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Biomedical subjects

S Linn

Publications and source records attributed to S Linn.

At least 181 records · Page 10Linked to original sources
PubMed1980

Apurinic/apyrimidinic endonucleases in repair of pyrimidine dimers and other lesions in DNA.

The characteristics of the nicks (single-strand breaks) introduced into damaged DNA by Escherichia coli endonucleases III, IV, and VI and by phage T4 UV endonuclease have been investigated with E. coli DNA polymerase I (DNA nucleotidyltransferase). Nicks introduced into depurinated DNA by endonuclease IV or VI provide good primer termini for the polymerase, whereas nicks introduced into depurinated DNA by endonuclease III or into irradiated DNA by T4 UV endonuclease do not. This result suggests that endonuclease IV nicks depurinated DNA on the 5' side of the apurinic site, as does endonuclease VI, whereas endonuclease III has a different incision mechanism. T4 UV endonuclease also possesses apurinic endonuclease activity that generates nicks in depurinated DNA with low priming activity for the polymerase. The priming activity of DNA nicked with endonuclease III or T4 UV endonuclease can be enhanced by an additional incubation with endonuclease VI and, to a lesser extent, by incubation with endonuclease IV. These results indicate that endonuclease III and T4 UV endonuclease (acting upon depurinated and irradiated DNA, respectively) generate nicks containing apurinic/apyrimidinic sites at their 3' termini and that such sites are not rapidly excised by the 3' leads to 5' activity of DNA polymerase I. However, endonuclease IV or VI apparently can remove such terminal apurinic/apyrimidinic sites as well as cleave on the 5' side of the unnicked sites. These results suggest roles for endonucleases III, IV, and VI in the repair of apurinic/apyrimidinic sites as well as pyrimidine dimer sites in DNA. Our results with T4 UV endonuclease suggest that the incision of irradiated DNA by T4 UV endonuclease involves both cleavage of the glycosylic bond at the 5' half of the pyrimidine dimer and cleavage of the phosphodiester bond originally linking the two nucleotides of the dimer. They also imply that the glycosylic bond is cleaved before the phosphodiester bond.

Apurinic Acid↗
PubMed1980

Coliphage HK243: biological and physicochemical characteristics.

Coliphage HK243 can form plaques on Escherichia coli C and K-12, but not B. The plaques are 1-2 mm in diameter and are opaque areas which clear upon exposure to chloroform vapor. During one-step growth, the eclipse and the latent periods are 20 and 30 min, respectively. Phage-infected cells continue to produce cell-free plaque-forming units for as long as 80 min after the end of the latent period, although at high multiplicities of infection (MOI) most cells lyse. No lysogenic bacteria have been found among survivors, so HK243 is considered a virulent phage. Some of the cells surviving a high MOI challenge are maltose negative and resistant to both HK243 and coliphage lambda. This fact has made possible the isolation of lambda-resistant mutants of lambda-lysogens. However, no serological cross-reaction between the phages lambda and HK243 has been detected. Genetic data involving three essential loci and a locus controlling plaque morphology suggest a circular linkage map. The virions are tadpole-shaped with an icosahedral head 68 nm long which is attached to a flexible tail 131 nm long. The phage has a linear, duplex DNA genome of molecular weight approximately 44 x 10(6) and a base composition of 33% adenine, 31% thymine, 16% guanine, and 20% cytosine.

Coliphages↗
PubMed1980

A microstereotactic approach to small CNS lesions. Part I. Development of CT localization and 3-D reconstruction techniques.

The authors describe a newly designed and utilized stereotactic methodology for the removal of central nervous system lesions as small as a few millimeters in diameter. These small lesions are detected and localized by non-invasive computerized axial tomography (GE 8800 scanner) with additional computer processing of the digital data by means of a PDP-1145 computer. Multiple computer algorithms have been developed to enhance regions of interest on CT scans by three-dimensional reconstruction and magnification techniques. This same data can then be used to calculate a stereotactic approach to a small CNS lesion. The stereotactic approach coordinates are then mated to a head fixation system modified from the Reichert-Mundinger stereotactic apparatus enabling information transfer from CT scan to the stereotactic surgical system. These small CNS lesions, as small as 5 mm, can be removed with the apparatus described herein under direct binocular 3-D vision with minimal tissue damage, through a small trephine craniotomy or burr hole. Newly designed instruments and instruments in the process of being developed are mounted on a micromanipulator attached to the Riecher-Mundinger frame for guidance at surgery. These new instruments include stereoendoscopes with xenon arc illumination, multiple tissue expanders for exposing the operative site, a radiation tracer probe, a rotary-sucker extractor, and multiple other small instruments for operating and removal of blood and tissue from small CNS lesions. The stereotactic frame accurately defines all areas of the cranium in three-dimensional coordinates, and its combination with the micromanipulator-instrument assembly enables the site of any small CNS lesion, the three-dimensional coordinates of which have been located by additional computer processing of the CT digital data, to be accurately approached by the stereotactic guide micromanipulator assembly. The instrument described herein allows removal of very small tumor burdens, and opens the possibility for successful secondary application of adjuvant immunotherapy to a CNS tumor site as described in part II of this paper.

Brain↗
PubMed1979

Further characterization of a depurinated DNA-purine base insertion activity from cultured human fibroblasts.

The purification from cultured human fibroblasts of a protein that binds specifically to partially depurinated DNA and inserts purines into those sites is described. The purine insertion, but not the binding, requires K+. The DNA binding can be saturated with increasing apurinic sites and is weakened by the presence of adenine or guanine. Base insertion into depurinated DNA is specific for adenine or guanine; none is observed with dATP or dGTP. When the depurinated DNA substrate is specifically cleaved with apurinic endonuclease, no purine insertion occurs. Guanine insertion does not occur into tRNA or depyrimidinated DNA, and thymine is not inserted into either depyrimidinated DNA or depurinated DNA. Purine insertion activity follows Michaelis-Menten kinetics with respect to purintes; the apparent Km values for both adenine and guanine are 5 microM. The enzyme binds the purine bases very tightly. Adenine binding saturates at less than 1 microM adenine, perhaps reflecting the low intracellular adenine concentration. The binding protein specific for UV-irradiated DNA (Feldberg, R.S., and Grossman, L. (1976) Biochemistry 15, 2402-2408) had no detectable purine or pyrimidine base insertion activity with depurinated or depyrimidinated DNAs.

Apurinic Acid↗
PubMed1979

Further characterization of a cell-free system for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the involvement of DNA polymerase alpha in DNA repair.

DNA repair synthesis can be specifically measured in osmotically opened, confluent cultured human fibroblasts after exposure to DNA damaging agents such that both induction and mediation of DNA repair synthesis can take place in this cell-free system. Alternatively, by utilizing osmotically shocked, log phase cells and altering the DNA precursors, pH and ionic strength, replicative DNA synthesis can be specifically monitored. Autoradiographic studies show that virtually all of the nuclei from the lysates of the confluent, UV-iradiated cells are lightly labeled in the fashion characteristic of DNA repair. By contrast, only a fraction of nuclei is labeled in a population of unperturbed, opened log phase cells and the labeling is heavy and characteristic of replicative synthesis. Furthermore, equilibrium density gradient sedimentation shows that DNA synthesis in lysates of log-phase cells is semiconservative, whereas that with UV-irradiated cells is repair synthesis. This open cell system has been used to study the enzymology of DNA repair. Thus, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerases beta and gamma, does not inhibit either replicative or repair synthesis. By contrast, aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits DNA repair and replicative synthesis in both intact and permeabilized cells. Finally, phage T4 UV-exonuclease stimulates repair synthesis, but only when phage T4 UV-endonuclease is also added to the UV-irradiated nuclei.

4-Nitroquinoline-1-oxide↗
PubMed1979

Survival of apurinic SV40 DNA in the d-complementation group of xeroderma pigmentosum.

The survival of depurinated Form I SV40 DNA was studied in normal human fibroblasts and in D-complementation Xeroderma pigmentosum (XP) fibroblasts. Survival was measured with an infective center assay. Heat-acid and methyl methanesulfonate (MMS) were used as depurinating agents. After 3 hrs of depurination by heat--acid treatment, infectivity in normal cells was less than 15% of the controls compared to more than 50% for the XP D cell strains. Similar results were obtained with MMS-treated DNA. These results are contrary to expectation since apurinic endonuclease activity, which is presumed to be involved in the repair of apurinic sites, is much lower in XP D cell strains than in normal cell strains. Our results indicate that another mechanism for the repair of apurinic sites could exist.

Cell Line↗
PubMed1979

DNA binding activity from cultured human fibrolasts that is specific for partially depurinated DNA and that inserts purines into apurinic sites.

A protein of molecular weight approximately 120,000 was isolated from cultured human fibroblasts and HeLa cells on the basis of its ability to bind specifically to apurinic DNA. After separation from apurinic endonuclease activity, the protein was found to incorporate purine, but not pyrimidine, bases specifically into depurinated DNA so as to protect the apurinic sites from alkali. Purine base insertion activity was sensitive to heating and freezing as well as to caffeine and EDTA; it required K+ but not a divalent cation. Guanine, but not adenine, was incorporated into depurinated poly(dG-dC), whereas adenine, but not guanine, was incorporated into poly(dA-dT). After incorporation into depurinated DNA, guanine could be reisolated as dGMP. Although this activity suggests an alternative pathway for DNA repair that is independent of nucleotide excision, other functions for such an enzyme are possible.

Apurinic Acid↗
PubMed1978

A cell-free assay measuring repair DNA synthesis in human fibroblasts.

Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of UV-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methyl methanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of UV-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro.

Carcinogens↗
PubMed1978

Recognition site of Escherichia coli B restriction enzyme on phi XsB1 and simian virus 40 DNAs: an interrupted sequence.

Methyl groups placed on varphiXsB1 replicative form DNA by the Escherichia coli B modification enzyme are located in the overlap between fragments Mbo II-3 and Alu I-2, a 61-base-pair DNA segment. Mutations that led to loss of susceptibility to restriction by E. coli B occurred within this segment at three positions spanning 14 nucleotides. A sequence difference between varphiXsB1 and varphiXam3cs70, a varphiX174 strain not restricted by E. coli B, occurs at one of these positions. The site on simian virus 40 DNA methylated by the modification enzyme is located in the 115-base-pair overlap between fragments Hae III-I and Alu I-G. The sequences of these segments of varphiXsB1 and simian virus 40 DNA and two regions of phage f1 DNA recognized by the E. coli B restriction enzyme [Ravetch, J. V., Horiuchi, K. & Zinder, N. D. (1978) Proc. Natl. Acad. Sci. USA 75, 2266-2270] contain a homology of nine bases in the configuration:5'-T-G-A... 8N... T-G-C-T... 9N... T-N-N-T-3'. The sequence 5'-T-G-A... 8N... T-G-C-T-3' may constitute the restriction enzyme recognition site since it does not occur in varphiXam3cs70 DNA and occurs only once in simian virus 40 DNA, and since all observed mutations leading to loss of the site occur at one of the bases specified by this sequence. Analysis of the sequence of varphiXam3cs70 showed that if no other residues are recognized, all seven of these bases are essential for recognition and the interval between the two groups of specified bases must be precisely eight.

Base Sequence↗
PubMed1978

Successive visual discrimination of forms in 10-month-old infants.

The purpose of this research was to assess the infant's ability to make successive discriminations in the size of a figure. The Ss were 90 10-month-old infants who were habituated to a block figure and dishabituated on different subtle size transformations of that figure. Dishabituation data revealed that the infants were able to detect transformations which increased the area of the habituated figure by 7 percent. There was no evidence of a differentiated sensitivity to detect a change in any special location of the original figure.

Differential Threshold↗