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Biomedical subjects

P Berg

Publications and source records attributed to P Berg.

At least 235 records · Page 13Linked to original sources
PubMed1979

Construction and analysis of viable deletion mutants of polyoma virus.

Viable mutants of polyoma with small deletions ranging in size from 2 to 75 base pairs were obtained by infecting 3T3 cells with polyoma DNA that had been cleaved once with HaeII endonuclease or with DNase-Mn2+ digestion. The HaeII endonuclease-cleaved DNA yielded mutants with deletions at map position 72--73, whereas the mutants generated by DNase I-Mn2+ digestion had deletions either at map position 72--73 or within the map coordinates 92 and 99. Both groups of mutants appeared to grow as well as wild-type virus in 3T3 cells. The deletions at map position 72--73 did not alter the virus's ability to transform rat cells. Hence, the region just to the early side of the origin of DNA replication is not essential for vegetative growth or transformation. But the mutants with deletions in the region between map coordinates 92 and 99, a segment thought to code for polyoma large and middle T antigens (Hutchinson et al., Cell 15:65--77, 1978; Smart and Ito, Cell 15:1427--1437, 1978; Soeda et al., Cell 17:357--370, 1979), transformed rat cells at 0.2 to 0.05 the efficiency of wild-type virus.

Animals↗
PubMed1979

The physical and genetic organization of a viral genome.

Mutants of SV40 with deletions of a few to several thousand base pairs have been constructed in vitro and cloned in cultured monkey cells. The location and size of these deletions has been determined by restriction endonuclease mapping and electron microscopic and enzymatic analysis of DNA heteroduplex molecules. Analysis of the phenotype of these deletion mutants permits us to specify the locations of the known SV40 genes, in particular, the novel organization of SV40s two early genes that are required for oncogenesis.

Antigens, Viral↗
PubMed1978

Altering the specificity of restriction endonuclease: effect of replacing Mg2+ with Mn2+.

In the presence of 100 mM Tris buffer (pH 7.5) and 1-10 mM Mg2+ EcoRI endonuclease cleaves DNA at a specific nucleotide sequence and in a characteristic way: -GAATTC-. But if Mg2+ is replaced by Mn2+, the specificity of the cleavage is relaxed and cleavages occur at many other sites; moreover, there appears to be a hierarchy of cleavage rates at the pseudo-EcoRI restriction sites. For example, SV40 DNA is cleaved only once in the usual digestion conditions, but with Mn2+ more than ten cleavages are made; the five most rapidly cleaved SV40 DNA map locations are 0/1.0 larger than 0.93 larger than 0.33 approximately equal to 0.42 larger than 0.29 approximately equal to 0.40 larger than 0.25. Mn2+ also alters the restriction specificity of HindIII but not HpaII endonuclease.

DNA Restriction Enzymes↗
PubMed1978

Organization and expression of early genes of simian virus 40.

The early region of simian virus 40 codes for at least two immunologically related polypeptides: large-T and small-t, with apparent molecular weights of 90,000-100,000 and 15,000-20,000, respectively. Because small-t shares methionine-containing tryptic peptides with large-T, the two polypeptides are probably coded, in part, by a common nucleotide sequence. To locate the coding sequences for large-T and small-t in the DNA, the production of these proteins was examined after infection of CV-1 cells with wild-type and deletion mutants of simian virus 40. We found that a deletion at the distal portion of the early region alters the structure of large-T but not of small-t; but deletions within the region between map coordinates 0.59 and 0.55 result in an alteration or absence of small-t and a normal large-T. These findings have been rationalized by a model that proposes the existence of two early mRNAs, one coding for large-T and the other for small-t. Both mRNAs span virtually the entire early region; but the mRNA coding for large-T lacks the nucleotide sequence between map coordinates 0.59 and 0.54. We suggest that small-t is translated from the larger of the two mRNAs, beginning at or near its 5' end and terminating at a termination codon at about map coordinate 0.54. Larger-T, on the other hand, is translated from the shorter mRNA, beginning at the same initiator codon, and, because of the deletion of the terminator codon at 0.54, translation proceeds to the terminator codon at or near map position 0.18.

Antigens, Viral↗
PubMed1978

Excision of DNA segments introduced into cloning vectors by the poly(dA-dT) joining method.

A method is described for excising cloned DNA segments that have been inserted into their vectors by poly(dA-dT) joins. The recombinant DNA is cleaved within the vector DNA portion by one or more restriction endonucleases to generate a linear DNA molecule with the insert DNA sequence flanked by the poly(dA-dT) joins. After denaturation, the single strands "snap back" because of the intrastrand poly(dA) and poly(dT) sequences to form circular structures with "tails" of vector DNA. The vector portion of the DNA is then digested by Escherichia coli exonuclease VII, while the insert portion remains resistant to attack. The resistant strands are annealed and purified by electrophoresis in agarose. The insert DNA segment free of contaminating vector sequences can be used as a hybridization probe and for insertion into a new vector since suitable cohesive termini are generated from the retained poly(dA) and poly(dT) tails by an appropriate exonuclease.

DNA, Bacterial↗
PubMed1978

New region of the simian virus 40 genome required for efficient viral transformation.

Viable mutants of simian virus 40 with deletions in three regions of the virus genome (map coordinates 0.21-0.17, 0.59-0.54, and 0.67-0.74) have been tested for their ability to transform rat fibroblasts to anchorage independence. Only those mutants whose deletions occur between 0.59 and 0.55 in the proximal part of the early region are defective in transforming ability. The most severely defective of these transform with less than one-hundredth the efficiency of wild type. They retain their defect when tested in Chinese hamster lung cells and when infection is initiated with viral DNA instead of intact virions. Complementation for transformation can be observed between these transformation-defective deletions and a simian virus 40 temperature-sensitive A mutant.

Cell Line↗
PubMed1978

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and Hölzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.

Cell Line↗
PubMed1978

[Not Available].

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Dentistry↗
PubMed1978

[Comparative studies of caries epidemiology using skulls from the Iron, Slavic, and Middle Ages as well as modern subjects (author's transl)].

The present paper compares the incidence and intensity of caries (DMF caries index) determined on skulls from the Iron, Slavic, and Middle Ages with corresponding results obtained for a modern civilized population. More specifically, the results reported in this paper were obtained from clinical and roentgenological examinations of 371 skulls from the ages referred to above as well as of 10,340 inhabitants of the city of Rostock. The results indicate a marked increase in cariosity between the Iron Age and the present time. This increase in the incidence and intensity of caries is considered to be due predominantly to the abrasion resulting from the then preparation and composition of food (which contained as much as 3 percent of stone dust), whereby an essential point of predilection for caries is removed.

Adolescent↗
PubMed1977

Effects of cytosine arabinoside, 6-aminonicotinamide, and 6-mercaptopurine riboside on ectoderm and mesoderm of mouse limb buds.

The effects of cytosine arabinoside, 6-aminonicotinamide, and 6-mercaptopurine riboside on the incorporation of [14C] glucose moieties and [32P] phosphate into acid-soluble material and lipids, RNA, DNA, and protein were measured in the dissected mesoderm and ectoderm of mouse limb buds at the 42-45 (day 11) somite stage. Due to the different proliferative capacities of the two tissues the incorporation of the precursors into mesodermal cells was considerably higher the than into ectodermal ones. Cytosine arabinoside inhibited the incorporation of the precursor moieties only into DNA, but very early after its application. This effect was more obvious in mesoderm than ectoderm. 6-Aminonicotinamide interfered only with glucose metabolism, whereas the incorporation of phosphate was not affected. 14C radioactivity in the various cell components was similarly reduced in mesoderm and ectoderm. 6-mercaptopurine riboside caused an increased incorporation of precursor material in all fractions studied in the mesoderm as well as in the ectoderm during the first 12 hours. This was succeeded by a dramatic decrease of incorporated 14C and 32P radioactivity. Differences of response in the tissues could not be detected with this drug. It is suggested that the malformations of the extrmities caused by these antimetabolites may be predominantly attributed to changes in the cell function rather than to gross effects on cell metabolism.

6-Aminonicotinamide↗
PubMed1977

Characterization of components released by alkali disruption of simian virus 40.

Treatment of simian virus 40 (SV40) particles at pH 9.8 in the presence of 1 mM dithiothreitol for 5 min at 37 degrees C disrupted the virions into a 60S DNA-protein complex and DNA-free 7S protein particles. The DNA-protein complex contained approximately equal amounts of DNA and protein, and appeared by electron microscopy to be relaxed circular structures with an average of 21 beads joined by short, thin bridges. The major protein components in the complex were host cell histones, but SV40-specific proteins VP3 and VP2 were also present. The 7S protein particles were almost exclusively VP1 and, in negatively stained samples, resembled the capsomer structures of intact virions.

DNA, Circular↗
PubMed1977

Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro.

Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.

Cell Line↗
PubMed1977

Structure and formation of circular dimers of simian virus 40 DNA.

Most of the viral DNA extracted from simian virus 40 (SV40)-infected African green monkey kidney cells consists of circular molecules about 5.3 kilobases in contour length. However, about 1% of the viral DNA was found to occur as closed circular dimers that appeared to be formed, preferentially, late in infection. The monomeric units of dimers were organized in a head-to-tail, tandem arrangement; moreover, the monomeric units were not defective; i.e., they lacked deletions or other rearrangements. After infections with dimer DNA, nondefective monomers were formed. These findings suggest that dimers are not intermediates in the production of defective SV40 genomes. The majority of the dimers formed in mixed infections with two mutants were homodimers, but about 5% of the circular dimers were heterodimers and must have arisen by intermolecular recombination.

Cell Line↗
PubMed1976

Isolation of a synaptic membrane fraction enriched in cholinergic receptors by controlled phospholipase A2 hydrolysis of synaptic membranes.

A procedure is described for the isolation of synaptic membrane fragments that retain such functionally important proteins as acetylcholine receptors, acetylcholinesterase, 3',5'-cyclic nucleotide phosphodiesterase, and (Na+ + K+)-ATPase. The method is based on the observation, made in brain slices, that junctional membranes are more resistant to phospholipase A2 attack than mitochondrial or plasma membranes. Hydrolysis by phospholipase A2 was controlled by addition of fatty acid-free bovine serum albumin. The membrane fraction obtained represents approximately a 15-fold enrichment of the postsynaptic marker proteins muscarinic and nicotinic acetylcholine receptor and 3',5'-cyclic nucleotide phosphodiesterase over an ordinary synaptic plasma membrane preparation, and is devoid of mitochondrial and microsomal contaminations. The membranes appear on the electron micrographs as rigid fragments (average length 2500-4000A), which do not form vesicles.

Animals↗
PubMed1976

Mercury poisoning in a dentist.

We examined a dentist with chronic elemental mercury poisoning electrophysiologically. Motor conduction in the upper and lower limbs was normal. Sensory nerve action potentials in the ulnar and median nerves were normal, but could not be elicited in the superfical peroneal nerves. Conduction velocity of the sural nerves was normal, but the action potential amplitude was abnormal. Following treatment with penicillamine, sensory conductions in the lower limbs returned to normal.

Dentistry↗