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On some genetic aspects of phage lambda resistance in E. coli K12.

Most mutations rendering E. coli K12 resistant to phage lambda, map in two genetic regions malA and malB.-The malB region contains a gene lamB specifically involved in the lambda receptor synthesis. Twenty-one independent lamB mutations studied by complementation belonged to a single cistron. This makes it very likely that lamB is monocistronic. Among the lamB mutants some are still sensitive to a host range mutant of phage lambda. Mutations mapping in a proximal gene essential for maltose metabolism inactivate gene lamB by polarity confirming that both genes are part of the same operon. Because cases of intracistronic complementation have been found, the active lamB product may be an oligomeric protein.-Previously all lambda resistant mutations in the malA region have been shown to map in the malT cistron. malT is believed to be a positive regulatory gene necessary for the induction of the "maltose operons" in the malA region and in the malB region of the E. coli K12 genetic map. No trans dominant malT mutation have been found. Therefore if they exist, they occur at a frequency of less than 10(-8), or strongly reduce the growth rate of the mutants.

Chromosomes, Bacterial↗

Isolation and classification of extranuclear mutants of Neurospora crassa.

Four extranuclear mutants, [exn-1], [exn-2],[exn-4], and [stp-C], were obtained from N-methyl-N'-nitro-N-nitrosoguanidine-treated conidia and mycelium of Neurospora crassa. The three exn mutants grow with a pronounced lag from conidia and ascospores and are female fertile, whereas [stp-C] has a stop-start growth phenotype and is female sterile. The mitochondria from all four mutants are deficient in cytochromes a+a(3) and b, but contain an excess of cytochrome c. On the basis of growth and fertility, nuclear suppressors and complementation in heteroplasmons, 16 of the extranuclear mutants now available in Neurospora can be divided into three groups. Group I consists of 8 female-fertile variants with both poky-like growth and cytochrome defects. Their slow growth is suppressed by the nuclear factor, f, but not by a second nuclear suppressor, su-1([mi-3]). They complement with group III mutants in mixed cytoplasmons. Group II is represented by a single variant, [mi-3]. It is phenotypically modified by the su-1([mi-3]) factor, but not by f. Its unique cytochrome spectrum shows a deficiency of cytochrome a, but c and b are present. It complements in heteroplasmons with group I and III mutants. Group III included 7 female-sterile variants with stopper growth phenotypes and the same cytochrome defects as group I. Group III mutants complement both with group I and II isolates, but they are unaffected by either f or su-1.

Crosses, Genetic↗

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. II. Properties of host and plasmid mutations.

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.

Coliphages↗

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli. I. Isolation and specificity of host and plasmid mutations.

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE(1)) were isolated following mutagenesis of E. coli K12 strain carrying the ColE(1) factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE(1) DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE(1) plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE(1) factor. Phenotypic class II mutations affected the maintenance of the ColE(1) and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE(1) replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE(1) plasmid.

Animals↗

On the origin of mitochondrial mutants: evidence for intracellular selection of mitochondria in the origin of antibiotic-resistant cells in yeast.

In wild-type Saccharomyces cerevisiae, erythromycin and certain other antibacterial antibiotics inhibit the formation of respiratory enzymes in mitochondria by inhibiting translation on mitochondrial ribosomes. This paper is concerned with the origin of mutant cells, resistant to erythromycin by virtue of having a homogeneous population of mutant mitochondrial DNA molecules. Such mutant cells are obtained by plating wild-type (sensitive) cells on a nonfermentable substrate plus the antibiotic. Colonies of mutant cells appear first about four days after the time of appearance of established mutant cells; new colonies continue to appear, often at a constant rate, for many days. Application of the Newcombe respreading experiment demonstrates that most or all of the mutant cells which form the resistant colonies on selective medium arise only after exposure of the population to erythromycin. It is suggested that this result is most probably due to intracellular selection for mitochondrial genomes. Resistant mitochondria arising from spontaneous mutation are postulated to be at a selective disadvantage in the absence of erythromycin; reproducing more slowly than wild-type sensitive mitochondria, they cannot easily accumulate in sufficient numbers in a cell to render it resistant as a whole. In the presence of erythromycin, resistant mitochondria can continue to reproduce while sensitive mitochondria cannot, until there is a sufficient number to make the cell resistant, i.e. to permit normal cell growth. The same phenomenon is seen with respect to chloramphenicol resistance. Intracellular selection is considered more likely than direct induction of mutation by the antibiotic, since mutant cells do not accumulate in the presence of erythromycin if the mitochondrial genome is rendered non-essential by growth on glucose or nontranslatable by chloramphenicol. Intra-cellular selection provides a mechanism for direct adaptation at the cell level, compatible with currently acceptable ideas of spontaneous mutation and selection at the organelle level.

Cell Count↗

Extrachromosomal element delta in Drosophila melanogaster. IX. Induction of delta-retaining chromosome lines by mutation and gene mapping.

[Delta b], symbolized as [delta(b)], is retained by S(b) chromosome lines and transmitted through the females to their progeny. Transmission through the males is not directly demonstrable (Minamori 1969a). [delta r], symbolized as [delta(r)], is retained by S(r) chromosome lines and transmitted biparentally (Minamori 1971). The multiplication of delta is suppressed at low temperature. All descendant lines derived from S(b)-carrying or S(r)-carrying flies in which the presence of delta cannot be demonstrated gradually accumulate their specific delta factors over many generations (Minamori 1969b, 1972). The delta factors and the sensitive chromosomes are inseparably associated. This observation led to the assumption that delta may be a copy of a chromosomal gene or a certain agent integrated into the chromosome (Minamori 1972). This assumption was examined in the present study by experiments designed to induce delta-retaining sensitive chromosomes, and to map the gene(s) responsible for delta-retention and/or for sensitivity to the killing action of delta factor. One sensitive chromosome which retained [delta(b)] (S(b) chromosome) was obtained in the presence of [delta(b)] out of 2492 insensitive chromosomes which retained no delta; in addition one S(b) chromosome was obtained in the presence of [delta(r)] out of 2131 insensitives. The latter finding suggests that S(b) might be induced by a mutation caused by [delta(b)] or [delta(r)], but not by integration of either delta into the chromosome. Four S(b) chromosomes and one sensitive chromosome which retained [delta(r)] (S(r) chromosome) were obtained out of 1970 insensitives when males carrying the chromosome were fed an alkylating mutagen, ethyl methane sulfonate (EMS). The location of delta-retaining genes was examined by crossing-over experiments employing eight S(b) and five S(r) chromosomes. The genes on these chromosomes were found to be located in the same region or near one another. The gene for [delta(b)], symbolized as Da(b), and the gene for [delta(r)], symbolized as Da(r), are assumed to be multiple alleles of a locus at 2-24.9. The sensitivity of the chromosomes was modified appreciably by recombination; hence, the genes controlling this trait are assumed to be a polygenic system. The findings obtained in this study lead to the hypothesis that delta may be produced by a chromosomal gene (Da) and transmitted extrachromosomally.

Animals↗

Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. I. Isolation and phenotypic characterization of nonmating mutants.

Nonmating mutants were also isolated from haploid strains of yeast of both mating types. The mutants were characterized with respect to their ability to produce and respond to specific yeast sex factors, their ability to mate at low frequencies, and the ability of the low-frequency diploids to sporulate. Loss of the ability to mate by either mating type was invariably accompanied by the loss of one or more, and in some cases, all, of the above capabilities. The results strongly indicate that the sex factors are functionally involved in the conjugation process.

Alleles↗

Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. II. Genetic analysis of nonmating mutants.

Rare diploids formed by sterile mutants have been studied by tetrad analysis. Sixteen classes of mutants representing at least five distinct genetic loci have been defined. One group of mutations, isolated only in alpha, maps at the mating-type locus, while none of the others shows any linkage to mating type. Some of the mutations are nonspecific for mating type, while others act only on a or alpha. In addition, mutations were found that prevent sporulation when heterozygous in diploids. These appear to be mutations of the mating-type alleles.

Alleles↗

Isolation of P22 specialized transducing phage followong F'-episome fusion.

A P22 specialized transducing phage has been constructed which carries the structural gene for aspartate transcarbamylase (ATCase). This gene (pyrB) was first brought close to the P22 attachment site by fusing an F' pyrB(+) episome to an F' prolac episome which carries a P22 prophage attachment site. A prophage was added to these fused F' episomes and the lysogen was UV-induced. The specialized transducing phage was isolated from the resulting lysate. The phage also carries argI, the structure gene for ornithine transcarbamylase.

Aspartate Carbamoyltransferase↗

Genetic control of chromosomal and plasmid recombination in Staphylococcus aureus.

Recombination-deficient mutants of Staphylococcus aureus have been isolated and found to have properties similar to those of recombination-deficient Escherichia coli. In addition, one Rec(-) mutant was found to be defective in the restriction and modification of DNA. There is a marked reduction ( approximately 10(4)-fold) in recombination between penicillinase plasmids in the Rec(-) mutants suggesting that these elements do not encode an efficient recombination system. There is, however, a demonstrable residuum of interplasmid recombination; evidence is lacking on whether this residuum is a plasmid or host function. In the absence of the generalized host recombination system it has been possible to demonstrate that interplasmid recombination occurs during vegetative bacteriophage growth and is presumably mediated by a phage-determined recombination system.

Chromosomes, Bacterial↗

Isolation and properties of selenomethionine-resistant mutants of Neurospora crassa.

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.

Biological Transport↗

The use of hybrid somatic cells as an approach to mitochondrial genetics in animals.

We have studied the fate of parental mitochondrial DNA (mtDNA) in hybrid somatic cells derived by Sendai virus-induced fusion of human cells and mouse or rat cells. Many hybrid cell strains were obtained which contained sequences from both human and rodent mtDNA after 40 to 60 population doublings. Some strains were subcloned and cultured further for up to 150 doublings; a large fraction of these strains contained both parental mtDNA sequences at that time. The relation between human and rodent mtDNA sequences was tested in some of the hybrid cell strains. In a high fraction of strains tested the human and rodent mtDNA sequences were linked to each other by what are most likely covalent bonds. This linkage may be described as "recombination" of mtDNA sequences from two different animals.

Animals↗

Genetic analysis of a double male strain of Escherichia coli K12.

The behavior of a double male strain of Escherichia coli K12 has been compared to that of its parents and a primary F' strain carrying F14 in order to determine whether the genome of the double male, responsible for the double origin mode of gene transmission, is composed of one or two linkage groups. F-prime plasmids carrying leu and pyrB and argG and metC have been detected following mating an F(-)recA(-) recipient with the double male. Their existence strongly supports the contention that the double male is composed of a single linkage group with two integrated sex factors. Data from acridine orange curing experiments places the frequency of double male cells in the population of growing cells in the two-chromosome configuration as less than one percent. Evidence for a unique origin and terminus of DNA replication deduced by the density labelling and transduction of double male DNA supports the contention that the double male is best considered a cell with a single chromosome carrying two integrated F plasmids.

Acridines↗

Recipient gene duplication during generalized transduction.

An Hfr13 Delta(proA-lac) deletion recipient, -Delta(proA-lac)-F-purE(+)-, has been utilized in a study of the origins of duplications formed during chromosome fragment integration. Among the Pro(-)Lac(+) transductants, some have duplications spanning the F locus. These transductants are, or segregate, strains with F' episomes carrying genes of the duplication. Some of the duplications include purE(+), a gene which is not coinherited with lac(+) during bacteriophage P1-mediated transduction. Thus recipient genes have been duplicated during recombinant formation. Crossing-over models including replication steps provide a basis for explaining the duplication process.

Chromosome Aberrations↗