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Biomedical subjects

N Sternberg

Publications and source records attributed to N Sternberg.

At least 55 records · Page 3Linked to original sources
PubMed1984

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.

Animals↗
PubMed1984

Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process.

We have constructed phage lambda and plasmid DNA substrates (lambda tk2 and ptk2) that contain two defective herpesvirus thymidine kinase (tk) genes that can be used to detect homologous recombination during the transfer of DNA into mouse L cells deficient in thymidine kinase activity. The recombination event reconstructs a wild-type tk gene and is scored because it converts Tk- cells to Tk+. Using this system, we have shown that (i) both intramolecular and intermolecular homologous recombination can be detected after gene transfer; (ii) the degree of recombination decreases with decreasing tk gene homology; and (iii) the efficiency of recombination can be stimulated 10- to 100-fold by cutting the tk2 DNA with restriction enzymes at appropriate sites relative to the recombining sequences. Based on the substrate requirements for these recombination events, we propose a model to explain how recombination might occur in mammalian cells. The essential features of the model are that the cut restriction site ends are substrates for cellular exonucleases that degrade DNA strands. This process exposes complementary strands of the two defective tk genes, which then pair. Removal of unpaired DNA at the junction between the paired and unpaired regions permits a gap repair process to reconstruct an intact gene.

Bacteriophage lambda↗
PubMed1983

Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination.

Bacteriophage P1 encodes its own site-specific recombination system consisting of a site at which recombination takes place called loxP and a recombinase called Cre. A number of lambda and plasmid substrates containing two loxP sites have been constructed. Using these substrates we have shown both in vivo and in vitro that a fully functional loxP site is composed of no more than 60 bp. In vitro, when an extract containing Cre is used, recombination between loxP sites on supercoiled, nicked-circle or linear DNA occurs efficiently. The most surprising result from the in vitro studies is that 50% of the products of recombination between loxP sites on a supercoiled DNA substrate are present as free supercoiled circles. The ability to produce free products starting with a supercoiled substrate suggests a rather unique property of Cre-mediated lox recombination, the implications of which are discussed in terms of possible effects of the protein on the topology of the DNA molecule.

Bacteriophage lambda↗
PubMed1983

Isolation and characterization of P1 minireplicons, lambda-P1:5R and lambda-P1:5L.

We have isolated two new classes of P1 miniplasmids, called lambda-P1:5R and lambda-P1:5L, by the in vivo extension of a cloned P1 fragment, EcoRI-5, which by itself is not capable of plasmid replication. The lambda-P1:5R plasmids contain EcoRI-5 plus a variable portion of the adjacent P1 EcoRI fragment 8. They have a copy number like that of P1 (about 1 per host chromosome), are faithfully segregated at cell division, and are subject to incompatibility exerted by either a single copy of P1 or a single copy of EcoRI-5. In contrast, the lambda-P1:5L plasmids contain EcoRI-5 and a portion of adjacent P1 DNA that includes at least P1 EcoRI fragments 15, 18, and 23 and a part of fragment 17. These plasmids have a copy number of about 15 per cell chromosome. Despite this they are segregated to daughter cells somewhat less faithfully than are lambda-P1:5R plasmids. They are sensitive to incompatibility exerted by a single copy of P1, but not to incompatibility exerted by a single copy of EcoRI-5. lambda-P1:5L plasmids are, however, sensitive to incompatibility exerted by multiple copies of EcoRI-5. These results show that the relative copy numbers of exerting and responding elements are important for the incompatibility phenotype and strongly suggest that lambda-P1:5L plasmids lack a repressor of replication that can be supplied in trans from P1 but not from EcoRI fragment 5. We suggest that P1 normally uses the 5R replicon and that the 5L replicon may be a backup system that ensures plasmid maintenance should the primary replication event fail to initiate.

Bacteriophage lambda↗
PubMed1982

P1 site-specific recombination: nucleotide sequence of the recombining sites.

Site-specific recombination between molecules of bacteriophage P1 DNA occurs at sites called loxP and requires the action of a protein that is the product of the P1 cre gene. Although recombination between two loxP sites is very efficient, recombination between loxP and a unique site in the bacterial chromosome (loxB) is inefficient and generates two hybrid lox sites called loxR and loxL. We present here the nucleotide sequences of all four lox sites. Analysis of these sequences indicates that (i) a region of extensive homology is not present at the loxP X loxB crossover point, in contrast to the 15-base pair common-core sequence in the bacteriophage lambda att sites, and (ii) the sites contain a region of dyad symmetry with 8- to 13-base pair inverted repeats separated by an 8- to 9-base pair sequence. The loxP X loxB crossover point falls in the sequence that separates the inverted repeats, and deletions that remove either the left or the right inverted repeat of loxP inactivate the site. These two observations are consistent with the conclusion that the region of dyad symmetry is important in los recombination. We have shown further that the loxP X loxP crossover point occurs in a 63-base pair sequence containing the loxP X loxB crossover point, suggesting that, despite the great difference in efficiencies of the two reactions, the crossover points may occur at the same place in both. Explanations for the different recombination properties of the various lox sites are discussed.

Base Sequence↗
PubMed1982

Plastic surgery and civilian casualties due to "terrorist" activities.

Between 1975 and 1979, 511 injuries were caused by "terrorist" explosions in Jerusalem; 340 patients were treated at the Shaare Zedek Medical Center. The injuries were studied and analyzed. The Department of Plastic Surgery bore the brunt of the work, receiving over one-third of all admissions. More than half the hospitalization days went to plastic surgery patients. It seems that when there are large numbers of casualties due to explosions, the plastic surgery service should be enlarged immediately to cope with the large proportion of victims who will be referred to it.

Adolescent↗
PubMed1982

Werner's syndrome as a cause of chronic leg ulcers.

Werner's syndrome is believed to be an autosomal-recessive inherited disorder. Among other manifestations of premature aging, patients with Werner's syndrome frequently develop chronic leg ulcers that heal poorly. We present a patient who suffered from this rare syndrome and developed typical leg ulcers. We wish to alert physicians to this syndrome so that its attendant problems can be considered when treatments are being planned for patients with chronic leg ulcers.

Chronic Disease↗
PubMed1982

Genetic and physical map of a P1 miniplasmid.

The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.

Base Sequence↗