Oliver C. Richards

Isolation and molecular weight of circular chloroplast DNA from Euglena gracilis

Abstract 1. Chloroplast DNA from Euglena gracilis has been examined by electron microscopy and by sedimentation analysis in order to establish its molecular size and in order to determine the feasibility of physical isolation of whole molecules. 2. Using bacteriophage λ DNA as an internal standard, a molecular weight of 9.2 · 10 7 is obtained for chloroplast DNA by comparison of contour lengths of circular λ and circular chloroplast DNA molecules found on identical grids in the electron microscope. Chloroplast DNA molecules were also found in a tightly twisted configuration; this is the first demonstration of supercoiled DNA structures in chloroplast preparations. 3. Sucrose gradient sedimentation analysis of chloroplast DNA with molecular wholes and halves of bacteriophage T7 DNA gives a molecular weight for chloroplast DNA identical to that obtained by electron microscopy. Furthermore, molecular whole and half molecules of chloroplast DNA can be effectively isolated by sucrose gradient sedimentation.

Enzymatic activity of poliovirus RNA polymerase synthesized in Escherichia coli from viral cDNA

Plasmids have been constructed that contain DNA sequences that direct the expression of the poliovirus RNA-dependent RNA polymerase, in the form of recombinant fusion proteins. Inclusion of an additional gene for the poliovirus protease results in cleavage of the fusion protein to yield a 52-kDa, enzymatically active, polymerase protein, apparently identical to the functional enzyme isolated from virus-infected HeLa cells. A large amount of polymerase protein accumulates as particulate or insoluble material in bacteria, and this protein has little or no activity. However, significant amounts of soluble, active enzyme are recovered, such that the resulting specific activity of crude bacterial extracts is greater than that obtained from virus-infected HeLa cells. Purification of the enzyme from Escherichia coli is readily accomplished, and yields a preparation that will copy poliovirion RNA as template, in the presence of oligo(U) primer. The availability of cloned DNA sequences encoding catalytically active RNA polymerase will allow genetic manipulations to initiate structure-function studies of this enzyme.

Effects of cycloheximide and of chloramphenicol on DNA synthesis in Euglena gracilis

Abstract 1. The independence of chloroplasts in synthesis of their DNA has been examined in Euglena gracilis by 32 Plabeling of the DNA and with the aid of the inhibitors, chloramphenicol and cycloheximide. Strain Z, containing chloroplast and mitochondrial DNA, and strain W3 BUL, containing mitochondrial DNA, were used. 2. Chloramphenicol has no effect on the labeling or on the distribution of chloroplast and mitochondrial DNA relative to nuclear DNA for at least 5.5 generations. Cell division is slowed in strain Z but is unaffected in strain W3 BUL. Since chloramphenicol inhibits protein synthesis on Euglena chloroplast ribosomes, continued synthesis of chloroplast DNA and continued cell division suggest that all unstable proteins essential for synthesis of chloroplast DNA are made on cytoplasmic ribosomes, or all proteins required for this synthesis turn over slowly. 3. After growth in the presence of cycloheximide, the specific activity of the nuclear DNA is 35 to 40 % of that of the mitochondrial DNA and 50 % of that of the chloroplast DNA. Under these conditions cell division is halted. Since control cultures did not show increased turnover of extranuclear DNA, synthesis of chloroplast and mitochondrial DNA must continue after synthesis of nuclear DNA has been virtually stopped by cycloheximide.

Synthesis and turnover of Euglena gracilis mitochondrial DNA

Abstract Replication of mitochondrial DNA was investigated by a density transfer experiment in a strain of Euglena gracilis lacking chloroplast DNA. DNA was uniformly labeled in a medium containing 32P-labeled inorganic phosphate and [3H]adenine in the presence of the heavy-density label and transferred to a medium containing 32P-labeled inorganic phosphate but no [3H]adenine following removal of the heavy-density label. Replication of nuclear DNA within these cells was used as an internal control. The densities and ratios of the peaks of nuclear DNA were those expected for a strict semiconservative replication. In contrast, replication of mitochondrial DNA was dispersive, as illustrated by the following results: (1) both native and denatured mitochondrial DNA exhibited a single density peak at 1.1 and 2.2 cell doublings after the density transfer. (2) The specific activity of 3H-labeled DNA varied across the peak of native or denatured DNA, indicating a heterogeneous population of molecules exhibiting different degrees of density and radioisotope labeling. This dispersive replication could involve either multiple recombination events or extensive turnover of the DNA or a mixture of both. Extensive dispersion of the sample obtained at 1.1 cell doublings after the density transfer is shown by the persistence of the same peak density for duplex DNA reduced to a molecular weight of 6 × 105 by shearing. Two measures of the rate of replication of mitochondrial DNA were obtained from the densities of native duplex DNA and the rate of decrease in 3H-specific activities of duplex DNA during the experiment. The average of these rates indicates that mitochondrial DNA replicates at least 1.5 times as fast as nuclear DNA. Since there is a constant ratio of mitochondrial DNA:nuclear DNA in a logarithmic culture, mitochondrial DNA was calculated to have a half-life of 1.8 cell doublings.

Formation of poliovirus RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA.

The poliovirus polymerase 3D was synthesized in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA inserted into several plasmid expression vectors. Cleavage was accomplished by the action of viral protease 3C sequences expressed in the same bacteria, either from a second plasmid or from the same plasmid, cloned so as to produce contiguous sequences in the same protein. In the case of two plasmids, protease 3C functioned in trans to cleave the fusion protein at or very near the normal Gln/Gly cleavage site. When protease and polymerase sequences were produced in the same protein, the protease sequences acted in the precursor form to release the polymerase from itself. Thus, cleavage can occur to generate polymerase 3D both as an intermolecular reaction and, very likely, also as an intramolecular event.

Temperature-sensitive polioviruses containing mutations in RNA polymerase

Site-directed mutagenesis was performed to change the wild-type residue (asparagine) to aspartate, histidine, or tyrosine at amino acid 424 of the poliovirus RNA polymerase, 3Dpol. The mutations were introduced into plasmids containing full-length viral cDNA and plasmids which direct the expression of 3Dpol in Escherichia coli. Mutant viruses, recovered after transfection of HeLa cells with RNA transcripts of the full-length clones, produced small plaques at 32 degrees. In addition, the plaquing efficiency was decreased for all three mutants at 37 degrees, compared to 32 degrees. The polyprotein processing of all mutant viruses was normal at the temperatures tested, suggesting that the mutant plaque phenotypes were not due to incorrect processing of viral proteins. Analyses of viral RNA synthesis in infected cells and of the polymerase activities of mutant enzymes produced in E. coli suggested the following: (1) The his424 mutant enzyme appeared to be defective in the initiation of plus-strand RNA synthesis in HeLa cells. (2) The asp424 mutant enzyme appeared unable to assume proper conformation for active polymerase function when synthesized at 37 degrees. (3) The tyr424 mutant enzyme was totally inactive when synthesized in E. coli at 37 degrees.

Expression of hepatitis A virus capsid sequences in insect cells.

A cDNA coding for hepatitis A virus (HAV) VP1 and portions of the flanking VP3 and P2 sequences was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter and translational start codon. Cells infected with recombinant virus produced high levels of a 55 kDa protein, identified as containing HAV VP1 by reactivity with anti-VP1 serum. The expressed protein also reacted on immunoblots with human HAV convalescent sera as well as sera from rabbits immunized with intact HAV. This protein was found predominantly in the cytoplasm of infected insect cells, probably as an insoluble aggregate.

Expression and characterization of poliovirus proteins 3BVPg, 3Cpro, and 3Dpol in recombinant baculovirus-infected Spodoptera frugiperda cells

As an initial step toward investigating the roles of poliovirus proteins in viral RNA replication, a baculovirus expression system was used to produce poliovirus proteins from the P3 region. Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus, vETL-PoV3A*BCD, which contains cDNA coding for poliovirus proteins 3D, 3C, 3B, and a portion of 3A protein sequence. Immunofluorescence microscopy revealed that the majority of 3D (polymerase) was in the cytoplasm of recombinant baculovirus-infected Sf9 cells. In the same cells, the 3C (protease) and 3B (VPg) proteins appeared to be located in distinct subcellular regions, possibly membrane structures, suggesting that the expressed polyprotein was cleaved to generate mature proteins. Processing of the polypeptide was confirmed by immunoblot analysis which demonstrated that 3Cpro sequences were active in cleavage of the polyproteins 3A*BCD and 3CD. Over 95% of the 3D sequences accumulated in the form of mature 3Dpol, with only low levels of 3CD remaining. The majority of 3Dpol remained in the supernatant after low speed centrifugation of sonicated cells. The 3Dpol had RNA-dependent RNA polymerase activity as measured by elongation of an oligo(U) primer using a poly(A) template. The protein 3CDpro was active in cleaving P1 protein. The yield and activities of the poliovirus proteins expressed will facilitate future biochemical studies.

Analysis of Theiler's Virus Isolates from Persistently Infected Mouse Nervous Tissue

The DA strain of Theilers virus causes a chronic progressive demyelination in mice following intracerebral inoculation. Virus was isolated from chronically infected mice, and then grown in cell culture, and the isolates were compared with the parent virus used for inoculation. No defective interfering particles or temperature-sensitive virus were recovered, and capsid proteins appeared identical by SDS-PAGE. One of three isolates had evidence of genomic mutation by Tl ribonuclease oligonucleotide fingerprinting. The significance of these findings with regard to the generation and maintenance of persistence and to adaptation to cell culture is discussed. Also of interest was the marked difference between the DA fingerprint and that of GD VII, a serologically related strain with different biological activity.