Genetic Map of Bacteriophage T3

About 200 amber mutants of phage T3 were found to lie in 14 different genes. These genes are homologous to known T7 genes. The genetic map of T3 is very similar to that of T7.     

Nonsense Mutants in the rII A Cistron of Bacteriophage T4

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.     

Bacteriophage T4 rIIB protein synthesis with a temperature-sensitive mutation in the rIIB initiation codon.

Summary In protein synthesis, the incorporation of an N-terminal formylmethionine residue is directed by an initiation codon. The most frequently used codon is AUG, although initiation at GUG and UUG codons has also been observed. The HD263 mutation is an AUG to AUA change in the rIIB initiation codon. Evidence is presented here that wild type and HD263 rIIB proteins, whether synthesized in vivo or in vitro, have identical fmet peptides. It is concluded that translation began at the AUA mutant initiation codon in vitro and in phage T4 infected cells.In the in vitro translation system used in these studies, the rIIB protein synthesized at 25° no longer contains the N-terminal formyl group whereas a large proportion of the formyl group is retained at 37°.Abbreviations used tss-mutation temperature-sensitive, synthesis mutation - PrIIB protein product of gene rIIB - PrIIB⁺ PHD263 and PHE122, rIIB proteins synthesized by rIIB⁺ phage, tss-mutant HD263 and amber mutant HE122 - fmet-tRNA N-formylmethionyl-tRNA_inf^met     

Revised Location of the rIII Gene on the Genetic Map of Bacteriophage T4

Two- and three-factor crosses showed that the T4 rIII gene is located between genes 31 and 30 rather than between genes 31 and 63.     

Genetic map of the beginning of the rIIB cistron of bacteriophage T4

Summary In this report the herefore uncharacterized mutants at the beginning of the rIIB cistron of bacteriophage T4 have been connected to the main part of the map and have been shown not to be in any way unusual. The temperature sensitive mutant HD263 appears to be the earliest mutant in the cistron.     

Streptomycin-Induced Phenotypic Suppression of Hydroxylamine-Induced Nonsense Mutants in the rII A Cistron of Bacteriophage T4

Twenty-one hydroxylamine-induced rII A cistron nonsense mutants were tested for streptomycin (SM)-induced phenotypic suppression by exposing Escherichia coli SBO (nonpermissive host) to phage in the presence and absence of SM. All nine amber, four of six ochre, and five of six opal mutants were phenotypically suppressible by SM. For suppressible mutants, the ratio of the average burst size in the presence of SM to size in the absence of SM ranged from 12 to 242 for the ambers, 3 to 33 for the ochres, and 4 to 14 for the opals. Increased susceptibility of the amber mutants to SM-induced phenotypic suppression relative to the susceptibility of the opal and ochre mutants may reflect a neighboring base effect, such that a 3′-terminal adenine inhibits misreading of a 5′-terminal uracil.     

Genetic Map of Bacteriophage phiX174

Bacteriophage X174 temperature-sensitive and nonsense mutations in eight cistrons were mapped by using two-, three-, and four-factor genetic crosses. The genetic map is circular with a total length of 24 × 10⁻⁴wt recombinants per progeny phage. The cistron order is D-E-F-G-H-A-B-C. High negative interference is seen, consistent with a small closed circular deoxyribonucleic acid molecule as a genome.     

Translational reinitiation in the rIIB cistron of bacteriophage T4

During translation of the bacteriophage T4 rIIB gene messenger RNA, premature termination sometimes results in translational reinitiation. The nucleotide sequence surrounding the true initiating AUG of the rIIB message has been determined recently. We have identified potential reinitiation codons within this sequence and determined which of these codons are utilized in reinitiation events. We have used the sequence to reinterpret the x reinitiation event described by Sarabhai & Brenner (1967).     

Genetic Mapping of the Amino-Terminal Domain of Bacteriophage T4 DNA Polymerase

The DNA polymerase of bacteriophage T4 is a multifunctional enzyme that harbors DNA-binding, DNA-synthesizing and exonucleolytic activities. We have cloned in bacterial plasmids about 99% of the structural gene for this enzyme (T4 gene 43). The gene was cloned in six contiguous 5'-terminal DNA fragments that defined seven intragenic mapping regions. Escherichia coli hosts harboring recombinant plasmids carrying the gene 43 subsegments were used in marker-rescue experiments that assigned a large number of ts and nonsense polymerase mutations to different physical domains of the structural gene. Conspicuously, only one missense mutation in a large collection of mutants mapped in the 5'-terminal 450 base-pair segment of the approximately 2700 base-pair gene. To test if this indicated a DNA polymerase domain that is relatively noncritical for biological activity, we mutagenized a recombinant plasmid carrying this 5'-terminal region and generated new conditional-lethal mutations that mapped therein. We identified five new ts sites, some having mutated at high frequency (nitrosoguanidine hot spots). New ts mutations were also isolated in phage genes 62 and 44, which map upstream of gene 43 on the T4 chromosome. A preliminary examination of physiological consequences of the ts gene 43 mutations showed that they exhibit effects similar to those of ts lesions that map in other gene 43 segments: some were mutators, some derepressed gene 43 protein synthesis and they varied in the severity of their effects on T4-induced DNA synthesis at nonpermissive temperatures. The availability of the gene 43 clones should make it possible to isolate a variety of lesions that affect different activities of the T4 DNA polymerase and help to define the different domains of this multifunctional protein.     

Gene rIIB from bacteriophage T4 in genetic recombination

The effect of the rIIB gene on genetic recombination in bacteriophage T4 was studied. Relationships between recombination frequency and the physical distance were determined in three series of isomarker two-factor crosses between rII mutants. In the first series of intergenic crosses (rIIa x rIIb), the rII gene function was restored owing to complementation. In the second series of crosses, identical to the first one, the rIIB gene function was suppressed, because the rIIa parent carried an additional amberlike mutation in the rIIB gene. The recombinants were scored by plating lysates on the amber-suppressor Escherichia coli strain, on which an amberlike mutation was not expressed phenotypically. In the third series, all crosses were intragenic (rIIb x rIIb). In two series of crosses in the absence of the rIIB function, the relationships between recombination frequency and the physical distance were identical, whereas enhanced recombination frequencies were observed in the rIIB+ background. The magnitude of the rIIB-related effect depended on distance, reaching the maximum in the region located 100 to 200 bp from the beginning of the rIIB gene. The possible role of the rIIB protein in genetic recombination is discussed.     

Correlation of the Genetic Map and the Endonuclease Site Map of Bacillus subtilis Bacteriophage SP02

By marker rescue of bacteriophage SP02 sus mutants with purified bacteriophage SP02 DNA fragments, 11 of the 17 known bacteriophage SP02 sus loci were assigned to discrete DNA fragments. The left-most genetic locus, susA, was found to reside near one bacteriophage SP02 terminus (EcoRI-C1 fragment), whereas the right-most genetic locus, susP, was found to reside near the other bacteriophage SP02 terminus (EcoRI-C2 fragment). The physical locations of the intervening genetic loci were found to be consistent with the previously determined genetic order. Evidence was also obtained which suggested that at least one end of a transforming DNA fragment is degraded during DNA uptake by the competent bacterium.     

The Genetic Constitution of Tandem Duplications of the rII of Bacteriophage T4d

Methods for genetically mapping the end points of tandem duplications of the rII region of bacteriophage T4D are presented. Analysis of ten duplications indicates (1) that the position of duplication end points and therefore the length of the duplicated segment differ in strains of independent origin; (2) that there is a direct relationship between segregation frequency and length; (3) that segregation is more frequent than expected on the basis of standard genetic mapping; and (4) that while duplications frequently include non-rII genetic material, frequently they do not include the entire rII region. The duplications studied range from less than two to about five cistrons in length.