Propidium: induction of petites and recovery from ethidium mutagenesis in Saccharomyces cerevisiae.

It was shown that petite induction in growing cells of $\text{Saccharomyces}$$\text{cerevisiae}$ by ethidium was strongly stimulated by the presence of propidium, a phenanthridinium dye of similar structure to ethidium. Propidium itself also induced petites in growing but not in resting cells. Furthermore, propidium could prevent petite induction in resting cells and caused recovery from ethidium induction with prolonged incubation. A possible mode of action of propidium in the ethidium-induced petite mutagenesis is discussed.     

The presence of collagenase in Kupffer cells of the rat liver

Non-chromosomal petites can be produced in $\text{Saccharomyces}$$\text{cerevisiae}$ by treatment with guanidine hydrochloride, a protein denaturing agent. Its efficiency in inducing petite mutants is comparable to the action of ethidium bromide. The high frequency of petite mutants observed is due to an induction effect rather than to a selection of preexisting mutants. Induction of petites by guanidine hydrochloride occurs even in non growing conditions, indicating that even parental cells are transformed in petites. Transformation depends upon the physiological properties of the cells, since repressed cells, cultivated in the presence of glucose, are more easily transformed than cells cultivated in ethanol.     

Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.     

The use of netropsin with CsCl gradients for the analysis of DNA and its application to restriction nuclease fragments of ribosomal DNA from Physarum polycephalum

Netropsin binds to DNA in caesium chloride density gradients and reduces the density of the DNA. The DNA is saturated at a netropsin/DNA weight ratio of about 6 and the change in density, deltarho, at saturation is given by deltarho = -109 (dA + dT content)1.87 mg/ml for the six DNAs tested covering dA + dT contents from 0.28 to 0.69. At lower netropsin/DNA ratios the observed density shifts are consistent with a two-site model for netropsin binding to DNA. Netropsin approximately doubles the resolution of Physarum polycephalum nucleolar satellite DNA from main-band DNA. The fragments of P. polycephalum nucleolar satellite DNA obtained with the restriction endonuclease HindIII do not separate on CsCl gradients, even in the presence of netropsin, which shows that the transcribed and non-transcribed sequences in this DNA have similar nucleotide compositions.     

Electron microscopy study of viral DNA packaging with lambda head mutants

In a previous study, various intermediates in λ DNA packaging were visualized after lysis of λ-infected cells with osmotic shock and sedimentation through a sucrose formalin cushion onto electron microscope grids. Along this line, a systematic screening for intermediates accumulated in all head mutants available was performed. λA^?-infected cells accumulate only empty spherical protein shells (petit λ) bound at an intermediate point along the DNA thread. In situ digestion experiments with restriction endonuclease EcoRI show that the petit λ-DNA complexes are formed at a fixed point on the DNA concatemer. In $\text{λNu1}{}^{\mathrm{?}}\text{-}\text{infected}$ cells, however, most petit λ was not bound to DNA. In Fec^? cells, which are defective in formation of concatemers but normal in head protein synthesis, most petit λ did not sediment onto the carbon film of the grid. In $\text{D}{}^{\mathrm{?}}$ mutant, petit λ, partially full heads and empty heads with released DNA were observed. λFI^?-infected cells also accumulate petit λ and partially full heads. The present studies suggest that protein pNu1 is required for complex formation between head precursors and DNA concatemers, $\text{p}\text{A}$ for the initiation of DNA packaging, $\text{p}\text{D}$ and $\text{p}\text{F}$I for the promotion of DNA packaging, and $\text{p}\text{D}$ for stabilization of head structures. The results obtained with other head mutants involved in formation of mature proheads and head completion confirm earlier results obtained by different techniques.     

Electron microscopic and renaturation kinetic analysis of mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Mitochondrial DNA isolated from a series of nine petite yeast strains and from the parent grande strain was characterized by electron microscopic and renaturation kinetic analysis. The mtDNA² from all strains contained a variety of branched molecules which may be intermediates of replication or recombination. Although no circles were observed in the grande mtDNA, all the petites contained circular mtDNA molecules. The size distribution of the circles conformed to an oligomeric series that was characteristic for each strain. In seven petites, the length series could be related to a single circle monomer size, ranging from 0.13 μm to 5.5 μm; and in two petites to two or more circular monomer lengths. In contrast to circular mtDNA, linear molecules showed no unique size distribution. Circle monomer lengths were linearly related to the kinetic complexity (κ₂ or $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of sheared total mtDNA in the seven petite strains that contained a predominant single series of circle lengths. Thus in each of these petite strains the circle monomer length defined the same DNA sequence present in the linear DNA molecules of non-unique length.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Induction of respiratory incompetent mutants by unsaturated fatty acid depletion in Saccharomyces cerevisiae

Constant levels of cellular unsaturated fatty acids were obtained by growing a fatty acid desaturase mutant of $\text{Saccharomyces cerevisiae}$ in glucose limited chemostat cultures supplemented with various concentrations of Tween 80. An increase in the frequency of cytoplasmic respiratory incompetent mutants was observed in cultures growing at low cellular levels of unsaturated fatty acids. This effect has been shown to result from an increase in the rate of mutation as the cellular unsaturated fatty acid level is decreased. The majority of induced petite mutants are ?° (contain no mitochondrial DNA).     

Excision repair of DNA base damage

Exposure of cells to exogenous physical and chemical agents can result in damage to the DNA bases. DNA damage can lead to mutation, malignant transformation and cell death and may possibly be involved in cellular aging. Structurally related base modifications are expected to have similar biological effects regardless of the agent responsible for their formation. The biological effects may be a consequence of the local distortion of the DNA conformation by the lesion rather than of the chemical properties of the modified base $\text{per se}$. It may be useful, therefore, to classify DNA base damage according to their effect on DNA conformation. The elucidation of the structures of the DNA lesions produced $\text{in situ}$ in the living cell represents a prerequisite for the correlation of specific lesions with the biological effects and for the study of the cellular repair processes.Excision repair represents an ubiquitous mechanism in cells for the removal of damaged residues from the DNA. The most specific first step in excision repair is the recognition of the damage by an endonuclease followed by incision of the damaged DNA strand in the proximity of the damage. Several “repair endonucleases” have been characterized from bacteria while the search for the corresponding mammalian enzymes is only beginning. The second, probably less specific step, is the exonucleolytic degradation of the damaged portion of the DNA leading to the removal of the damaged residue. In $\text{E. coli}$ the removal of both cyclobutane-type photodimers and γ-ray products of the 5,6-dihydroxy-dihydrothymine type is accomplished by the 5′→3′ exonuclease associated with polymerase I. All three $\text{E. coli}$ polymerases appear to participate in the rebuilding of the degraded portion of the DNA. Studies on the corresponding enzymes in mammalian cells have been initiated. The last step of exicison repair involves the sealing of a phosphodiester bond of the DNA backbone and is accomplished by the enzyme polynucleotide ligase in bacterial and mammalian cells.     

Netropsin suppresses the rise of activity of an intracellular proteolytic system in sporulatingBacillus megaterium

Intracellular catabolism of proteins labeled at the end of the exponential growth proceeded in two phases during sporulation. The first phase was induced by starvation and took place also in cells whose sporulation was inhibited by netropsin. The second phase of degradation, which was triggered at the onset of the irreversible sporulation phase, was inhibited by netropsin. Intracellular proteolytic activity determined in disintegrated cells, i.e., primarily the activity of the cytoplasmic Ca²⁺-dependent serine proteinase(s) at the first place, was increasing throughout the sporulation process and reached its maximum during the irreversible sporulation phase. Its increase was suppressed by netropsin. Fractionation of the cell sap by HPLC revealed a similar distribution of proteolytic activities in the extract from control and netropsin-inhibited cells. The antibiotic thus probably affected the activation, not the formation of the cytoplasmic serine proteinase(s). Netropsin also inhibited an increase of proteolytic activity in the membrane fraction, probably owing to the presence of two different proteolytic enzymes.     

Selective inhibition of sporulation of Bacillus subtilis by netropsin

At low concentrations, the basic-polypeptide antibiotic, netropsin, did not inhibit growth, over-all RNA synthesis, replication of phage Øe, or synthesis of some catabolite-repressed enzymes in $\text{Bacillus subtilis}$ 168. Cells developed normally until t₂ of sporulation, but no refractile spores were formed in the presence of the antibiotic. The selective inhibition of sporulation by netropsin may be related to the base composition or sequence of some sporulation specific genes.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Possible involvement of DNA polymerases alpha and beta in bleomycin-induced unscheduled DNA synthesis in permeable HeLa cells

DNA polymerases involved in bleomycin-induced unscheduled DNA synthesis in some permeable human cells and rodent cells were studied by using selective inhibitors (aphidicolin, 2′,3′-dideoxythymidine-5′-triphosphate and N-ethylmaleimide) for DNA polymerases. The results suggest that both DNA polymerases α and β are involved in bleomycin-induced unscheduled DNA synthesis in permeable HeLa-S3 cells and probably in some other permeable human cells (HEp-2, KB and WI-38 VA-13 cells). Bleomycin-induced unscheduled DNA synthesis in some permeable rodent cells ($\text{SR-}\text{C3H}\text{He}$, $\text{Balb}\text{c}$ 3T3, 3Y1 and XC cells) is mostly attributed to DNA polymerase β.     

Specificity of DNA uptake in genetic transformation of gonococci

Genetic transformation of gonococci to streptomycin resistance was inhibited by homologous DNA or by DNA from related $\text{Neisseriae}$, but not by high concentrations of heterologous DNAs. Gonococci were capable of adsorbing large quantities (up to about 50 μg per 10⁸ cells) of both homologous and heterologous DNA, which could not be eluted by strong shearing forces. Treatment with externally added DNase removed virtually all the heterologous DNA while a small fraction of the homologous DNA, not influenced by the presence of excess heterologous DNA, remained cell-bound in a form resistant to nuclease treatment. Competing homologous DNA suppressed nuclease-resistant binding. These findings suggest that gonococci have two types of DNA binding components at their surface. Competence of gonococci for genetic transformation undergoes a rapid decay if the cells are incubated with homologous (but $\text{not}$ with heterologous) DNA.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

DNA strand scission by the antitumor protein neocarzinostatin

The antibiotic protein, neocarzinostatin, induces the scission of DNA strands $\text{in vivo}$ and $\text{in vitro}$. HeLa cell DNA prelabelled with [¹⁴C] thymidine is cut into large pieces with a peak at 80–90S when cells are incubated with 0.5 to 5.0 μg/ml of highly purified neocarzinostatin. Incubation of the antibiotic (0.5 μg/ml) with [³H] SV40 DNA in the presence of 2-mercaptoethanol results in the conversion of superhelical DNA I to nicked circular duplex DNA II. At high levels of drug, smaller fragments of linear DNA are produced. Strand breaks are detected in both neutral and alkaline sucrose gradients, indicating that drug susceptibility is not due to alkali-labile bonds.     

Informational content of mitochondrial DNA from a "low density" petite mutant of yeast

Mitochondrial DNA from wild-type yeast $\text{Saccharomyces cerevisiae}$ contains the genetic information for some mitochondrial tRNAs including tRNA_ser and tRNA_phe. In a “low density” petite mutant, mitochondrial DNA still retains the information for tRNA_ser, while the information for tRNA_phe is lost.The permanence of genetic information in this DNA containing only 3.6% G+C supports previous results concerning its intramolecular heterogeneity. An irregular distribution of G+C content along the molecule was further demonstrated by annealing experiments performed with DNA fragmented by sonication and fractionated on CsCl density gradient. These experiments show that the heavy fractions of the gradient preferentially anneal with mitochondrial seryl-tRNA.     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

Yeast mutants requiring ergosterol as only lipid supplement

Mutants of $\text{Saccharomyces cerevisiae}$ were isolated which required ergosterol or cholesterol as the only lipid supplement. They also required methionine, were petite, and showed complete absence of respiratory cytochromes. Revertants of these strains grew without ergosterol and methionine, were grande, and had respiratory cytochromes. Most revertants did not make ergosterol and were nystatin resistant. Sterol analysis and enzyme assays suggested a block in sterol formation after lanosterol.