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 Identification of Sites Influencing the Folding and Subunit Assembly of the P22 Tailspike Polypeptide Chain Using Nonsense Mutations

Genetics ◽  
1987 ◽  
Vol 117 (2) ◽  
pp. 157-171
Author(s):  
Bentley Fane ◽  
Jonathan King
Keyword(s):  
Polypeptide Chain ◽  
Acid Sites ◽  
Temperature Sensitive ◽  
Primary Defect ◽  
Subunit Assembly ◽  
Temperature Range ◽  
Lethal Phenotype ◽  
Different Temperatures ◽  
P22 Tailspike ◽  
Phage Growth

ABSTRACT Amber mutations have been isolated and mapped to more than 60 sites in gene 9 of P22 encoding the thermostable phage tailspike protein. Gene 9 is the locus of over 30 sites of temperature sensitive folding (tsf) mutations, which affect intermediates in the chain folding and subunit association pathway. The phenotypes of the amber missense proteins produced on tRNA suppressor hosts inserting serine, glutamine, tryosine and leucine have been determined at different temperatures. Thirty-three of the sites are tolerant, producing functional proteins with any of the four amino acids inserted at the sites, independent of temperature. Tolerant sites are concentrated at the N-terminal end of the protein indicating that this region is not critical for conformation or function. Sixteen of the sites yield temperature sensitive missense proteins on at least one nonsense suppressing host. Most of the sites with ts phenotypes map to the central region of the gene which is also the region where most of the tsf mutations map. Mutations at 15 of the sites have a lethal phenotype on at least one tRNA suppressor host. For nine out of ten sites tested with at least one lethal phenotype, the primary defect was in the folding or subunit association of the missense polypeptide chain. This analysis of the tailspike missense proteins distinguishes three classes of amino acid sites in the polypeptide chain; residues whose side chains contribute little to folding, subunit assembly or function; residues critical for maintaining the folding and subunit assembly pathway at the high end of the temperature range of phage growth; and residues critical over the entire temperature range of growth.

METHODS FOR ANALYSIS OF THE C CISTRON OF TEMPERATE PHAGE 16–3 OF RHIZOBIUM MELILOTI

Genetics ◽  
1980 ◽  
Vol 94 (2) ◽  
pp. 265-276
Author(s):  
LÁszló Orosz
Keyword(s):  
Fine Structure ◽  
Rhizobium Meliloti ◽  
Point Mutations ◽  
Cumulative Effect ◽  
Temperate Phage ◽  
Deletion Mutants ◽  
Temperature Sensitive ◽  
Structure Mapping ◽  
Repressor Protein ◽  
Sensitive Allele

ABSTRACT A series of clear mutants of the temperate phage 16-3 of Rhizobium meliloti were isolated that included various point and deletion mutants ofthe Ccistron, coding for the phage repressor. It was observed that recombinant genotypes, such as c+ and ti (temperature-sensitive allele), which form turbid plaques, can be detected quantitatively as lysogenic colonies and scored even at frequencies as low as 10-6. Point mutations, deletions and the autonomy of intracistronic second-site mutations were characterized by this method. Further analysis has shown that each possible pair from three ti mutants gave non-conditional clear recombinants. It was shown that these latter bear the two initial ti mutations, suggesting a cumulative effect of two conditional mutations on the structure of the repressor protein. The double mutants were utilized in fine-structure mapping of the Ccistron.

scholarly journals A genetic map of several mutations affecting the mucopeptide layer ofEscherichia coli

1972 ◽  
Vol 20 (1) ◽  
pp. 65-74 ◽  
Author(s):  
H. J. W. Wijsman
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Fine Structure ◽  
Cell Division ◽  
Genetic Map ◽  
Genetic Relationship ◽  
Ofescherichia Coli ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Temperature Sensitive Mutants

SUMMARYSeveral temperature-sensitive mutants ofEscherichia coliwere isolated which lyse at the restrictive temperature. Some of these possess a biochemically defined lesion in cell-wall mucopeptide synthesis. Three genes, termedmurC, EandF, have been localized between theaziandleumarkers. From transductional data a fine structure map was constructed of themurmutations, establishing the order of the genes. The genetic relationship between these cell wall genes and neighbouring genes involved in cell division is discussed.

Transduction of Streptococcus pyogenes K 56 by Temperature-Sensitive Mutants of the Transducing Phage A 25

1969 ◽  
Vol 24 (12) ◽  
pp. 1556-1561 ◽  
Author(s):  
Horst Malke
Keyword(s):  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Donor Strain ◽  
Wild Type ◽  
Differential Survival ◽  
Group A ◽  
Phage Multiplication ◽  
Time Required ◽  
Ts Mutations ◽  
Virulent Group

Experiments aimed at increasing the efficiency of transduction by the virulent group A streptococcal phage A 25 were carried out using temperature-sensitive phage mutants. Lysates of phages carrying ts mutations proved to transduce more efficiently than wild type lysates, the order of increasing effectiveness being ts+ < tsl ~ ts2 < tsl — 2. Differential survival of the recipient, K 56, appeared to be the principal reason for the improvement of transduction when cells decapsulated by hyaluronidase were transduced at the restrictive temperature. In transduction of encapsulated recipient cells, in which the superiority of ts lysates was less pronounced, the possibility was considered that mutant lysates might contain higher fractions of transducing particles than wild type lysates produced under parallel conditions.The transducing activity of wild type lysates depended strongly on the temperature at which the phage had multiplied on the donor strain, the frequency of transduction increasing with decreasing temperature. Since the minimal latent period of the phage varied roughly in the same way with temperature, a connection is suggested between the time required for phage multiplication and the proportion of transducing particles formed.

scholarly journals GENETIC FINE STRUCTURE OF THE BRONZE LOCUS IN MAIZE

Genetics ◽  
1986 ◽  
Vol 113 (4) ◽  
pp. 1021-1036
Author(s):  
Hugo K Dooner
Keyword(s):  
Thermal Stability ◽  
Fine Structure ◽  
Physical Map ◽  
Chromosome 9 ◽  
Intragenic Recombination ◽  
Coding Region ◽  
Structure Mapping ◽  
Regulation Of Expression ◽  
Average Value ◽  
Genetic Fine Structure

ABSTRACT The bronze (bz) locus in maize, located in the short arm of chromosome 9 (9S), is the structural gene for the anthocyanin biosynthetic enzyme UFGT. The gene has been cloned and its physical map has been oriented relative to the centromere of 9S. We report here the genetic fine structure mapping of several biochemically characterized EMS-induced bz-E mutations, derived from the Bz-W22 isoallele, and Ds insertion bz-m mutations, derived from the Bz-McC isoallele. Two UFGT-, CRM+ mutants (bz-E2 and bz-E5), which genetically identify coding sequences in the gene, and three UFGT-, CRM-  bz-E mutants were mapped against the Ds insertion mutants bz-m1 and bz-m2(DI) by selecting Bz intragenic recombinants from heterozygotes of the type bz-E/bz-m. The exclusive occurrence of one recombinant outside marker class allowed the unambiguous placement of the mutants in a genetic fine structure map. Peculiarly, the two CRM+  bz-E mutants lie upstream of the three CRM-  bz-E mutants and at a considerable genetic distance. The UFGT allozymes encoded by the progenitor alleles Bz-W22 and Bz-McC differ in two properties, thermal stability and activity. The sites responsible for these properties were mapped as unselected markers among the Bz intragenic recombinants. The thermal stability site, which also identifies a coding region of the gene, mapped very close to the CRM+  bz-E mutant sites. The site responsible for variation in activity, which probably identifies a region involved in regulation of expression of the bz locus, mapped at the 5' or proximal end of the locus. It was found to be inseparable from the Ds insertion in bz-m1 that lies very close to the 5' end of the transcribed region.—Evidence was obtained that the insertion of Ds within the bz gene has a suppressing effect on intragenic recombination. Additional data are also presented supporting our observation that Ds affects the pattern of intragenic recombination at bz.—Based on the total genetic length of the bz gene and on the physical size of the transcribed region, we estimate that one unit of recombination at bronze corresponds to 14 kb of DNA. This estimate is more than 100 times smaller than the average value for the whole genome and implies that there may be regions, such as bronze, that serve as hotspots for recombination.

scholarly journals Fine structure genetic and physical map of the gene 3 to 10 region of the bacteriophage P22 chromosome.

Genetics ◽  
1991 ◽  
Vol 127 (4) ◽  
pp. 637-647 ◽  
Author(s):  
S Casjens ◽  
K Eppler ◽  
L Sampson ◽  
R Parr ◽  
E Wyckoff
Keyword(s):  
Physical Map ◽  
Dna Packaging ◽  
Temperature Sensitive ◽  
Genetic Dissection ◽  
Bacteriophage P22 ◽  
Neighboring Gene ◽  
Particle Assembly ◽  
Protein Functions ◽  
Mutant Phenotypes ◽  
Gene 4

Abstract The mechanism by which dsDNA is packaged by viruses is not yet understood in any system. Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging. Only five phage encoded proteins, the products of genes 3, 2, 1, 8 and 5, are required for packaging the virus chromosome inside the coat protein shell. We report here the construction of a detailed genetic and physical map of these genes, the neighboring gene 4 and a portion of gene 10, in which 289 conditional lethal amber, opal, temperature sensitive and cold sensitive mutations are mapped into 44 small (several hundred base pair) intervals of known sequence. Knowledge of missense mutant phenotypes and information on the location of these mutations allows us to begin the assignment of partial protein functions to portions of these genes. The map and mapping strains will be of use in the further genetic dissection of the P22 DNA packaging and prohead assembly processes.

scholarly journals Fine structure mapping and properties of mutations suppressing thelonmutation inEscherichia coliK-12 and B strains

1977 ◽  
Vol 30 (3) ◽  
pp. 273-286 ◽  
Author(s):  
Ben F. Johnson
Keyword(s):  
Escherichia Coli ◽  
Fine Structure ◽  
Growth Conditions ◽  
Temperature Sensitive ◽  
Structure Mapping ◽  
E Coli ◽  
Uv Sensitivity ◽  
Division Process ◽  
Increased Sensitivity ◽  
Derivatives Of

SUMMARYMutations,sulAandsulB, that suppress the UV sensitivity conferred by thelonmutation have been isolated and precisely positioned on the linkage map ofEscherichia coli. TheE. coliB strains Bs-3 and Bs-8 have been shown to possesssulAmutations. Also theE. coliK12 strain J6271 that possesses a suppressor of thelonmutation, previously designated assuf, has been shown to be asulAmutation. A series of methylmethane sulphonate resistant derivatives of anE. coliK12lonstrain has been isolated and genetically characterized. In addition tosulAmutations, a second suppressorsulBwas identified and located betweenleuandazigenes on the chromosome. NeithersulAorsulBmutations result in increased sensitivity to the antibiotics ampicillin, rifampicin, or actinomyein D, nor do they have any significant effect upon the overproduction of mucopolysaccharide caused by thelonmutation. Under some growth conditions thesulBmutation causes cells to be temperature sensitive for the cell division process at 42 °C.

Sign in / Sign up

Export Citation Format

Share Document