From growth arrest to growth suppression.

Since the introduction of the cell cycle concept two approaches to study growth regulation of cells have been proposed. One claims that cells are naturally quiescent, requiring a stimulatory encouter with growth factors for induction of cell division. The other considers cellular multiplication as the natural steady-state; cessation of multiplication is thus a restriction imposed on the system. In the latter case emphasis is mainly on the signals involved in arrest of multiplication. This Prospect focuses on specific events occurring in mammalian cells at growth arrest, senescence, and terminal differentiation, specifically emphasizing the growth inhibitory factors, tumor suppressor genes, and other signals for growth suppression.     

RecE independent deletions of recombinant plasmids in Bacillus subtilis

Fragments from the Bacillus bacteriophage φ105 have been cloned in recE⁺ and recE^? bacteria lysogenic and nonlysogenic for the phage. Recombination between homologous DNA in the plasmid and the prophage occurs only in the rec⁺ strain at a low frequency of around 4%. After prolonged cultivation with selective pressure on the antibiotic resistance gene of the vector, the bacteria contained only plasmids with various deletions. This process is recE independent and occurs irrespective of whether base pair homology exists between chromosomal and plasmid DNA. The rate of spontaneous curing of the plasmid decreases in parallel to the appearance of deletions, presumably due to higher stability of the small plasmids.     

Transformation reveals a chromosomal locus of the gene(s) for methicillin resistance in Staphylococcus aureus.

The localization of the gene(s) mediating methicillin (mecr) in Staphylococcus aureus was determined by transformation with deoxyribonucleic acid (DNA) from a natural mecr strain (DU 4916) and transformation obtained with DNA from this strain. Streptomycin resistance genes (strr) and novobiocin resistance genes (novr) were used concurrently as representatives for chromosomal genes; penicillinase (PI254) and tetracycline plasmids were used as examples of medium- and small-size extrachromosomal genes, respectively. Superinfection of the lysogenic recipients with the competence-inducing phage phi11 or 83A enhanced transformation for all markers. Phenotypic expression of cadmium (cadr), tetracycline (tetr), or methicillin resistance (mecr) did not appear to require a host recombination system since a recA1 mutant could serve as the recipient provided it was superinfected with a competence-inducing phage. There was, furthermore, no requirement for preexisting plasmids for phenotypic expression. Ultraviolet irradiation of transforming DNA enhanced at low doses the transformation frequency for chromosomal genes strr and novr but not for mecr, cadr, or tetr. The gene(s) for mecr was transformed with chromosomal DNA after sodium dodecyl sulfate-sodium chloride extraction and after neutral sucrose gradient centrifugation of bulk DNA from wild-type strain DU 4916 and the transformats. No cavalently closed circular DNA or open circular DNA carrying the methicillin resistance gene(s) could be detected in the wild type or the transformants either by ethidium bromide-cesium chloride gradient centrifugation or by zonal rate centrifugation of cells directly lysed on top of the gradients. The mecr gene(s) is thus probably of chromosomal nature but possibly under recombinational control of phage genes, since transfer of mecr is independent of the recA1 gene(s) but can be accomplished in this strain after superinfection with a competence-inducing phage. Ultraviolet light inactivation of transforming DNA shows first-order kinetics for mecr transformability similar to that observed for both transfecting and plasmid DNA.     

Attachment and Eclipse of Adenovirus

The attachment and eclipse of adenovirus have been studied with the aid of highly purified ¹⁴C-threonine and ³²P-labeled adenovirus type 2 in KB cells in suspension cultures. Adenovirus particles and infectivity appear to attach at the same rate. The attachment rate appears to be highly dependent on the cell concentration and less dependent on virus concentration within the multiplicity range from 0.15 to 3 plaque-forming units per cell, probably corresponding to 4.5 to 90 particles per cell. Subsequent to attachment, 5 to 8% of the ¹⁴C label is eluted from the cell at a structure level, corresponding to free hexon. The ³²P activity is rapidly associated with the cells and is converted within 20 to 30 min to 65 to 85% deoxyribonuclease-susceptible material. This process is unaffected by actinomycin and puromycin. The deoxyribonuclease-sensitive material is, however, associated with ¹⁴C label for an extended period after infection, and does not sediment as free deoxyribonucleic acid in sucrose gradients. The implications of these findings on the penetration mechanism of animal viruses are discussed.     

Further characterization of the phosphate moiety of the adenovirus type 2 DNA-binding protein

The adenovirus type 2 DNA-binding protein is phosphorylated. Alkaline phosphatase treatment removes phosphate groups resulting in a decrease in molecular weight from 72000 to 70000. The dephosphorylated protein binds to single-stranded and double-stranded DNA as well as the phosphorylated protein does. Controlled chymotrypsin treatment cleaves the DNA-binding protein into two subspecies of Mr about 45000 and 25000. The 45000-Mr polypeptide contains most of the methionine residues but no phosphate and binds to DNA. The 25000-Mr polypeptide contains all the phosphate groups and shows no binding to DNA. Isoelectric focusing gels show heterogeneity of the DNA-binding protein and 15 subspecies with different charges can be observed after partial dephosphorylation by alkaline phosphatase. After extensive dephosphorylation two or three basic species with a molecular weight around 70000 are observed. Quantitative immunoprecipitation from cells labeled to equilibrium with inorganic 32PO4 gives a molar ratio of phosphate to protein of 4--7 and direct chemical determination of the phosphate residues yields 4 mol Pi/mol protein. These results suggest that there exist subspecies of the protein moiety of the adenovirus DNA-binding protein. The DNA-binding protein isolated from infected cells after a short 'pulse' of [35S]methionine has a molecular weight which corresponds to that of the dephosphorylated protein. After a 'chase' period the molecular weight increases to 72000, but alkaline phosphatase treatment converts it to a species with the same molecular weight as the newly synthesized DNA-binding protein, indicating that the modification of the protein is due to phosphorylation.