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Biomedical subjects

E L Winnacker

Publications and source records attributed to E L Winnacker.

72 records · Page 4Linked to original sources
PubMed1980

Recombination in adenovirus. I. Analysis of recombinant viruses under non-selective conditions.

A method is described, based on an approach previously employed for the mapping of adenovirus ts mutants (Grodzicker et al. 1975), for the identification of recombinant viruses in the progeny of mixed infections of human HeLa cells with wild-type adenovirus types 2 and 5 under non-selective conditions. Differences in restriction enzyme sites for the endonuclease HpaI could be used to detect new recombinant DNA fragments in a yield of 3 to 6% after single mixed infections and yields up to 25% following three successive mixed infections. The recombinant nature of the new fragments was proved by the clonal isolation of recombinant viruses in similar yields. Analysis of one cloned recombinant pointed to the occurrence of multiple crossover events at different sites along the virus chromosome. The results are discussed in terms of a biochemical approach towards the study of the mechanism of general genetic recombination in human cells as well as with respect to the evolution of adenoviruses.

Adenoviruses, Human↗
PubMed1980

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.

Adenoviruses, Human↗
PubMed1976

Two complementary strand-specific termination sites for adenovirus DNA replication.

Adenovirus type 2 DNA, specifically labeled at the termini for DNA replication, was prepared by isolation of viral DNA molecules which were completed during short pulses with 3H-thymidine. The distribution of radioactivity in the two complementary strands at the termini for DNA replication was determined by liquid phase hybridization and gelelectrophoresis. At the right-hand terminus, nearly all radioactivity was found in the viral h strand, whereas at the left-hand terminus, most radioactivity was confined to the viral l strand. The results suggest that both molecular ends serve as origins and termini for replication of adenovirus type 2 DNA.

Adenoviridae↗
PubMed1976

Isolation and characterization of temperature-sensitive mutants of adenovirus type2.

Fourteen temperature-sensitive mutants of human adenovirus type2, which differed in their plaquing efficiencies at at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts206 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a high-molecular-weight form of viral DNA.

Adenoviridae↗
PubMed1975

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA.

Adenosine Triphosphate↗
PubMed1975

Adenovirus type 2 DNA replication. II. Termini of DNA replication.

Complete, mature adenovirus type 2 DNA molecules were isolated from virus-infected HeLa cells, pulse-labeled at 20 h postinfection in [3H]thymidine pulses shorter than the time necessary for one round of viral DNA replication. After digestion with the restriction endonucleases Eco RI, Hpa I, and Hind III, a temporal order of synthesis of different regions of the viral genome was established from the relative specific radioactivities in the restriction enzyme fragments. A comparison with the physical order of these fragments revealed the existence of two termini of DNA replication towards both the molecular right and left ends, respectively, of the viral chromosome.

Adenoviridae↗
PubMed1973

RNA-linked short DNA fragments during polyoma replication.

During in vitro incubation, nuclei from polyoma-infected cells elongate the daughter strands of the replicative intermediate of polyoma DNA. This process is now shown to involve the transient formation of short fragments (4-5 S), a process that is stimulated by the addition of ribonucleoside triphosphates. The presence of stretches of RNA at the 5'-end of short DNA chains was determined from Cs(2)SO(4) equilibrium centrifugation and from the finding that isotope from alpha-(32)P-labeled deoxynucleoside triphosphates was recovered in 2'(3')-ribonucleotides after alkaline hydrolysis. Transfer occurred preferentially with [alpha-(32)P]dCTP as substrate. Starvation for deoxynucleotides by in vivo treatment with hydroxyurea resulted in the accumulation of short fragments that are deficient in RNA. Our results suggest that a late step during the discontinuous synthesis of polyoma DNA is selectively inhibited when deoxynucleotides are in short supply.

Adenosine Triphosphate↗
PubMedDate not supplied

Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli.

This paper reports the expression of an artificial functional polypeptide in bacteria. The gene of a designed 24-residue DDT-binding polypeptide (DBP) was inserted between the BamHI and PstI cleavage sites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, was cloned in Escherichia coli JM109. After induction by isopropyl-beta-D-thiogalactopyranoside a fusion protein was expressed in which DBP was linked to the COOH-terminus of beta-galactosidase. DBP, which is stable to trypsin, was obtained by tryptic digestion of the fusion protein and subsequent fractionation of the tryptic peptides by reversed-phase h.p.l.c. Recombinant and chemically synthesized DBP showed identical chromatographic properties, amino acid composition, and chymotryptic digestion patterns. Both the beta-galactosidase-DBP fusion and isolated recombinant DBP bound DDT. The fusion protein was 25 times as potent as the designed 24-residue DBP in activating a cytochrome P-450 model system using equimolar catalytic amounts of the two proteins.

Amino Acid Sequence↗
PubMedDate not supplied

[Human cloning. The biological fundamentals and an ethical-legal assessment].

Following a description of the cloning process and how this might be used in humans, the authors examine the possibility of human cloning in the light of recognised ethical principles. They also address the question of whether current national and international laws are sufficient to prevent such practices.

Animals↗