Degradation of Escherichia coli B Deoxyribonucleic Acid After Infection with Deoxyribonucleic Acid-Defective Amber Mutants of Bacteriophage T7

The degradation of bacterial deoxyribonucleic acid (DNA) was studied after infection of Escherichia coli B with DNA-negative amber mutants of bacteriophage T7. Degradation occurred in three stages. (i) Release of the DNA from a rapidly sedimenting cellular structure occurred between 5 and 6 min after infection. (ii) The DNA was cleaved endonucleolytically to fragments having a molecular weight of about 2 x 10(6) between 6 and 10 min after infection. (iii) These fragments of DNA were reduced to acid-soluble products between 7.5 and 15 min after infection. Stage 1 did not occur in the absence of the gene 1 product (ribonucleic acid polymerase sigma factor), stage 2 did not occur in the absence of the gene 3 product (phage T7-induced endonuclease), and stage 3 did not occur in the absence of the gene 6 product.     

Isolation of Bacteriophage T4 Mutants Defective in the Ability to Degrade Host Deoxyribonucleic Acid

A method was devised for identifying nonlethal mutants of T4 bacteriophage which lack the capacity to induce degradation of the deoxyribonucleic acid (DNA) of their host, Escherichia coli. If a culture is infected in a medium containing hydroxyurea (HU), a compound that blocks de novo deoxyribonucleotide biosynthesis by interacting with ribonucleotide reductase, mutant phage that cannot establish the alternate pathway of deoxyribonucleotide production from bacterial DNA will fail to produce progeny. The progeny of 100 phages that survived heavy mutagenesis with hydroxylamine were tested for their ability to multiply in the presence of HU. Four of the cultures lacked this capacity. Cells infected with one of these mutants, designated T4nd28, accumulated double-stranded fragments of host DNA with a molecular weight of approximately 2 x 10(8) daltons. This mutant failed to induce T4 endonuclease II, an enzyme known to produce single-strand breaks in double-stranded cytosine-containing DNA. The properties of nd28 give strong support to an earlier suggestion that T4 endonuclease II participates in host DNA degradation. The nd28 mutation mapped between T4 genes 32 and 63 and was very close to the latter gene. It is, thus, in the region of the T4 map that is occupied by genes for a number of other enzymes, including deoxycytidylate deaminase, thymidylate synthetase, dihydrofolate reductase, and ribonucleotide reductase, that are nonessential to phage production in rich media.     

Effect of Elevated Temperature on Extended Enzyme Synthesis Induced by Bacteriophage T4 Amber Mutants Unable to Synthesize Deoxyribonucleic Acid

The extended synthesis of early enzymes by the deoxyribonucleic acid-negative amber mutants of bacteriophage T4 after infection of the nonpermissive host Escherichia coli B was prevented by incubating the infected cells at 44 C. This effect did not occur if the incubation temperature was 43 C or less or if the cells were grown and infected in broth rather than minimal medium (C medium). Once early enzyme synthesis had ceased at 44 C, lowering the incubation temperature to 37 C did not occasion resumption of synthesis. Experiments with chloramphenicol at 44 C indicated that increased degradation of early enzymes is an unlikely explanation for the effect. Examination of pulse-labeled ribonucleic acid and polysomes made at 37 and 44 C in infected cells revealed some differences, but at present there is no obvious way in which these differences may be related to the effect on enzyme formation. There was no discernible difference between the ribosomal ribonucleic acid and ribosomes at the two temperatures, nor was there a difference in the cell-free amino acid-incorporating systems isolated from cells infected at the two temperatures as judged by polyuridylic stimulation of phenylalanine incorporation. Incubation of cells infected with T4amN82 at 44 C with protein synthesis blocked by 5-methyltryptophan for 15 min did not prevent the typical pattern of enzyme synthesis at 44 C when the block was reversed by excess l-tryptophan. The relation of this and other observations relative to the effect at 44 C on the synthesis of early enzymes is discussed.     

Properties of Bacteriophage T4 Mutants Defective in Gene 30 (Deoxyribonucleic Acid Ligase) and the rII Gene

In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.     

Mutants of Escherichia coli with Cold-Sensitive Deoxyribonucleic Acid Synthesis

Ten cold-sensitive mutants defective in deoxyribonucleic acid (DNA) synthesis at 20 C have been identified among 218 cold-sensitive mutants isolated from a mutagenized population of Escherichia coli K-12. Four of the ten mutant alleles, dna-339 dna-340, dna-341, and dna-342, cotransduce with serB(+) and hence may be dnaC mutants. Two of these, dna-340 and dna-341, are recessive to their wild-type allele. The gene product of their wild-type allele is trans acting. Complementation tests have demonstrated that dna-340 and dna-341 are in the same cistron. The mapping of the remaining six mutations is in progress. In an attempt to determine whether LW4 and LW21 were initiator mutants, cultures of these strains were starved of an essential amino acid at 37 C and then incubated at 15 C with the essential amino acid. The amount of DNA synthesis observed under these circumstances was insignificant. These data are consistent with the idea that LW4 and LW21 are initiator mutants. However, attempts to integratively suppress LW4 and LW21 with F' factors were unsuccessful. To resolve the question of whether or not LW4 and LW21 are initiator mutants, more specific tests and criteria are required. Cultures of LW4 and LW21 were toluene treated and used to measure in vitro DNA synthesis. If the cells were incubated either at 15 or 20 C before toluene treatment, they were capable of markedly less DNA synthesis than if preincubation had not occurred. The amount of in vitro DNA synthesis is directly proportional to the amount of DNA synthesis occurring during preincubation in vivo; i.e., more DNA synthesis is observed at 20 than at 15 C. The fact that the cold-sensitive mutants are unable to synthesize DNA when supplied with deoxyribonucleoside triphosphates, DNA precursors, is evidence they are not defective in precursor synthesis.     

Mutants in a Nonessential Gene of Bacteriophage T4 Which Are Defective in the Degradation of Escherichia coli Deoxyribonucleic Acid

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine ((3)H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the (3)H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4.     

T3 and T7 Bacteriophage Deoxyribonucleic Acid-Directed Enzyme Synthesis In Vitro

The T3 phage enzymes S-adenosyl methionine cleaving enzyme and lysozyme and the T7 lysozyme were synthesized in a deoxyribonucleic acid (DNA)-dependent, cell-free system derived from uninfected Escherichia coli. The data presented suggest that these enzymes are encoded in that portion of the DNA which is transcribed early after infection.     

Defective Concatemer Formation in Cells Infected with Deoxyribonucleic Acid-Delay Mutants of Bacteriophage T4

Nonpermissive host cells infected with phage T4 mutants in genes 52, 39, and 58 through 61 are shown to form short intracellular single-stranded deoxyribonucleic acid in contrast to wild-type infected cells, which form dimers and trimers of T4 genome size.     

Replicative Intermediates of Bacteriophage T7 Deoxyribonucleic Acid

After infection with bacteriophage T7, parental and newly synthesized deoxyribonucleic acid (DNA) exhibit an extremely fast sedimentation rate in neutral sucrose gradients. This fast-sedimenting component (intermediate I) has a sedimentation constant of about 1,500S and contains T7 DNA as determined by DNA-DNA hybridization experiments. Pulse-chase experiments indicate that the fast-sedimenting material is metabolically active and serves as a precursor to the formation of T7 DNA. Intermediate I contains about 2.5 to 7% of the total ³H-labeled protein formed between 3 and 9.5 min after T7 infection. Treatment of intermediate I with Pronase results in the release of the DNA from the complex. At early times after infection, a second intermediate (intermediate II) can be detected which contains both parental and newly synthesized DNA sedimenting slower than intermediate I but 2 to 3 times as fast as mature T7 DNA. Intermediates I and II containing parental DNA are formed after infection of the nonpermissive host with an amber mutant in gene 1, a gene whose expression is necessary for the synthesis of most T7 proteins. The two intermediates are also observed when infection with T7 wild type is carried out in the presence of chloramphenicol.     

Defective Deoxyribonucleic Acid Replication of T4rII Bacteriophage in Lambda-Lysogenic Host Cells

Sedimentation of the replicative deoxyribonucleic acid through alkaline sucrose gradients showed that rII single chains reached the half-mature size at a time when wild-type molecules formed long chains (dimers and trimers of genome size). Long rII single chains could be observed on substitution of tris(hydroxymethyl)aminomethane buffer for Na⁺K⁺ phosphate in the growth medium.     

Mutants of the N-3 R-Factor Conditionally Defective in hspII Modification and Deoxyribonucleic Acid-Cytosine Methylase Activity

The N-3 drug resistance (R) factor specifies a deoxyribonucleic acid (DNA)-cytosine methylase and a DNA restriction-modification (hspII) system. We have isolated three independent mutants that are conditionally defective in their ability to modify bacteriophage lambda and to methylate DNA-cytosine residues. The ratio of 5-methylcytosine to N(6)-methyladenine in bacterial DNA and in the DNA of phages lambda and fd was determined after labeling with [methyl-(3)H]methionine at various growth temperatures. Although the ability of the wild-type N-3 factor to modify phage lambda and to methylate DNA-cytosine residues was unaffected with increasing temperature, two of the mutants exhibited a parallel loss in modification and cytosine methylation ability. The ability of the third mutant to carry out these functions was dependent on the presence or absence of an amber suppressor mutation in the host genome. These results offer further support for the notion that hspII modification is mediated by a DNA-cytosine methylase. Evidence is also presented that the modification methylase is responsible for the in vivo methylation of phage fd DNA (which is not subject to hspII restriction in vivo).     

Intramolecular Heterogeneity of the Deoxyribonucleic Acid of Temperate Bacteriophages

Spectral changes accompanying the thermal denaturation of phage deoxyribonucleic acid suggested that λ is not unique in possessing large-scale intramolecular heterogeneity and nucleotide clustering; instead, λ seems to share this property with other enteric phages.     

Inhibition of Bacterial Conjugation by Ribonucleic Acid and Deoxyribonucleic Acid Male-Specific Bacteriophages

Both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) male-specific phages, with an F-specific host range, inhibited the bacterial mating process of Escherichia coli. DNA phages prevented the formation of mating pairs but had no effect on mating pairs once they were formed. A step in RNA phage infection, prior to RNA penetration, prevented the formation of mating pairs and, in addition, prevented a fraction of existing mating pairs from completing the mating process. These findings are compatible with the hypothesis that donor cells have a single surface structure involved in both conjugation and male-phage adsorption and that this element is the F pilus.     

Effect of Nalidixic Acid on the Growth of Deoxyribonucleic Acid Bacteriophages

The effect of nalidixic acid on the growth of various deoxyribonucleic acid (DNA) bacteriophages has been investigated by one-step growth experiments. The Escherichia coli bacteriophages T5, lambda, T7 and phiR are strongly inhibited by nalidixic acid, whereas T4 and T2 are only partially inhibited. The Bacillus subtilis bacteriophages SP82, SP50, and phi29 are relatively unaffected by nalidixic acid. There is no correlation between those bacteriophages which can grow in the presence of nalidixic acid and the presence of an unusual base in the phage DNA.     

Equal Incorporation of Both Parental Bacteriophage T7 Deoxyribonucleic Acid Strands into Intracellular Concatemeric Deoxyribonucleic Acid

After infection of Escherichia coli B with radiolabeled T7 bacteriophage, the parental deoxyribonucleic acid label was found in both polynucleotide chains of the intracellular T7 concatemer.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Role of Gene 46 in Bacteriophage T4 Deoxyribonucleic Acid Synthesis

In an attempt to establish whether Escherichia coli B infected with N130 (an amber mutant defective in gene 46) is recombination-deficient, the postinfection fate of (14)C-labeled N130 parental deoxyribonucleic acid (DNA) was followed, its amount in complex with the host cell membrane being determined in sucrose gradients after mild lysis of the infected cells. The parental DNA was found to undergo gradual detachment from the membrane during infection. Pulse-chase experiments similarly showed that newly synthesized DNA is normally attached to the host cell membrane and is detached by endonucleolytic breakage at a late stage of infection. The conclusion is that only attached DNA molecules are replicated by membrane-bound replicase, whereas those detached by endonucleolytic breakage are not. It thus seems that the gene 46 product controls the activity of a nuclease whose main function is recombination of DNA nicked by endonuclease, thereby attaching it to the host cell membrane. The rate of T4 DNA synthesis is apparently governed by the efficiency of recombination. Supporting evidence was found in experiments with the double mutant N130 x N134 (genes 46, 33).