John Spizizen

Early blocked asporogenous mutants of Bacillus subtilis 168

SummaryA number of mutants (abs)-resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168 have been isolated from an early blocked asporogenous mutant (spoA12). At least four classes were recognized according to their phenotypic properties. Genetic analysis has shown that these mutants were neither partial revertants nor suppressor mutants of the spoA gene. Both nonsense and missense mutants of the spoA gene are reverted partially by a secondary mutation which is resistant to antibiotic of B. subtilis 168. Another asporogenous mutant, spoB, whose locus is closely linked to pheA, is also affected by the same abs mutation. The nature of abs mutants is discussed.

Studies on Bacillus subtilis bacteriophage φ15

Abstract The growth characteristics and physicochemical properties of Bacillus subtilis bacteriophage φ15 have been investigated and compared with φ29. Differences in growth characteristics of these phages were found in various stages of spore germination and outgrowth which may account for the absence of plaque formation by φ15 on wild-type cells. Both φ15 and φ29 genomes were incorporated into spores when cells were infected at a certain critical time during host cell development. φ15 is morphologically indistinguishable from φ29. The DNA of φ15 is a linear duplex molecule having a mean length of 6.1 μm. The S value of this DNA is 23 ± 0.4 S. These values correspond to a molecular weight of approximately 12 × 106 daltons. At least seven structural proteins of φ15 were recognized in SDS-acrylamide gel electrophoresis. The sum of the molecular weights of φ15 proteins is approximately 354,000 daltons. This corresponds to about 59% of the coding capacity of double-stranded DNA of φ15. φ15 DNA was found to hybridize with φ29 DNA.

Extrachromosomal DNA in Bacillus thuringiensis var. kurstaki, var. finitimus, var. sotto, and in Bacillus popilliae.

Abstract Four entomopathogenic bacteria contained extrachromosomal deoxyribonucleic acid (DNA) molecules of various sizes. Bacillus thuringiensis var. kurstaki contained twelve elements banding on agarose gels that ranged from 0.74 to > 50 × 106 daltons, three of which were giant extrachromosomal DNA elements. B. thuringiensis var. sotto contained one giant extrachromosomal DNA element with a molecular size of about 23.5 × 106 daltons and two lesser elements of 0.80 and 0.62 × 106 daltons. B. thuringiensis var. finitimus harbored two giant DNA elements corresponding to >50 × 106 daltons and two lesser bands with relative small size (0.98 and 0.97 × 106 daltons). B. popilliae contained no giant extrachromosomal DNA elements but did contain two smaller elements corresponding to 4.45 and 0.58 × 106 daltons. The possible use of extrachromosomal DNA elements that prove to be autonomous replicons for recombinant DNA studies is discussed.

In vitro‐labeled DNA for detecting viral genomes in multiple sclerosis: I. papovaviruses

Papovaviruses appear to be neurotropic and one, JC virus, is implicated as the Cause of one type of demyelinating disease, progressive multifocal leukoencephalopathy. To investigate whether human papovaviruses play a role in multiple sclerosis, radioactively labeled DNA from BK virus, human papilloma virus, and simian virus 40 was used as a probe in order to detect related unlabeled DNA sequences in DNA isolated from multiple sclerosis brain and/or spinal cord. Labeled viral probes were denatured and DNA allowed to reassociate in the presence of excess unlabeled DNA from multiple sclerosis tissue or from controls. The reassociation rate of the probe is proportional to the concentration of viral DNA present, and an increase in the reassociation rate of the probe over that of control reactions would indicate the presence of unlabeled viral DNA in multiple sclerosis cellular DNA. However, addition of DNA derived from multiple sclerosis patients did not increase rates of reassociation of viral probes. Known human papovaviruses probably have no role in the pathogenesis of multiple sclerosis.

Further Characterization of Herpes Virus Persistence

Infection of cell cultures with herpes simples virus type 1 (HSV-1) under standard culture conditions yielded persistently infected cells capable of continued growth in the presence of virus and of forming colonies in agarose. The ability of an infected culture to yield cells able to survive HSV-1 infection was directly related to the presence of S phase cells (cells actively engaged in DNA synthesis) at the time of infection. Only when very high multiplicities of infecting virus (greater than 10) were used did cultures fail to yield persistently infected cells capable of colonial growth in agarose. Cell clones derived from colonies grown in agarose established cell cultures which possessed all the characteristics of HSV-1 persistently infected cultures. When cultures were cloned a second time in agarose, as a rare event, cell cultures which did not immediately liberate infectious virus could be isolated. These cell cultures, however, possessed an increased resistance to superinfection by HSV-1. On continued cultivation they reverted to persistence as exhibited by the sudden onset of virus cytopathic effects and release of infectious virus.

An effect of temperature on the Bacillus subtilis transfer RNA's which respond to codons beginning with U and a correlation with cytokinin activity

1. 1. The fraction of unaminoacylated tRNAs from Bacillus subtilis that requires 1 M NaCl and 10% ethanol for elution from benzoylated DEAE-cellulose (BD-cellulose) contains amino acid acceptor activities for phenylalanine, serine, tyrosine, leucine, cysteine, and tryptophan. Codons beginning with U code for only the amino acids in this set. The amount of each of these tRNA species which binds to BD-cellulose is dependent on temperature. At 70°, more of these tRNA species require the addition of ethanol for elution. 2. 2. The chromatographic behavior of cytokinin activity is dependent on temperature in a similar manner. At room temeprature, some cytokinin activity is found in both 1 M NaCl eluted fractions and the fraction which requires 1 M NaCl and 10% ethanol for elution. At 70°, however, all cytokinin activity is retarded until ethanol is added to the eluant. 3. 3. The same effect caused by running the BD-cellulose column at 70° can be achieved by pretreating the tRNAs at 70° for 10 min and then running the column at room temperature. 4. 4. An explanation of this phenomenon is discussed in terms of molecular structure.