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PubMed1981

Interactions between phage lambda replication proteins, lambda DNA and minicell membrane.

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.

Bacteriophage lambda↗
PubMed1984

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.

Base Sequence↗
PubMed1988

A spliced Epstein-Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear viral genome.

Epstein-Barr virus (EBV) immortalizes B-lymphocytes efficiently in vitro and in most or all of the cells a latent infection is established. Only a few viral genes are expressed during latency and these apparently maintain cell immortalization by EBV. A 1.9 kb cDNA clone of an EBV mRNA has been isolated from a B95-8 cDNA library and this cDNA corresponds to a new member of the group of EBV latent cycle genes. On Northern blots two polyadenylated transcripts 1.7 and 2.0 kb in length have been detected by the cDNA clone in B95-8 cells as well as in latently infected cell lines and in various cell lines derived from Burkitt's lymphomas. The cDNA sequence showed that this gene consists of nine exons and showed that this gene consists of nine exons and that the transcription unit crosses the terminal repeats. Thus, the intact gene is created upon infection by the circularization of the linear viral DNA molecule at the terminal repeats. One major open reading frame is present in the cDNA apparently coding for a 53 kd protein. A strongly hydrophilic part of the protein is followed by a very hydrophobic C-terminus, perhaps suggesting a membrane protein. The gene is unrelated to the other latent cycle genes, the EBV nuclear antigens (EBNA 1-4), the leader protein (LP), and the latent membrane protein (LMP) genes. This is the first example of a viral gene whose coding sequence is only created by joining of the ends of a linear virus.

Amino Acid Sequence↗
PubMed1991

A microcomputer program to analyze the CD spectrum of proteins and nucleic acids--use of LOTUS 1-2-3 spread sheet.

A user friendly interactive computer program, CIRDIC, is developed which calculates the molar ellipticity and molar circular dichroic absorption coefficients from the CD spectrum. This, in combination with LOTUS 1-2-3 spread sheet, will give the spectra of above parameters vs wavelength. The code is implemented in MicroSoft FORTRAN 77 which runs on any IBM compatible PC under MSDOS environment.

Algorithms↗
PubMed2026

Exploring the Mitochondrial Genomes of Phoebe Species (Lauraceae): Structural Dynamics and Functional Conservation.

Plant mitochondrial genomes (mitogenomes) vary markedly in size and architecture despite generally slow rates of sequence evolution. Phoebe is an ecologically and economically valuable genus of Lauraceae, yet its mitogenome diversity remains poorly characterized. In this study, we newly sequenced, assembled, and annotated the mitogenomes of three nationally protected Class II wild plants (P. bournei, P. chekiangensis, P. zhennan) from China and compared their mitogenomic characteristics. The three assemblies were resolved into representative circular configurations ranging from 808 to 864 kb, with similar GC contents and conserved protein-coding capacity. Each mitogenome contained distinct 41 protein-coding genes, 27-28 transfer RNAs, and three ribosomal RNAs. Synteny analysis revealed extensive changes in homologous-block order and orientation despite substantial sequence homology among the three species. Abundant repeats occurred predominantly in noncoding regions, while plastid-derived fragments documented historical intracellular DNA transfer. The three species exhibited similar codon usage and predicted RNA-editing patterns, whereas low synonymous divergence limited inference from pairwise ratios. Phylogenetic analysis based on mitochondrial protein-coding genes recovered Phoebe as a well-supported monophyletic lineage. These results reveal substantial structural divergence accompanied by conserved nucleotide composition and coding capacity, providing valuable data for further understanding the evolutionary variation of plant mitogenomes of Phoebe and the Lauraceae.

Phoebe↗
PubMed1975

Incidence and nature of cytoplasmic hepatitis B antigen in hepatocytes.

Hepatitis B antigen (HB Ag) in the hepatocytic cytoplasm is detected by immunofluorescence after reaction with fluoresceinated antiserum to HB Ag or by electron microscopy as numerous 20- to 30-nm. tubular and circular structures in dilated cisternae of excess endoplasmic reticulum. On light microscopy, these hepatocytes can be recognized because their cytoplasm has a ground-glass appearance and stains with Gomori's aldehyde fuchsin. Aldehyde fuchsin-positive ground-glass hepatocytes were detected in all 14 asymptomatic carriers of HB Ag and in 16 of 60 HB Ag-seropositive patients with chronic hepatitis, but not in HB Ag-seropositive acute viral hepatitis or in various other HB Ag-seronegative liver diseases. These cells are helpful in identifying on light microscopy HB Ag carriers and a portion of patients with HB Ag-positive chronic hepatitis. Nuclear HB Ag did not stain with aldehyde fuchsin. Nucleic acids were not detected in the ground-glass cytoplasm by special stains at the light or electron microscopic level. We suggest that the tubular and circular structures in the hepatocytic cytoplasm are coat material of the hepatitis B virus or virally coded host cell reaction product rather than the complete hepatitis B virus.

Acute Disease↗
PubMed1975

Size, conformation and purity of chloroplast DNA of some higher plants.

1. Chloroplast DNA of Antirrhinum majus, Oenothera hookeri, Beta vulgaris and Spinacia oleracea band at the same buoyant density of 1.697 g-cm-3 in neutral CsCl equilibrium gradients. The corresponding nuclear DNAs band at 1.691, 1.703, 1.695 and 1.695 g-cm-3, respectively. The purity of chloroplast and nuclear DNA can be assessed objectively only in the cases of Antirrhinum and Oenothera. 2. Electron microscopic analysis of chloroplast DNA, purified in CsCl or CsCl/ethidium bromide gradients, revealed up to 80% circular molecules. Of these about 15% were of supertwisted conformation. Best yields of circular molecules were recovered when populations of unbroken chloroplasts were subjected to DNAase and phosphodiesterase treatment, and when the DNA was purified from viscous lysates by centrifugation into a CsCl cushion. Treatment of plastids with DNAase alone did not guarantee complete degradation of nuclear DNA. 3. The average contour length of the open circular chloroplast DNA molecules was basically similar for all four plants. They were 45.9 plus or minus 2.1 mum for Antirrhinum, 45.7 plus or minus 1.9 mum for Spinacia, 44.9 plus or minus 1.7 mum for Beta and 45.2 mum for Oenothera. This is comparable to the size derived for the coding capacity of chloroplast DNA from reassociation experiments. As much as 15% of the total population of circles in chloroplast DNA of Spinacia were circular dimers.

Centrifugation, Density Gradient↗
PubMed1985

The perception of a dotted line in noise: a model of good continuation and some experimental results.

A formal model is proposed, describing how the perceptual interpretation of dot figures is guided by the Gestalt rule of good continuation. The algorithm will be restricted to figures with a collinear dot array (line) embedded in a background of randomly placed dots. The model, CODE-2, is an elaboration of the model, CODE-1, of grouping dots on the basis of the Gestalt rule of (relative) proximity, and consists of the introduction of non-circular symmetric gaussian distribution functions for the representation of the orientation dependent strength of interaction between collinear dots. Supra-threshold contours of the function, resulting from a superposition on each dot of the gaussian functions, are assumed to predict the perceptual grouping of the dots. A quantitative measure for the perceptual salience of dotted lines was defined as the contrast between the internal coherence of the line dots, and their interference with the noise dots. For 20 stimuli the CODE-2 grouping of the dots is reported, together with the results of a line-in-noise latency experiment. There was a significant correlation between the predicted saliences and the experimental results. The results support the usefulness of representing good continuation between collinear dots by non-circular symmetric gaussian distribution functions.

Adult↗
PubMed1987

The nucleotide sequence of a geminivirus from Digitaria sanguinalis.

The encapsidated single-stranded circular DNA of a geminivirus isolated from Digitaria sanguinalis has been sequenced. The data obtained are consistent with there being one DNA circle of 2701 nucleotides. Comparison of the nucleotide sequence with those of maize streak virus (MSV) and wheat dwarf virus showed 64 and 47% DNA homology, respectively. The sequence has four potential coding regions for proteins of greater than 10 kDa, two in the viral (+) sense and two in the complementary (-) sense. Each of these potential coding regions has a highly homologous counterpart among the seven open reading frames previously described for MSV. Virion DNA contained, in addition to the circular single-stranded DNA, a population of small DNA molecules similar to those associated with MSV particles. A comparison with MSV DNA of the region complementary to these small DNA molecules revealed conserved sequences, which may have a role in defining the limits of these primer-like molecules.

Base Sequence↗
PubMed1982

A transcriptionally active, covalently closed minichromosome of cauliflower mosaic virus DNA isolated from infected turnip leaves.

Purified nuclei from turnip leaves infected by cauliflower mosaic virus (CaMV) have been shown to contain a fraction of CaMV DNA that consists of covalently closed circular molecules; possesses a nucleosome structure, based on sensitivity to micrococcal nuclease; and contains nuclear RNA polymerase II that selectively transcribes the coding strand of CaMV DNA in vitro. Our results suggest that the transcriptionally active CaMV DNA is in the form of a minichromosome and that this DNA does not contain the site-specific discontinuities characteristic of the virion.

Chromosomes↗
PubMed1990

The quantitative study of the orientation of collagen in compact bone slices.

We describe and validate a simple method for the study of the proportion of collagen fibers (and apatite crystals) in a bone slice parallel with the plane of section. Viewed between crossed circular polarizers, all bone areas with collagen lying more nearly in the plane of section (i.e., transverse [TS] collagen) appear bright whatever its direction in this plane; longitudinal [LS] collagen appears dark, but not as dark as the background. The degree of brightness increases with section thickness, which must therefore be standardized--we chose 100 microns plane parallel sections. We transferred the circularly polarized light [CPL] image via a CCD TV camera to an image analyzing computer. Color-coded maps of the CPL image were used to compare regions within and between sections. The new analytical procedure makes more detailed studies of the fine-structural orientation in compact bone possible, but does this have any significance? To answer this question, a bone in which the in vivo strain pattern had been clearly documented was chosen for particular study. Transverse mid-diaphyseal sections of the equine radius showed a distribution of CPL bright areas which correlated closely with previously reported strain patterns.

Animals↗