Ethel S. Tessman

Mutants of bacteriophage S13 blocked in infectious DNA synthesis

In cells that are multiply infected with wild-type S13, infectious replicative form DNA is found at the earliest time measured (two minutes) and increases in amount until about eight minutes, when it reaches a plateau. Synthesis of infectious single-stranded DNA and of infectious phage particles begins at about eight minutes. The addition of 30 μg/ml. chloramphenicol before infection prevents the normal shut-off of replicative form synthesis. Suppressible (su) mutants of complementation group IV of phage S13 are blocked in synthesis of infectious replicative form about three minutes after infection of the restrictive host. Su mutants of the other four complementation groups synthesize as much replicative form as wild type but fail to form infectious single-stranded DNA. Mixed infection together with wild-type phage has little or no effect on the ability of group IV mutants to synthesize infectious replicative form. Synthesis of early and late replicative form in a wild-type infected culture is differentially affected by 100 μg/ml. of chloramphenicol. Early replicative form synthesis is only mildly affected by 100 μg/ml. chloramphenicol; late replicative form synthesis is severely inhibited but eventually shows some recovery. It is concluded that early replicative form synthesis is under host control, whereas late replicative form synthesis is under phage control.

COMPLEMENTATION GROUPS IN PHAGE S13.

Abstract Four complementation groups have been found for phage S13 using two types of conditional lethal mutants, temperature-sensitive ( t ) and suppressible ( su ) mutants. Each complementation group contains both t and su mutants. Genetic crosses show that the mutants of each group are located in a separate region of the genetic map. Many group I t mutants are affected in their host range and adsorption properties and are therefore believed to lie in a genetic region determining the structure of the phage protein coat. su mutants of group IV are unable to lyse the restrictive host, E. coli C, while su mutants of groups I, II, and III lyse E. coli C completely. su mutants of all four groups produce little or no phage coat antigen in E. coli C. su mutants of groups I, II, and III are completely rescued by an su + phage in mixed infection of the restrictive host, but group IV su mutants are very poorly rescued. An su mutant has been isolated which appears to be an O 0 or polar mutant, since it is defective in the functions of both groups III and IV, yet does not seem to be a double mutant. Mapping experiments show that map distances are roughly additive for groups I, III, and IV.

The eight genes of bacteriophages φX174 and S13 and comparison of the phage-specified proteins☆

Abstract Complementation tests between phages S13 and φX174 show that the two phages have at least eight homologous essential genes. Gel electrophoresis of the phage-specified proteins give results which are very similar for the two phages.

Early replicative form DNA of bacteriophage S13: I. Sucrose gradient analysis of replicative form made by gene IV mutants

Abstract Early replicative form of bacteriophage S13 is the term applied to the small amount of double-stranded DNA synthesized immediately after infection under the following conditions: (1) by wild-type or mutant phage in the presence of at least 100 μg/ml. chloramphenicol, a dose very inhibitory to protein synthesis; or (2) by gene IV mutant phage in the absence of chloramphenicol. The present results show that early replicative form consists of both 21 s (twisted double-stranded ring) and 16 s (untwisted double-stranded ring) components.

Direction of Translation in Bacteriophage S13

Suppressible (amber-type) mutants in gene IIIa of bacteriophage S13 show polarity in reducing the activity of only the neighboring gene IIIb. The polarity implies that the genome is translated in the direction of IIIa to IIIb. From the known homology of the messenger RNA with the single-stranded DNA, the orientation of the DNA with respect to the genetic map can also be inferred.

A promoter site and polarity gradients in phage S13

Abstract Acrylamide gel analysis shows that the polar effect of nonsense mutations in gene F of phage S13 extends to adjacent genes G and H, but not to genes A and B, which follow H in the direction of translation. Therefore, a promoter site must be located before gene A, but not before genes G or H. Degree of polarity was compared with genetic map position for genes F and G. Gene F generates a polarity gradient which is strongly dependent on distance to the end of the gene; in gene G distance to the end of the gene appears to have no effect on degree of polarity.

Protein synthesis by a mutant of phage S13 blocked in DNA synthesis

Abstract Gene IV amber mutants of phage S13 are known to be blocked in the synthesis of progeny replicative form DNA (RF). Protein synthesis by a gene IV mutant has been studied in a nonpermissive host cell strain that permits phage particle formation by the standard phage at doses of ultraviolet radiation that severely depress host protein synthesis. Using gel electrophoresis analysis, it was found that the mutant formed normal amounts of all the six major protein peaks that are observed for the standard phage. (Three of the gel peaks correspond to protein-coat components.) Thus the block in DNA synthesis caused by the gene IV mutation does not switch off either transcription or translation of the majority of the phage genes. Furthermore, parental RF alone is sufficient to produce normal amounts of phage-specific protein.

Direction of translation in phage S13 as determined from the sizes of polypeptide fragments of nonsense mutants

Abstract The direction of translation for phage S13 was determined by comparing the sizes of polypeptide fragments of gene I nonsense mutants with the genetic map order of the mutants.

Reinitiation mutants of gene B of bacteriophage S13 that mimic gene A mutants in blocking replicative form DNA synthesis.

Abstract Several mutations in gene B of phage S13 appear to shorten the B protein by elimination of an N-terminal fragment, without destroying the B protein function. The shortened B protein resulting from each of these mutations can block the unique DNA-nicking properties of the S13 gene A protein. Because of the block in gene A function, normal gene B protein may have a function in phage DNA synthesis in addition to its known role in catalyzing capsid assembly. From gel electrophoresis the mutant B protein is estimated to be shorter than the normal S13 B protein by 1720 ± 70 daltons and is therefore believed to be an internal reinitiation fragment. The reinitiated fragments are functional and are made in about twice the amount of the normal B protein. The phage mutants which yield the reinitiation fragments are double mutants, each phage containing the same gene B nonsense mutation and each appearing to contain a different compensating gene B mutation. Various data support the assumption that the compensating mutations are frame-shifts, including the fact that suppression does not restore the normal-sized B protein. The reinitiation is assumed to occur at a pre-existing out-of-phase initiator codon, near the nonsense triplet; the correct reading frame would then be restored by each of the several different compensating mutations. The position of the normal S13 B protein in the gel electrophoresis pattern has been located both by elimination and shifting of the B peak, using appropriate amber mutants. The molecular weight of the S13 B protein is about 17,200, and is 2100 daltons less than the B protein of phage φX174; the S13 B protein can nevertheless substitute for the φX 174 B protein. Thus substantial portions of the B protein can be deleted without destroying its function.

Radiological mapping of functional transcription units of bacteriophages φX174 and S13. The function of proximal and distal promoters

Abstract We have found that the nearest promoter is not always the primary promoter for making translatable message. The technique of ultraviolet mapping was used to determine the location of promoter sites for translated mRNA coded for by bacteriophages φX174 and S13. The method is based on the theory that the “target size” for u.v. inactivation of expression of a gene is proportional to the distance between the promoter and the 3′ end of the gene. This method has revealed an expected and some unexpected locations for the promoters responsible for gene expression. Ultraviolet-survival curves for expression of phage genes were interpreted in the following way. The contiguous genes D , F , G and H are expressed as a unit under the control of a promoter located near gene D . However, gene B (and probably the adjacent genes K and C ) are controlled by a promoter distant from gene B , possibly in the region of gene H , rather than from a promoter located just before gene B . Likewise, gene A is controlled by a promoter distant from gene A .