Genetics of host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli

Lederberg, Seymour (Brown University, Providence, R.I.). Genetics of host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli. J. Bacteriol. 91:1029-1036. 1966.-The locus for the host specific restriction and modification of deoxyribonucleic acid in Escherichia coli has been mapped by matings between mutants for these characters in strains K-12, C600, and B. Linkage analysis and kinetics of marker transfer indicate that a single or closely linked multiple chromosomal site located about 4 min counterclockwise to leucine is responsible for these activities. Secondary factors which affect the quantitative level of restriction also were detected. Wild-type recombinants were isolated in crosses between rm(-) (restriction or modification, or both) mutants. The expression in zygotes of the restrictionless character of a rm(-) donor is masked by a separate, physiological impairment of restriction, which results from mating and is independent of the modification state of the donor. The relevance of the restriction character to mating incompatibilities in these and other bacterial strains is considered.     

Protection of DNA damage by dietary restriction.

Dietary restriction is known to retard the aging processes and delay the onset of age-related neoplastic diseases. The mechanisms underlying these remarkable actions of nutritional intervention are not known in spite of recently intensified research efforts. However, the last couple of years' research on dietary restriction produced strong evidence indicating that its effective antiaging actions might be related to its ability to modulate free radical damage. In the present study, DNA damage and attenuation of the damage by dietary restriction were assessed by measuring 8-hydroxydeoxyguanosine 8-OH dG) in both nuclear DNA (nuDNA) and mitochondrial DNA (mitDNA) fractions. The data show that substantially more damage (approximately 15 times) occurred in mitDNA compared to nuDNA. More interestingly, the DNA damage was significantly attenuated in dietarily restricted rats.     

Mechanism of foreign DNA selection in a bacterial adaptive immune system

In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary "nonself" DNA sequences for destruction, while avoiding binding to "self" sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for nonself target recognition and binding. Combining a 2.3 ? crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved three base pair motif that is required for nonself target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade.     

Protection of mice against bacterial infection by oral administration of bacterial lipopolysaccharide

The effect of orally administered bacterial lipopolysaccharide (LPS) on host resistance against bacterial infections was studied. LPS orally given for 5 consecutive days prior to infection caused no apparent toxic effect and protected mice against Pseudomonas aeruginosa and Listeria monocytogenes infections.     

Detection in situ of foreign DNA in eukaryotic cells

A simple technique is described that allows mixed populations of eukaryotic cells to be screened for clones containing multiple copies of a particular DNA. Essentially, eukaryotic cells are transferred to either nitrocellulose of Whatman 541 filters, and their DNA is immobilised in situ. Exposure of the filters to a 32P-labeled DNA "probe" results in detectable hybridisation only at the positions of clones containing multiple copies of the DNA. Using Whatman 541 paper, a portion of the cells, evenly distributed throughout the mixed population is retained on the culture dish, and can be propagated further for subsequent cell cloning. The technique has allowed rapid distinction of clones of transformed rat cells that contain a single or only a few copies per cell of polyoma viral DNA from clones maintaining multiple copies. The technique has also been used to distinguish between clones of mouse L-cells containing multiple and only a few copies of 0X174 DNA. In this manner the technique allows rapid detection of cells amplifying a particular species of DNA. Finally, the method can be used to detect cells assimilating many copies of a foreign DNA, even in the absence of a co-transfected selectable marker.     

Direct visualization of plasmid DNA in bacterial cells

The direct visualization of plasmid DNA inside Escherichia coli cells is demonstrated using phase-fluorescence microscopy of DAPI (4',6-diamidino-2-phenylindole)-stained bacteria. Small as well as large plasmids could be detected, both in minicells and in cells of larger size. For large plasmids, even single molecules appeared to be within the detection limit. The fluorescence generated from monomers of small plasmids was probably below this limit, and for these plasmids the observed signals may represent aggregates. The distribution of the fluorescence foci might reflect specific plasmid positioning during partition and/or replication.     

Introduction of host-controlled modification and restriction systems of Bacillus subtilis IAM1247 into Bacillus subtilis 168.

Bacillus subtilis IAM1247 had two modification and restriction systems (Bsu1247I and Bsu1247II), the former producing an isoschizomer of PstI endonuclease. A transformant clone was isolated which had Bsu 168, BsuR, and Bsu1247I systems coexisting within a genome.     

Virus-mediated transfer of foreign DNA into taste receptor cells

Foreign genes can be transferred into taste cells via adenoviral vectors. The present study was undertaken to characterize the subpopulation of taste cells that are susceptible to adenovirus infection and to determine whether another viral vector, derived from herpes simplex 1 (HSV-1), infects the same subpopulation of taste cells. Using an adenovirus containing the gene for enhanced green fluorescent protein (EGFP) under the control of the human cytomegalovirus (CMV) immediate early promoter, we found that EGFP was present in blood group antigen H immunoreactive (ir) taste cells, but not in gustducin-ir or PGP 9.5-ir cells. Infection of taste buds with an HSV-1 vector containing EGFP also resulted in a subpopulation of EGFP-positive taste cells. However, both gustducin-ir and PGP 9.5-ir taste cells expressed the marker protein. In conclusion, this study shows that both adenoviral and HSV-1 vectors can be used to transfer foreign genes into the cells of isolated rat taste buds and that different viruses can be used to target specific subpopulations of taste cells.