheromone, the ligand is internalized and its binding sites are lost from the cell surface in a time-, energy-, and temperature-dependent manner. This report presents direct evidence for alpha-factor-induced internalization of cell surface receptors. First, membrane fractionation on Renografin density gradients indicated that the alpha-factor receptors were predominantly found in the plasma membrane peak before alpha-factor treatment and then appeared in membranes of lesser buoyant density after alpha-factor exposure. Second, receptors were susceptible to cleavage by extracellular proteases before alpha-factor treatment and then became resistant to proteolysis after exposure to pheromone, consistent with the transit of receptors from the cell surface to an internal compartment. The median transit time in both assays was approximately 8 min. The ultimate target of the internalized receptors was identified as the vacuole, since the membranes containing internalized receptors cofractionated with vacuolar membranes, since the turnover of receptors was stimulated by alpha-factor exposure, and since receptor degradation was blocked in a pep4 mutant that is deficient for vacuolar proteases. The carboxy-terminal domain of the receptor that is required for ligand internalization was also found to be essential for endocytosis of the receptor. A receptor mutant, ste2-L236H, which is defective for pheromone response but capable of ligand internalization, was found to be proficient for receptor endocytosis. Hence, separate structural features of the receptor appear to specify its signal transduction and internalization activities.     

DNA supercoiling in gyrase mutants.

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.     

Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12.

The RecG protein of Escherichia coli is a structure-specific DNA helicase that targets strand exchange intermediates in genetic recombination and drives their branch migration along the DNA. Strains carrying null mutations in recG show reduced recombination and DNA repair. Suppressors of this phenotype, called srgA, were located close to metB and shown to be alleles of priA. Suppression depends on the RecA, RecBCD, RecF, RuvAB, and RuvC recombination proteins. Nine srgA mutations were sequenced and shown to specify mutant PriA proteins with single amino acid substitutions located in or close to one of the conserved helicase motifs. The mutant proteins retain the ability to catalyze primosome assembly, as judged by the viability of recG srgA and srgA strains and their ability to support replication of plasmids based on the ColE1 replicon. Multicopy priA+ plasmids increase substantially the recombination- and repair-deficient phenotype of recG strains and confer similar phenotypes on recG srgA double mutants but not on ruvAB or wild-type strains. The multicopy effect is eliminated by K230R, C446G, and C477G substitutions in PriA. It is concluded that the 3'-5' DNA helicase/translocase activity of PriA inhibits recombination and that this effect is normally countered by RecG.