Enrique Cerdá-Olmedo

Mutagenesis of the replication point by nitrosoguanidine: Map and pattern of replication of the Escherichia coli chromosome☆

Abstract The majority of the mutations induced by nitrosoguanidine in Escherichia coli are located in the replication point region of the chromosome. The treatment with nitrosoguanidine of successive samples from a synchronized culture produces a maximum of a given type of mutant at the time the corresponding gene is being replicated. This permits the construction of a replication map of the chromosome that describes the pattern of the replication of the genes. This map is in general agreement with the standard genetic map. The origin of replication is fixed at a determined position of the chromosome, that corresponds roughly to 50 minutes of the standard conjugation map and is the same for all strains tested (a 15 T − , a K12 Hfr and a K12 F − ). The direction of replication is clockwise, by reference to the same standard, and the cycles of DNA replication, under the conditions studied, succeed each other without delay or overlap. As an application of the method to the study of the control of DNA replication, a genetic demonstration is presented that thymine starvation leads to the initiation of a new round of DNA replication, even if the chromosome is already being replicated at the time thymine is withheld.

Mutants of Phycomyces with abnormal phototropism.

SummaryA collection of mutants of Phycomyces blakesleeanus with abnormal phototropism is divided into phenotypic groups on the basis of the results of five routine tests of sensory function. The three classes of mutants found permit the formulation of a network of sensory pathways, linking the three types of receptor (light, chemical, gravity) with the three types of response (sporangiophore tropisms, initiation of sporangiophores, induction of β-carotene synthesis). A model with a photoreceptor, early and late linear transducers, a logarithmic transducer and a growth-controlling element is proposed for the information channel of the phototropic response. Mutagenesis and the possible functional relationship between carotenes and phototropism are discussed.

Diazomethane as the active agent in nitrosoguanidine mutagenesis and lethality

Summary1.Two mutant strains have been derived from Escherichia coli TAU-bar; one selected for resistance to nitrosoguanidine at pH 7.5 and the other for resistance to diazomethane. The comparative study of these strains shows that diazomethane, a decomposition product of nitrosoguanidine, is the principal agent involved in both lethal and mutagenic effects of nitrosoguanidine above pH 5. Nitrosoguanidine is the lethal agent at pH 5 and it is possibly a lesser mutagen itself.2.There are two types of chromosomal effects, lethal (susceptible to repair replication) and mutagenic (non-repairable), both due mostly to diazomethane.3.The expression of mutations was found to be independent of DNA replication or repair. The optimal conditions for nitrosoguanidine mutagenesis are discussed.

Segregation of heterokaryons in the asexual cycle ofPhycomyces

SummaryArtificial heterokaryons between carotene mutants ofPhycomyces blakesleeanusBgff. have been prepared by squeezing cytoplasm out of two different mutant sporangiophores and allowing the fused droplets to regenerate.These heterokaryons are used to study the distribution of nuclei at different stages of the asexual life cycle. It is proposed that the nuclear ratio is constant in all parts of the mycelium, sporangiophores and sporangia, and that random samples of nuclei are packaged into spores. This model permits quantitative predictions regarding the proportions of phenotypes in the asexual progeny and these predictions are corroborated by experiments. The nuclear ratio remains constant during repeated mycelial transfers.

Carotenoid mutants of Gibberella fujikuroi

SummaryThe orange pigment neurosporaxanthin colours the mycelia of wild Gibberella fujijuori (Fusarium monifliforme) grown in the light, but is barely detectable in the dark. We have isolated carotenoid mutants from conidia exposed to N-methyl-N′-nitro-N-nitroso-guanidine and other mutagens. Specific blocks in the pathway are represented by white mutants accumulating phytoene and red mutants accumulating torulene; there are also mutants without carotenoids or with complex carotenoid mixtures. Regulatory mutants overproduce neurosporaxanthin, both in the light and in the dark. Other mutants contain considerable neurosporaxanthin in the dark, but less than in the light. The results bring out similarities between the carotenoid biosynthetic pathways of Gibberella and Phycomyces, and significant differences in their respective regulations.

Regulation of carotene synthesis in Phycomyces

SummaryThreeindependent mutations of Phycomyces blakesleeanus resulting in overaccumulation of β-carotene are recessive and belong to the same complementation group. The corresponding gene has been named carS. Evidence is presented that gene carS is not the same as gene carA, previously defined by mutations blocking carotene production. Vitamin A increases carotenogenesis in wild types and in carS mutants to about the same extent. Intersexual heterokaryosis increases carotenogenesis most prominently in carS genetic backgrounds (up to 300 times the production of the wild type in the same conditions). Vitamin A, intersexual heterokaryosis and carS mutations are thought to stimulate carotenogenesis through different mechanisms. It is suggested that the carS gene product participates in end-product regulation of the pathway.

Mutants and intersexual heterokaryons of Blakeslea trispora for production of β-carotene and lycopene

ABSTRACT The industrial production of β-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of β-ionone and other chemical activators. We have obtained improved strains by mutation and heterokaryosis. We chose wild strains on the basis of their growth and carotene content in single and mated cultures. Following exposure of their spores to N-methyl-N′-nitro-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having β-carotene as the main product. Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains. The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex. These contained up to 39 mg of β-carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of β-carotene per g (dry mass). β-Ionone did not increase the carotene content of these strains. Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures.

Photoinduced accumulation of carotene in Phycomyces

Blue light stimulates the accumulation of beta-carotene (photocarotenogenesis) in the fungus Phycomyces blakesleeanus. To be effective, light must be given during a defined period of development, which immediately precedes the cessation of mycelial growth and the depletion of the glucose supply. The competence periods for photocarotenogenesis and photomorphogenesis in Phycomyces are the same when they are tested in the same mycelium. Photocarotenogenesis exhibits a two-step dependence on exposure, as if it resulted from the additon of two separate components with different thresholds and amplitudes. The low-exposure component produces a small beta-carotene accumulation, in comparison with that of dark-grown mycelia. The high-exposure component has a threshold of about 100 J· m−2 blue light and produces a large beta-carotene accumulation, which is not saturated at 2·106 J·m−2. Exposure-response curves were obtained at 12 wavelengths from 347 to 567 nm. The action spectra of the two components share general similarities with one another and with those of other Phycomyces photoresponses. The small, but significant differences in the action spectra of the two components imply that the respective photosystems are not identical. Light stimulates the carotene pathway in the carB mutants, which contain the colourless precursor phytoene, but not beta-carotene. Carotenogenesis is not photoinducible in carA mutants, independently of their carotene content. This and other observations on various car mutants indicate that light prevents the normal inhibition of the pathway by the carA and carS gene products. The chromophore(s) for photocarotenogenesis are presumably flavins, and not carotenes.

Macromolecular action of nitrosoguanidine in Escherichia coli

Abstract 1. The effects of the mutagen N-methyl-N′-nitro-N-nitrosoguanidine on macromolecules and their synthesis have been studied in several strains of Escherichia coli. 2. Nitrosoguanidine treatment at pH 5.0 results in some alteration of DNA that is recognized and repaired by the same mechanism involved in the dark repair of DNA damaged by ultraviolet light. This alteration, if unrepaired, is lethal to the cell. This is the principal cause of lethality in strain Bs-1 which is deficient in the dark repair mechanism. 3. Nitrosoguanidine inactivates the enzyme β-galactosidase within the cells. This action on proteins, however, is not sufficient to account for lethality. 4. Nitrosoguanidine exhibits a pronounced inhibitory effect on protein synthesis and a much lesser effect on RNA synthesis. This effect involves the reduction in rate of synthesis of proteins and the production of small amounts of non-functional proteins. Several indications exist that this is the principal cause of lethality on strains that can repair DNA damage. 5. The presence of streptomycin enhances the survival of nitrosoguanidine-treated streptomycin-resistant cells, suggesting that nitrosoguanidine causes misreading of the genetic code that can be suppressed by streptomycin.

Sexual Activation of Carotenogenesis in Phycomyces blakesleeanus

SUMMARY: Sexual stimulation increases the β-carotene content of the fungus Phycomyces blakesleeanus. The same effect is observed in single cultures exposed to natural and synthetic trisporates and in intersexual heterokaryons. Synthetic racemic (9Z)-methyl trisporate B, a trisporate precursor made by cultures of the (+) mating type, stimulates carotenogenesis only in (-) cultures. Synthetic racemic (9Z)-methyl trisporate C is less effective and the corresponding all-(E) isomers and other related compounds are inactive. Sexual stimulation of carotenogenesis is additive with the stimulations induced by light, retinol, dimethyl phthalate, and mutation of the gene carS.