Isolation and preliminary characterization of R6K plasmid deletion mutants.

Several deletion mutants of R6K have been isolated by mutagen treatment of bacterial host carrying wild type of the plasmid and search for clones that lost ampicillin or streptomycin resistance. The molecular weight of the mutants as estimated by agarose gel electrophoresis was 15 times 10(6) minus 23 times 10(6) compared to 26 times 10(6) for the parental plasmid. The mutants were characterized in respect of the level of resistance to ampicillin and frequency of conjugational transfer. Some of the mutants were found to differ in Eco RI digestion pattern from the wild type.     

Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number.

We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.     

Selection and characterization of ColE1 plasmid mutants that exhibit altered stability and replication.

This report describes a method for isolating mutants of plasmid ColE1 that exhibit unstable maintenance and altered replication characteristics. It also describes the initial characterization of four mutants isolated by that method. A chimeric plasmid, pHSG124, containing a ColE1 derivative and a temperature-sensitive replication derivative of pSC101 was mutagenized in vitro, using hydroxylamine. By adjusting the growth conditions of transformants containing the mutagenized chimeric deoxyribonucleic acid, it was possible to rapidly screen colonies and identify those that had a high probability of carrying ColE1 mutants that exhibit unstable maintenance. Of those mutants, some exhibited altered copy number or accumulated catenated structures. Evidence is presented which suggests that the mutations in three of the mutants are probably located in the HaeII A fragment of ColE1.     

Isolation and complementation of temperature-sensitive replication mutants of Staphylococcus aureus plasmid pC194

Temperature-sensitive replication (Tsr) mutants have been isolated from the Staphylococcus aureus plasmid pC194. For three of the four mutant plasmids tested (pSAO801, pSAO802, and pSAO804) the segregation kinetics suggested a complete block of plasmid replication at 43 degrees C. The replication defects of three mutant plasmids: pSAO802, pSAO803, and pSAO804 could be complemented by recombinant plasmids carrying a segment from either the wild type or the other mutant, pSAO801. There was no complementation when the segment carried by the recombinant plasmid was derived from one of the three complementable mutants. These data were taken as evidence for the involvement of a diffusible, plasmid-encoded product, RepH, in pC194 replication. The complementation of the fourth Tsr mutant, pSAO801, could not be tested due to an abnormal susceptibility of this mutant to the incompatibility expressed by recombinants carrying segments derived from pC194 or its mutants. A single mutation was found to be responsible for both pSAO801 instability and its altered incompatibility properties but the nature of the defect has not yet been elucidated.     

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.     

Studies on plasmid replication. V Replicative intermediates.

A procedure has been developed for the study of rapidly labeled intermediates in plasmid replication in normally growing bacteria. Pulse-labeled cells are enzymatically lysed on top of a neutral sucrose gradient and centrifuged so that the chromosomal DNA forms a pellet and the plasmids (and other smaller DNA molecules) form bands in the gradient. Analysis of penicillinase plasmid replication in Staphylococcus aureus has revealed that although pulse-labeled intermediates sediment faster than the 60 S circular duplex monomeric plasmid molecules, they do not have stable superhelical structure. The conversion of partially polymerized molecules, having a sedimentation coefficient of about 58 S, to fully polymerized terminal forms appears to involve a progressive change in sedimentation rate from 58 S to 67 S. Conversion of the presumably dimeric terminal forms to mature closed circular monomers is a slow and rate-limiting multi-step process (taking some 3 to 4 min at 37 °C).     

Isolation and characterization of pLS 1 plasmid mutants with increased copy numbers

Abstract Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids.     

Isolation and characterization of ColE2 plasmid mutants unable to kill colicin-sensitive cells

Summary After transfer from a mutagenized host, twenty one ColE2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production. Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene for colicin E2. Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors.Studies with a nonsense mutant producing only a small colicin E2 fragment (ColE2-421) suggest that colicin E2 is not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production. The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33°, 37° and 43°. One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.     

Isolation, propagation and characterization of replication requirements of reiteration mutants of Simian Virus 40.

We have identified five reiteration mutants from serially-propagated, defective stocks of Simian Virus 40 and DAR virus (an SV40³ variant of human origin). The genomes of these mutants contain tandem repeats of specific segments of the SV40 genome. In order to propagate individual reiteration mutants, the monomer DNA segments from the mutant genomes are separated from wild-type SV40 DNA after cleavage by certain bacterial restriction endonucleases which produce short cohesive termini at their cleavage sites. These monomer segments, which are one-third, one-fourth, or one-fifth the size of wild-type SV40 DNA, are then circularized in vitro using bacteriophage T4 polynucleotide ligase and used to infect African green monkey kidney cells in the presence of wild-type or temperature-sensitive mutant DNAs as helpers. While wild-type SV40 and late temperature-sensitive mutants can serve as helpers in the replication and amplification of these minicircular DNAs, early temperature-sensitive mutant genomes are unable to do so at the nonpermissive temperature. The minicircular DNAs are amplified in vivo through an arithmetic series of oligomers. Encapsidation of reiterated molecules between 70 and 100% the size of wild-type SV40 DNA is observed, although reiterated viral DNA molecules much larger than unit size are formed in vivo.     

Molecular biology of rotaviruses. III. Isolation and characterization of temperature-sensitive mutants of bovine rotavirus

Twenty-six temperature-sensitive (ts) mutants of United Kingdom tissue culture-adapted bovine rotavirus were isolated and characterized. Fourteen of these mutants were determined to be ts both by efficiency of plating and by virus yield at the nonpermissive temperature of 39.5 degrees C as compared with that at the permissive temperature of 32 degrees C. The remaining mutants were only ts by the criterion of efficiency of plating. High-frequency recombination (gene reassortment) was observed when some pairs of mutants were crossed, and this allowed the classification of the mutants into five separate recombination groups. Groups III and V have prototype ts mutants (ts34 and ts115, respectively) that do not synthesize RNA or polypeptides at 39.5 degrees C. The other groups, I, II, and IV, have prototype mutants (ts17, ts7, and ts6, respectively) that synthesize both RNA and polypeptides at 39.5 degrees C, although ts17 does so only at a reduced level.     

Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication

Thermosensitive deoxyribonucleic acid replication-defective mutants have been isolated by using an autoradiographic selection method. The mutants have been analyzed genetically and biochemically. Some of the mutants show thermosensitivity of in vitro deoxyribonucleic acid replication. These can be classified into three groups according to their behavior in in vitro complementation assays. This classification is congruent with that obtained by genetic mapping by using cotransduction frequencies with selected markers in P1 transduction analysis.     

Isolation and characterization of unusual gin mutants.

Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation.     

Studies on mammalian chromosome replication : III. Organization and replication of diplochromosomes

The process of DNA synthesis in normal and endoreduplicating mammalian cells are very similar. Both types of chromosomes are replicated in defined units termed chromosomal replicons, and in the same sequences along their lengths. The sister chromosomes of the diplochromosomes are replicated synchronously in identical patterns. The present observations suggest that organization and sequences of chromosome replication are genetically programmed.     

SOS induction by thermosensitive replication mutants of miniF plasmid

Summary MiniF, a 9.3 kb fragment of the dispensable F plasmid, carries genes necessary for its replication and partition as well as for the expression of an SOS signal. The arrest of replication of a thermo-sensitive miniFts at 42°C induced SOS functions such as prophage , sfiA expression, W-reactivation of UV-irradiated phage . Two miniF ts9 and ts17 mutations were located within the KpnI fragment (43.6–46.9) in the minimal oriS replicon. Blocking miniF replication by incBC ⁺ incompatibility genes situated in trans on a second plasmid also induced SOS functions. In contrast, if miniFts17 plasmid escaped the replication block at 42°C by being inserted into pR325, there was no SOS induction. SOS induction by the arrest of miniF replication required the miniF lynA ⁺ locus in cis, the host recA ⁺ and lexA ⁺ genes. We found that SOS induction was increased greatly near the stationary phase and that cell viability declined. During host cell exponential growth, miniFts9 and miniFts17 plasmids were lost rapidly, although SOS induction persisted for several cell generations. We postulate that lynA expresses a persistent product that may lead to the unwinding of chromosomal DNA.     

Proteinase yscD mutants of yeast. Isolation and characterization

Mutants of the yeast Saccharomyces cerevisiae, devoid of proteinase yscD activity, were isolated by screening for the inability of mutagenized cells to hydrolyze Ac-Ala-Ala-Pro-Ala-beta-naphthylamide in situ. One of the selected mutants bears a thermolabile activity pointing to the gene called PRD1 as being the structural gene for proteinase yscD. All mutants isolated fell into one complementation group. The defect segregates 2:2 in meiotic tetrads indicating a single gene mutation, which was shown to be recessive. Diploids heterozygous for PRD1 display gene dosage. The absence of proteinase yscD did not affect mitotic growth under rich or poor growth conditions, neither mating nor ascopore formation. Also growth of mutant cells after a nutritional shift-down was not altered. Inactivation of enzymes tested which are subject to carbon-catabolite inactivation, a process proposed to be of proteolytic nature, is not affected by the absence of proteinase yscD. Protein degradation rates in growing cells, in cells under conditions of differentiation or heat shock, showed no obvious alteration in the absence of proteinase yscD activity. Also inactivation of alpha-factor pheromone was not affected by proteinase yscD absence. Normal growth of mutant cells on glycerol indicates that the enzyme is not involved in any vital event in mitochondrial biogenesis.     

Isolation and genetic characterization of ethanol-resistant reovirus mutants.

To better understand the mechanism(s) by which viruses respond to chemical or physical treatments, we isolated a series of mutant strains of reovirus type 3 Dearing that exhibit increased ethanol resistance. Following exposure to 33% ethanol for 20 min, the parental strain exhibited a 5 log10 decrease in infectivity. The mutant strains, however, exhibited a 2 to 3 log10 decrease in titer following identical treatment. Through the use of reassortant viruses, we mapped this increased ethanol resistance mutation to the M2 gene segment, which encodes a major outer capsid protein, mu1C. Sequence analysis of mutant M2 genes revealed that six of seven unique mutants possessed single-point mutations in this gene. In addition, the change in six of seven mutants caused a predicted amino acid change in a 35-amino-acid region of the gene product between amino acids 425 and 459. The identification of ethanol resistance mutations within a discrete region of this outer capsid protein identifies that portion of the protein as important in reovirus stability. The presence of viral particles possessing altered stability also suggests that subpopulations of viruses may possess altered environmental stability, which, in turn, could affect viral transmission.