The temperature sensitive (TS) mutations of the bacteriophage T4 B

1966 ◽  
Vol 59 (3) ◽  
pp. 283-293 ◽  
Author(s):  
S. I. Alikhanian ◽  
C. N. Grinberg ◽  
V. N. Krylov ◽  
A. N. Maisourian ◽  
M. G. Oganesian
Keyword(s):  
Bacteriophage T4 ◽  
Temperature Sensitive ◽  
Ts Mutations

scholarly journals Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 213-220 ◽  
Author(s):  
L J Reha-Krantz
Keyword(s):  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Rnase H ◽  
Ribonuclease H ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Dna Polymerase I ◽  
E Coli ◽  
Intragenic Complementation ◽  
Polymerase I

Abstract Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.

A MAJOR ROLE FOR BACTERIOPHAGE T4 DNA POLYMERASE IN FRAMESHIFT MUTAGENESIS

Genetics ◽  
1983 ◽  
Vol 103 (3) ◽  
pp. 353-366
Author(s):  
Lynn S Ripley ◽  
Nadja B Shoemaker
Keyword(s):  
Dna Polymerase ◽  
Dna Sequences ◽  
Frameshift Mutation ◽  
Bacteriophage T4 ◽  
Base Substitution ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Frameshift Mutagenesis ◽  
Substitution Mutations ◽  
Polymerase Mutants

ABSTRACT T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis. Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes. Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others. The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation. A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum. Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4.

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

Genetics ◽  
1987 ◽  
Vol 115 (3) ◽  
pp. 393-403
Author(s):  
Melanie B Hughes ◽  
Arthur M F Yee ◽  
Myra Dawson ◽  
Jim Karam
Keyword(s):  
Dna Polymerase ◽  
Structural Gene ◽  
Base Pair ◽  
Hot Spots ◽  
Bacteriophage T4 ◽  
Multifunctional Protein ◽  
Amino Terminal ◽  
Preliminary Examination ◽  
Amino Terminal Domain ◽  
Ts Mutations

ABSTRACT 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.

scholarly journals Genetic definition of two functional elements in a bacteriophage T4 host-range "cassette".

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 471-479
Author(s):  
M Snyder ◽  
W B Wood
Keyword(s):  
Host Range ◽  
Bacteriophage T4 ◽  
Temperature Sensitive ◽  
Tail Fiber ◽  
Neighboring Gene ◽  
Distal Half ◽  
Bacterial Surface ◽  
Fiber Assembly ◽  
Proximal Half ◽  
Carboxy Terminal

Abstract Gene 37 of T4 encodes the major subunit of the distal half of the tail fiber. The distal tip of the fiber, comprised of the carboxy-terminal ends of two molecules of gene 37 product (gp37), carries the principal determinant of the phage host range. The gp37 carboxyl termini recognize the bacterial surface during infection, and, in addition, include a site required for interaction with the product of gp38 during distal half-fiber assembly. In the absence of interaction with gp38, gp37 polypeptides do not dimerize. Eleven temperature-sensitive mutants with defects located near the promoter-distal end of gene 37 were tested at nonpermissive temperatures for production of an antigen that is diagnostic of distal half-fiber assembly. Six of the mutations prevent distal half-fiber assembly. The other five allow assembly of distal half fibers, which combine with proximal half fibers and attach to phage particles, but the resulting phage do not adsorb to bacteria. These two classes of mutations define two adjacent but separate genetic regions, corresponding to two different functional domains in gp37. These two regions and the neighboring gene 38 comprise a functional unit that can be considered as a host-range "cassette," with features that are strikingly similar to corresponding functional units in other unrelated as well as related phages.

TEMPERATURE-SENSITIVE MUTANTS BLOCKED IN THE FOLDING OR SUBUNIT ASSMBLY OF THE BACTERIOPHAGE P22 TAILSPIKE PROTEIN. I. FINE-STRUCTURE MAPPING

Genetics ◽  
1980 ◽  
Vol 96 (2) ◽  
pp. 331-352 ◽  
Author(s):  
Donna H Smith ◽  
Peter B Berget ◽  
Jonathan King
Keyword(s):  
Fine Structure ◽  
Polypeptide Chain ◽  
Cell Attachment ◽  
Physical Map ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Structure Mapping ◽  
Bacteriophage P22 ◽  
P22 Tailspike ◽  
Ts Mutations

ABSTRACT As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.—None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of thes ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein.

scholarly journals In Vivo Bypass of Chaperone by Extended Coiled-Coil Motif in T4 Tail Fiber

2004 ◽  
Vol 186 (24) ◽  
pp. 8363-8369 ◽  
Author(s):  
Yun Qu ◽  
Paul Hyman ◽  
Timothy Harrah ◽  
Edward Goldberg
Keyword(s):  
Bacteriophage T4 ◽  
Coiled Coil ◽  
Critical Factor ◽  
Coiled Coils ◽  
Temperature Sensitive ◽  
Tail Fiber ◽  
Type Gene ◽  
In Vivo Screen

ABSTRACT The distal-half tail fiber of bacteriophage T4 is made of three gene products: trimeric gp36 and gp37 and monomeric gp35. Chaperone P38 is normally required for folding gp37 peptides into a P37 trimer; however, a temperature-sensitive mutation in T4 (ts3813) that suppresses this requirement at 30°C but not at 42°C was found in gene 37 (R. J. Bishop and W. B. Wood, Virology 72:244-254, 1976). Sequencing of the temperature-sensitive mutant revealed a 21-bp duplication of wild-type gene 37 inserted into its C-terminal portion (S. Hashemolhosseini et al., J. Mol. Biol. 241:524-533, 1994). We noticed that the 21-amino-acid segment encompassing this duplication in the ts3813 mutant has a sequence typical of a coiled coil and hypothesized that its extension would relieve the temperature sensitivity of the ts3813 mutation. To test our hypothesis, we crossed the T4 ts3813 mutant with a plasmid encoding an engineered pentaheptad coiled coil. Each of the six mutants that we examined retained two amber mutations in gene 38 and had a different coiled-coil sequence varying from three to five heptads. While the sequences varied, all maintained the heptad-repeating coiled-coil motif and produced plaques at up to 50°C. This finding strongly suggests that the coiled-coil motif is a critical factor in the folding of gp37. The presence of a terminal coiled-coil-like sequence in the tail fiber genes of 17 additional T-even phages implies the conservation of this mechanism. The increased melting temperature should be useful for “clamps” to initiate the folding of trimeric β-helices in vitro and as an in vivo screen to identify, sequence, and characterize trimeric coiled coils.

scholarly journals ISOLATION AND CHARACTERIZATION OF CONDITIONAL ALLELES OF BACTERIOPHAGE T4 GENES uvsX AND uvsY

Genetics ◽  
1984 ◽  
Vol 107 (4) ◽  
pp. 505-523
Author(s):  
Mark A Conkling ◽  
John W Drake
Keyword(s):  
Bacteriophage T4 ◽  
Null Alleles ◽  
Chain Elongation ◽  
Complementation Test ◽  
Temperature Sensitive ◽  
Isolation And Characterization ◽  
Defect Sensitivity ◽  
Dna Chain ◽  
Ionizing Radiations

ABSTRACT The bacteriophage T4 uvsW, uvsX and uvsY gene functions are required for wild-type levels of recombination and for normal survival and mutagenesis after treatments with ultraviolet (UV) and ionizing radiations. The ability of uvsX and uvsY mutations to suppress the lethality of gene 49 mutations was used to select temperature-sensitive and amber alleles of these two genes. (uvsW mutations do not suppress gene 49 mutations.) A simple and powerful complementation test was developed to assist in assigning uvs mutations to genes. The amber alleles of uvsX and uvsY behave as simple null alleles, fully suppressing a gene 49 defect, enhancing UV killing and abolishing UV mutagenesis. However, the properties of the ts alleles of uvsX and uvsY demonstrated that suppression of a gene 49 defect, sensitivity to UV-induced inactivation and UV mutability can be partially uncoupled. These results prompt the hypothesis that radiation mutagenesis occurs during DNA chain elongation past template damage within a recombinational intermediate rather than within a conventional replication fork.

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