Tryptophan-transducing bacteriophages: in vitro studies with restriction endonucleases HindII + III and Escherichia coli ribonucleic acid polymerase.

The HindII + III restriction enzyme fragmentation pattern of various lambda-phi80trp deoxyribonucleic acid molecules is presented. An analysis of deoxyribonucleic acid molecules carrying deletions ending within the trp regulatory elements and a deoxyribonucleic acid molecule carrying a deletion within trpE indicates that a fragment of 8.3 X 10(5) daltons contains at least part of the trp promoter, the entire trp leader region, and part of the trpE gene. The observation that ribonucleic acid polymerase, when present in the HindII + III digestion mixture, results in the fusion of this 8.3 X 10(5)-dalton fragment to the preceding bacterial fragment suggests that HindII + III cuts within trpP.     

Estimated prevalence of Aerococcus viridans and Anophryoides haemophila in American lobsters Homarus americanus freshly captured in the waters of Prince Edward Island, Canada.

The Canadian lobster industry holds lobsters Homarus americanus in captivity for various periods to supply markets with live product year-round. Mortality during holding results in considerable losses, estimated at 10 to 15 % yr(-1) by the industry. This study examined the prevalence of Anophryoides haemophila and Aerococcus viridans, causative agents of 'bumper car' disease and gaffkemia, respectively, in lobsters freshly captured in the waters of Prince Edward Island during the spring and fall fishing seasons of 1997. A total of 116 lobsters were sampled in the spring, and 138 in the fall. A. haemophila was not detected in the spring, while the prevalence was 0.72 % in the fall with a 95% confidence interval (CI) of 0.02 to 3.97% and an overall prevalence of 0.39% (95% CI: 0.01 to 2.17%). The prevalence of A. viridans was estimated at 6.9% (95% CI: 3.0 to 13.14%) in the spring, 5.8% in the fall (95% CI: 2.54 to 11.10%), and 6.30% overall (95% CI: 3.64 to 10.03%). Because of the reduced interest in food of diseased lobsters, and compromised metabolism in the case of gaffkemia, these prevalence estimates are likely underestimates of the true prevalence of gaffkemia and 'bumper car' disease in the wild populations of lobster around Prince Edward Island.     

Comparative analysis of mitochondrial genome data for Necator americanus from two endemic regions reveals substantial genetic variation

Necator americanus is a blood-sucking, intestinal nematode of major human health importance in many tropical and subtropical regions of the world. The aim of the present study was to compare the complete mitochondrial genome sequence from one N. americanus individual from Togo with another from China, in order to estimate the magnitude of genetic variability for different mitochondrial genes and non-coding regions. For the 12 protein genes, this comparison revealed sequence differences at both the nucleotide (3-7%) and amino acid (1-7%) levels. The most conserved of these was the nad4L gene, whereas the nad1 gene was least conserved at both the nucleotide and amino acid levels. Nucleotide differences were also detected in 14 of the 22 transfer RNAs (trns) (1-13%), the AT-rich region ( approximately 8%), non-coding regions (8-25%) and in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rrn) ( approximately 1%). Comparison of the rrnL sequences among multiple individual worms revealed nine unequivocal nucleotide differences between N. americanus from the two countries. Consistent with previous studies, these findings provide evidence for substantial genetic variation within N. americanus, which may have implications for the transmission and control of hookworm disease.     

Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division.

The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.     

Ultraviolet Sensitivity of Bacillus subtilis Spores upon Germination and Outgrowth

A strain of Bacillus subtilis, UVSSP-42-1, which produces ultraviolet (UV)-sensitive spores and vegetative cells, was found to possess germinated spores 25 times more UV resistant than the resting spores. This relative resistance achieved upon germination was associated with the transition of the heat-resistant refractile spores to the heat-sensitive phase-dark forms. Several generations of outgrowth were required before the cells attained the level of UV sensitivity characteristic of the vegetative cell. The UV sensitivity of germinated spores was compared with other strains with various combinations of mutations affecting deoxyribonucleic acid repair capabilities. The presence of hcr and ssp mutations which are known to abolish the removal of photoproducts from deoxyribonucleic acid did not alter significantly the sensitivity of the germinated forms. However, the addition of the recA mutation and, to some extent, the pol mutation increased the UV sensitivity of the germinated spores. These results indicate that deoxyribonucleic acid repair mechanisms dependent on the recA gene are active in the germinated spores. The chemical nature of the damage repaired by the recA gene product is not known. This study indicates that the life cycle of sporulating bacilli consists of at least three photobiologically distinct forms: spore, germinated spore, and vegetative cell.     

Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids.

Three I-like conjugative plasmids, ColIdrd1, R144drd3, and R64drd11, which are derepressed for functions involved in conjugation, were found to suppress at least partially the phenotype of temperature-sensitive dnaG mutants of Escherichia coli K-12, as judged from the kinetics of deoxyribonucleic acid synthesis at elevated temperature in newly formed and established plasmid-containing strains. In contrast, the corresponding wild-type plasmids and three F-like derepressed conjugative plasmids, F101, R100drd1, and R1drd16, all failed to suppress. Suppression is presumably caused by a different plasmid-determined function from that which promotes survival of ultraviolet-irradiated bacteria, because both the wild-type I-like plasmids and their drd mutants protected irradiated bacteria. One possible interpretation of these results is that the product of a gene carried by certain I-like plasmids can substitute for the bacterial dnaG gene product during ongoing deoxyribonucleic acid replication.     

Bacterial Cell Division Regulation: Characterization of the dnaH Locus of Escherichia coli

The dnaH locus is the fourth gene to be identified as required for deoxyribonucleic acid polymerization in Escherichia coli. A temperature-sensitive mutant defective in this gene exhibited an abrupt decrease in rate of deoxyribonucleic acid synthesis when shifted to 42 C. The locus mapped in the proC-purE region of the chromosome by conjugation and was co-transducible with purE. dnaH(+) is carried on the F'(13) episome and is dominant over the dnaH(-) mutation.     

Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization.

A mutation in the Escherichia coli gene for single-strand binding protein results in temperature-sensitive deoxyribonucleic acid replication (R. R. Meyer, J. Glassberg, and A. Kornberg, Proc. Natl. Acad. Sci. U.S.A. 76:1702-1705, 1979). The mutant (ssb-1) is also more sensitive to ultraviolet irradiation and about one-fifth as active in recombination. Single-strand binding protein is thus implicated in repair and recombination as well as in replication. The mutation in ssb is located between uvrA and melA at 90.8 min on the genetic map. The ssb gene appears to be allelic with lexC, a gene with a proposed role in regulating inducible deoxyribonucleic acid repair.     

Mating Reaction in Saccharomyces cerevisiae. IV. Retardation of Deoxyribonucleic Acid Synthesis

When a and a type haploid cells of Saccharomyces cere-visiae were mixed and cultured, deoxyribonucleic acid synthesis was retarded but ribonucleic acid and protein syntheses were not. It was found that culture filtrate of a type cells inhibited deoxyribonucleic acid synthesis of a type cells and that of a type cells inhibited that of a type cells. Thus, sex-specific diffusible substances secreted by opposite mating type cells are thought, at least partly, to be responsible for the retardation of deoxyribonucleic acid synthesis.     

Identification of a sporulation locus in cloned Bacillus subtilis deoxyribonucleic acid.

A cloned deoxyribonucleic acid from the purA-cysA region of the Bacillus subtilis chromosome was shown to contain the spoVC locus, a gene whose product is required for sporulation. This is the first demonstration of a spo locus in cloned B. subtilis deoxyribonucleic acid.     

Superinfection in Bacteriophage S13 and Determination of the Number of Bacteriophage Particles Which Can Function in an Infected Cell

Bacteriophage S13 shows exclusion of superinfecting homologous phage, but the exclusion is only partial. The superinfecting phage can form infectious replicative form deoxyribonucleic acid (RF), can direct protein synthesis, and can form progeny particles even at a superinfection time as late as 60 min after the first infection. Exclusion is also only partial for the closely related phage phiX174. Seven min after the first infection, the exclusion mechanism begins to operate, requiring continuous phage-specified protein synthesis. The gene A protein (required for synthesis of progeny RF) appears to be involved in the exclusion mechanism. In superinfection experiments, it was found that at least 40 phage particles per cell can replicate and can carry out protein synthesis, though the number of sites for binding of RF to the membrane is only about 15 per cell. The results suggest that attachment of RF to a binding site is not required for protein synthesis. Evidence is presented that non-attached parental RF can serve as a template for single-stranded deoxyribonucleic acid synthesis.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Isolation and Characterization of Mutants of Colicin Plasmids E1 and E2 After Mu Bacteriophage Infection

Cells colicinogenic for the colicin plasmids E1 or E2 (Col E1 and Col E2, respectively) were selected for a loss of colicin production after infection with bacteriophage Mu. Extrachromosomal deoxyribonucleic acid that was larger than the original colicin plasmids was found in such cells. A small insertion mutant in Col E1 deoxyribonucleic acid affecting active colicin production without affecting either expression of colicin immunity or Col E1 deoxyribonucleic acid replication was found. Cells carrying this Col E1 plasmid mutant do not exhibit the lethal event associated with colicin E1 induction, suggesting that synthesis of active colicin is required for killing during induction. The altered Col E2 plasmid, containing an insertion at least as large as phage Mu, was maintained unstably in the mutants examined.     

Intracisternal A-particle genes in Mus musculus: a conserved family of retrovirus-like elements.

The structural organization of intracisternal A-particle genes has been studied, using isolates from a mouse gene library in lambda phage Charon 4A. The predominant gene form among the isolates was 7.3 kilobases (kb) in length. R-loops between the 7-kb (35S) A-particle genomic ribonucleic acid and several of these genes were colinear, with no visible evidence of intervening deoxyribonucleic acid sequences. One recombinant was found with an A-particle gene that contained a 1.7-kb deletion. Using the deletion as a reference, the deoxyribonucleic acid and ribonucleic acid homology regions were localized with respect to one another and to the restriction map: the 5' terminus of the ribonucleic acid was several hundred base pairs within the 5' end of the deoxyribonucleic acid homology region. Restriction endonuclease fragments encompassing the 5' and 3' regions of one 7.3-kb gene were separately subcloned into pBR322. Heteroduplexes between the two subclones revealed an approximately 300-base pair segment of terminally redundant sequences. The cloned 3' fragment hybridized with restriction fragments from the 5' end of several other A-particle genes, demonstrating the presence of common (though not necessarily identical) terminally repeated sequences. A-particle genes varied in the occurrence of specific restriction sites at characteristic internal loci. However, heteroduplexes between several variant 7.3-kb genes showed continuous homology regions even when spread under stringent hybridization conditions. The relative abundance of restriction site variants was highly conserved in 12 laboratory strains of Mus musculus, in embryonic and adult tissues of a single inbred strain, and in the SC-1 cell line of feral mouse origin, but appeared to differ in a feral Japanese substrain, Mus musculus molossinus. Some evidence suggests that subsets of A-particle genes may have similar flanking sequences. The results are discussed in terms of the evolution of this multigene family.     

Excision of bacteriophage lambda from a site in the arabinose B gene.

A lambda lysogen with the prophage inserted into the arabinose B gene of Escherichia coli strain K-12 has been prepared. Induction of the phage from this lysogen yields viable phage at a frequency 4 X 10(-6) that found for induction of lysogens with phage inserted at the normal attachment site. Over 30% of the phage particles induced from the insertion in ara are arabinose-transducing phage. The excision end points of 62 independently isolated, nondefective araC-transducing phage containing less than the entire araC gene were genetically determined and were found to be randomly distributed through the araC gene. The amount of arabinose deoxyribonucleic acid contained on four selected transducing phage was determined by electron microscopy of deoxyribonucleic acid heteroduplexes, providing a physical map of the araC gene. The efficiency with which these phage transduce araC and araB point mutations was found to be approximately proportional to the homology length available for recombination.