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D T Denhardt

Publications and source records attributed to D T Denhardt.

At least 145 records · Page 8Linked to original sources
PubMed1978

Most short DNA molecules isolated from 3T3 cells are not nascent.

The population of short DNA molecules (less than 10(3) nucleotides) in 3T3 cells has been studied using in vivo and in vitro pulse labeling techniques and in vitro end-labeling. There is a large number of molecules of less than 100 nucleotides present in equal numbers in both Go and S phase cells. In S phase cells, most of these molecules are not replicating intermediates because they do not become density-labeled after a moderate period of substitution of BrdUMP, although they are detected by end-labeling in vitro. This population includes the nascent Okazaki pieces that can be labeled in a short pulse with [3H]dThd or [3H]dTTP, however, these represent less than 10% of the total population. Alkaline hydrolysis of the molecules that had been end-labeled with 32P using [gamma32P]ATP and polynucleotide kinase did not reveal significant release of [32P] 2'(3'), 5' ribonucleoside diphosphates.

Cell Line↗
PubMed1977

Ultraviolet light irradiation of PM2 superhelical DNA.

Superhelical PM2 DNA can be photochemically modified by u.v. irradiation. The variation of S20,w with dose shows the following characteristics. There is a linear increase from 28 to 31s produced by a low dose of u.v. irradiation (4,000 ergs/mm2). A plateau in S20,w occurs between 4,000 and 10,000 ergs/mm2. The S20,w then increases when irradiation is increased to 56,000 ergs/mm2. Thymine dimers are introduced proportional to dose throughtout the range of exposure to u.v. light. Sedimentation velocity-dye titrations reveal anomolous behavior, i.e. apparent increases in superhelix density (sigma). However, the dye-buoyant density procedure showed no change in sigma under the same conditions. The most satisfactory model for the data is preferential photochemical modification of premelted (possibly hairpin) sites as a greater rate than the introduction of photoproducts into duplex sites. The origin of the anomoly in the sedimentation velocity dye titrations is still unclear.

Bacteriophages↗
PubMed1976

Site of cleavage of superhelical phiX174 replicative form DNA by the single strand-specific Neurospora crassa endonuclease.

Experiments with the Neurospora crassa single strand-specific endonuclease have provided evidence for the existence of regions of partially single-stranded character in covalently closed superhelical replicative form DNA of phiX174. The nuclease converts the superhelical molecules to either singly hit relaxed circular or doubly hit linear molecules. We show that the initial cleavage of phiX174 superhelical DNA is a "nick" bounded by a 5'-phosphate and a 3'-hydroxyl; no nucleotides are excised as evidenced by the ability of T4-polynucleotide ligase to reform the phosphodiester bond. The nick can be found in either strand of the double-stranded DNA and is either randomly distributed or at least can be found at any one of many possible locations in the genome. Thus, the regions in phiX174 superhelical molecules that are sensitive to the N. crassa nuclease do not occur at highly specific sites in the genome.

Amino Acids↗
PubMed1976

Multiple structures of adeno-associated virus DNA: analysis of terminally labeled molecules with endonuclease R-Hae III.

The double-stranded form of adeno-associated virus (AAV) DNA has about 20 sites sensitive to endonuclease R.Hae III from Haemophilus aegypitus; the fragments produced fall into about 13 size classes, 8 of which contain single fragments. The location of the Hae III-produced AAV fragments relative to the three EcoR1 fragments was determined. Using revised figures for the molecular weights of the Hae III cleavage products of phiX174 replicative form DNA, we calculated that AAV DNA contains about 4,000 nucleotides. After Hae III digestiion of duplex DNA terminally labeled with 32P using polynucleotide kinase, the majority of fragments containing a 5' 32P label were about 40 nucleotides in length, and fragments of similar size were generated from each end, suggesting that the Hae site closest to the end is within the terminal repetition. Two more-slowly-migrating cleavage products also bore 5' 32P end label. These three terminally labeled species were also generated from single-stranded AAV DNA by digestion with Hae III, and evidence that one may have a nonlinear ("rabbit-ear") structure is presented. The predominant 5' terminal base was identified as thymine for both the plus and minus strands of AAV. Single-stranded AAV molecules could not be efficiently covalently circularized by incubation with polynucleotide ligase or ligase plus T4 DNA polymerase.

Base Sequence↗
PubMed1975

Physical map and strand polarity of specific fragments of adenovirus-associated virus DNA produced by endonuclease R-EcoRI.

Cleavage of adenovirus-associated virus type 2 (AAV2) DNA linear duplex monomers with the restriction endonuclease R-EcoRI yielded three fragments, A, B, and C, having approximate mol wt of 1.6 X 10(6), 1.1 X 10(6), and 1.3 X 10(5), respectively. Radioactive labeling the 5' termini of AAV DNA before cleavage with R-EcoRI showed that A and B were terminal fragments and C was internal. Separation of the complementary strands of fragments A and B showed that A contained the 5' terminus of the minus strand and the 3' terminus of the plus strand, and conversely for fragment B. The physical map of the AAV R-EcoRI fragments can thus be unambiguously determined and is drawn with B at the left-hand and A at the right-hand end. On this map, transcription of stable AAV mRNA from the minus strand proceeds from left to right, beginning in fragment B and terminating in fragment A. The asymmetry in distribution of thymidine between the AAV DNA plus and minus strands is preferentially located in fragment A, which represents the right-hand half of the duplex molecule. These experiments enable preparative separation of all four single-strand termini of AAV DNA and provide a basis for orientation of fragment maps derived by cleavage with other restriction enzymes.

DNA Restriction Enzymes↗
PubMed1974

Structure of nascent phiX174 replicative form: evidence for discontinuous DNA replication.

varphiX174 RF (replicative form) II DNA, labeled in vivo with [methyl-(3)H]thymidine, was isolated from Escherichia coli polA (DNA polymerase I-deficient) and polA(+) cells during RF replication. [(32)P]dCMP was incorporated into the gaps present in the RF II DNA with [alpha-(32)P]dCTP and T4 DNA polymerase. Sedimentation in alkaline sucrose gradients revealed that much of the incorporated (32)P was present in a heterogeneous collection of fragments shorter than unit length. Inclusion of polynucleotide ligase in the gap-filling reaction increased the average size of the (32)P-labeled fragments. Gel electrophoresis of the products formed by digestion of the (32)P-labeled RF II molecules with the restriction nuclease, endonuclease R, indicated that in the population of RF II molecules gaps could occur anywhere in the genome. Competition-annealing experiments provided evidence that the majority of the label incorporated into gaps was present in the minus strand. RF II molecules isolated from polA(+) cells were enriched for gaps in a unique region of the genome in comparison with RF II molecules isolated from polA cells. The presence of multiple gaps in the minus strand implies that it is synthesized by a discontinuous mechanism during varphiX RF replication.

Centrifugation, Density Gradient↗
PubMed1974

Isolation of repeated and self-complementary sequences from E. coli DNA.

We have used the single-strand specific nuclease from Neurospora crassa and chromatography on methylated albumin-kieselguhr to purify and characterize repeated and self-complementary sequences from Escherichia coli DNA. Approximately 0.5% of the genome renatures spontaneously at zero time and another 2% renatures somewhat more rapidly than the total DNA. The early renaturing DNA has a base composition and a Tm similar to the total DNA and contains on the average 100 base pairs; the self-complementary DNA also has a base composition like E. coli but contains a mean of 170 base pairs. No evidence was obtained for the presence of a highly redundant sequence.

Base Composition↗
PubMed1974

The rep mutation. III. Altered structure of the replicating Escherichia coli chromosome.

The rep gene function of Escherichia coli is essential for the replication of P2 and phiX174 double-stranded deoxyribonucleic acid (DNA). Compared with isogenic rep(+) strains, rep mutants show the following characteristics: larger cell size, more DNA per cell, and a slightly lower DNA/mass ratio. The replicating rep chromosomes show a steeper gradient of marker frequencies and contain more replicating forks per chromosome. The nucleoid body of rep mutants sediments faster and contains more DNA. We deduce that the rep function is required for the "normal" replication of the E. coli chromosome and that in its absence the E. coli chromosome replicates in an altered manner, perhaps involving slower-moving replicating forks.

Autoradiography↗
PubMed1974

Mechanism of replication of phi x174 single-stranded DNA. IX. Requirement for the Escherichia coli dnaG protein.

Escherichia coli NY73, possessing a temperature-sensitive mutation in the dnaG locus, was rendered sensitive to bacteriophage phiX174 by P1 transduction. phiX174 reproduces in this strain at 30 C but not at 40 C. All three stages of phiX174 replication, parental replicative form (RF) synthesis, RF replication, and progeny single-stranded DNA synthesis, are thermolabile in this mutant. Competition-annealing data show that both plus- and minus-strand synthesis are equally inhibited after shift up to 40 C during RF replication. We conclude that the dnaG gene product is required for the synthesis of both strands of phiX RF during RF replication and of the complementary strand and viral progeny strands during stages I and III, respectively.

Bacterial Proteins↗