A comparative study of three closely related cytoplasmic polyhedrosis viruses

A cytoplasmic polyhedrosis virus (CPV) from the pine caterpillar, Dendrolimus spectabilis, was compared with Japanese isolates of closely related viruses from the silkworm, Bombyx mori, and gypsy moth, Lymantria dispar. The sizes of the viral RNA genome segments were almost identical, although the CPVs from D. spectabilis and L. dispar could be distinguished from the silkworm virus by a small size difference (0.03 × 10⁶ daltons) in one segment. The same viruses were also distinguishable by RNA homology differences of 25–50% measured by the reannealing of ³H-labeled single-stranded viral messenger RNA (synthesized in vitro) to heat-denatured viral double-stranded RNA. Antigenic differences were also detected by gel immunodiffusion tests. CPVs of D. spectabilis and L. dispar were indistinguishable by these criteria.     

Extrachromosomal DNA in Bacillus thuringiensis var. kurstaki, var. finitimus, var. sotto, and in Bacillus popilliae

Four entomopathogenic bacteria contained extrachromosomal deoxyribonucleic acid (DNA) molecules of various sizes. Bacillus thuringiensis var. kurstaki contained twelve elements banding on agarose gels that ranged from 0.74 to > 50 × 10⁶ daltons, three of which were giant extrachromosomal DNA elements. B. thuringiensis var. sotto contained one giant extrachromosomal DNA element with a molecular size of about 23.5 × 10⁶ daltons and two lesser elements of 0.80 and 0.62 × 10⁶ daltons. B. thuringiensis var. finitimus harbored two giant DNA elements corresponding to >50 × 10⁶ daltons and two lesser bands with relative small size (0.98 and 0.97 × 10⁶ daltons). B. popilliae contained no giant extrachromosomal DNA elements but did contain two smaller elements corresponding to 4.45 and 0.58 × 10⁶ daltons. The possible use of extrachromosomal DNA elements that prove to be autonomous replicons for recombinant DNA studies is discussed.     

Messenger ribonucleoprotein particles in unfertilized sea urchin eggs

The properties of poly(A)-containing messenger ribonucleoprotein particles (mRNPs) from unfertilized sea urchin eggs isolated under various ionic conditions were studied. Poly(A)-containing RNPs of eggs sediment with a modal value of 60–65 S under all conditions used. However, buoyant densities vary strikingly with conditions of particle preparation. Deproteinized poly(A)-containing mRNA has an average molecular weight of about 1 × 10⁶. RNPs prepared in 0.35 M Na⁺ in the absence of Mg²⁺ contain an average of 0.25 × 10⁶ daltons of protein, while particles prepared in 0.05 M Na⁺ in the absence of Mg²⁺ contain 0.35 to 11 × 10⁶ daltons of protein per RNA molecule. Particles prepared in 0.35 M Na⁺ plus 5 mM Mg²⁺ contain 1.4 × 10⁶ daltons of protein suggesting that Mg²⁺ may be necessary for maintenance of RNP intergrity if high Na⁺ concentrations are used to prevent nonspecific RNA-protein interactions. Particles prepared in 0.35 M K⁺ contain 0.9 × 10⁶ daltons of protein in both Mg²⁺ and EDTA. Mg²⁺ does not cause significant aggregation of particles, since the size of RNA extracted from RNPs is proportional to RNP sedimentation rate. Monovalent cation concentrations normally used in analysis of RNPs by sedimentation cause deproteinized poly(A)-containing RNA to sediment with abnormally high sedimentation coefficients, indicating that high sedimentation rates alone do not indicate that RNA is contained in an RNP.     

Biosynthesis of Rauscher leukemia viral proteins

Antisera to disrupted Rauscher leukemia virus (RLV) or to the purified Rauscher viral 30,000 dalton polypeptide were used to specifically precipitate newly synthesized intracellular viral polypeptides from extracts of infected NIH Swiss mouse cells (JLS-V16). Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of extracts from cells pulse-labeled for 10–20 min with ³⁵S-methionine showed that immune precipitates contained none of the nonglycosylated internal structural polypeptides of mature viruses. The major viral-specific polypeptides labeled in 10 min included polypeptides of 180,000, 140,000, 110,000, 80,000, and 60,000 daltons with minor polypeptides of 65,000, 50,000, and 40,000 daltons. Labeling the intracellular virus-specific polypeptides with ¹⁴C-glucosamine indicated that the 180,000, 110,000, 80,000, and 60,000 dalton polypeptides were glycosylated, and all but the 110,000 dalton polypeptides are contained in the mature virions. Based on pulse-chase experiments, it appears that at least 3 of the large polypeptides (140,000, 65,000, and 50,000 daltons) are precursors to the three major internal structural polypeptides of the mature virions.     

The delta-endotoxin of Bacillus thuringiensis. V. On the occurrence of endotoxin fragments in hemolymph

Leucine-³H labeled crystals of Bacillus thuringiensis δ-endotoxin were fed to last-instar larvae of spruce budworm, Choristoneura fumiferana, eastern forest tent caterpillar, Malacosoma disstria, and silkworm, Bombyx mori. Radioactivity was detected in hemolymph 1 min after feeding in the first two species, but not until 3–5 min after feeding in silkworm larvae. Most of the radioactivity from hemolymph of all three species eluted from gel filtration columns at the same elution volume indicating similar moleculear weights (<1800 daltons).     

Molecular complexity of Paramecium symbiont lambda deoxyribonucleic acid: evidence for the presence of a multicopy genome

DNA was isolated from highly purified symbiont lambda particles of Paramecium aurelia. Renaturation kinetic data revealed a molecular size of 0.71 × 10⁹ daltons. Analytical complexity estimated from chemical data was 7.5 × 10⁹ daltons. These values correspond to about ten copies of the genome per lambda particle. The relationship of symbiont lambda particles to bacteria and cellular organelles, i.e. chloroplasts, mitochondria and kinetosomes, is discussed.     

Stabilization and functional studies of high-molecular-weight murine lymphotoxins

High levels of lymphotoxin-like activity (LT) were found in supernatants from secondarily stimulated immune mouse splenocytes activated with concanavalin A (Con A) in vitro. Splenocytes obtained from C57Bl/6 mice immune to the P815 mastocytoma were restimulated in vitro with mitomycin C-treated P815 cells, and then stimulated with Con A. High levels of unstable LT activity are rapidly (2–4 hr) released by these lectin-stimulated splenocytes. The introduction of a crosslinking agent, glutaraldehyde, was found to stabilize this LT activity and allowed us to perform more defined biochemical studies and to examine the functional activities of the LT classes. The lytic activity in these supernatants resided in the high-molecular-weight classes, termed Complex (Cx > 200,000 daltons) and alpha-heavy (α_H 130,000–160,000 daltons). It was found that the Cx and α_H LT classes from the secondarily stimulated immune splenocytes cause lysis of allogeneic target cells, P815 and EL-4, in a 16-hr ⁷⁵Semethionine release assay, and in some cases, this lysis was specific for the sensitizing target cell.     

On the origin of plastids

The buoyant density in CsCl of ribosomes from chloroplasts of the green algaChlorella pyrenoidosa and two species of higher plants,Pisum sativum andChenopodium album, has been studied. From the relative protein content it was calculated that 70S ribosomes from chloroplasts are much smaller than 80S cytoplasmic ribosomes (3.0–3.1×10⁶ and 4.0×10⁶ daltons) and slightly larger than 70S ribosomes from abcteriaE. coli 2.5×10⁶ daltons). Chloroplast ribosomes from pea seedlings were analyzed by two-dimensional polyacrylamide gel electrophoresis. They appear to contain 71 proteins. This indicates that chloroplast ribosomes contain a larger number of proteins than do the ribosomes fromE. coli and other species of Enterobacteriaceae. Further study will permit a probable evaluation of the validity of Mereschkowsky's hypothesis that the photosynthetic plastids of eukaryotic plant cells are the evolutionary descendants of endosymbiotic blue-green algae.     

Characterization of sub-genomic double-stranded RNAs from virus-infected plants

Double-stranded RNAs (sub-RFs) smaller than the double-stranded RNAs (RFs) corresponding to genomic RNAs of tobacco mosaic (TMV) and cowpea chlorotic mottle (CCMV) viruses were isolated from infected plants and characterized. Seven of the 12 sub-RFs of TMV that were found ranging in size from 3.00 – 0.42 × 10⁶ daltons corresponded to twice the size of the 7 sub-genomic mRNAs reported by Goelet and Karn (8). Six sub-RFs of CCMV were found ranging from 0.98 – 0.39 × 10⁶ daltons with the most abundant species corresponding to twice the size of RNA 4. The kinetics of incorporation of ³H-uridine into sub-RFs were different from that into RFs. Incorporation into sub-RFs was slow and linear whereas that into RF turned over rapidly.     

Bovine adrenocortical self-phosphorylating protein kinase,SPK 380: Purification and characterization

Recently, we described the partial purification and characterization of a novel adrenocortical cyclic nucleotide-independent protein kinase, PK 380, that catalyzes the phosphorylation of an endogenous peptide (120,000 daltons) and a serine residue(s) of the α subunit (38,000 daltons) of the eucaryotic initiation factor eIF-2 (Y. Kuroda, W. C. Merrick, and R. K. Sharma, 1982, Arch. Biochem. Biophys.213, 271–275). In the present communication we describe the purification to apparent homogeneity and characterization of this protein kinase (SPK 380). As shown by sucrose density sedimentation, the native enzyme has a molecular weight of 356,000. The protein is composed of three identical subunits of M_r 120,000. Polyacrylamide-gel isoelectric focusing electrophoresis revealed a single peak with pI 4.5. SPK 380 self-phosphorylated a histidine residue(s) of its 120,000-dalton peptide. This reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mm Mn²⁺ or 10 mm Mg²⁺) were essential for optimum activity. Thiol reagents (N-ethylmaleimide, p-chloromercuriphenylsulfonic acid) inhibited the kinase, indicating a sulfhydryl-group requirement for enzyme activity.     

Comparative analysis of the alkali-liberated components of the Hyphantria cunea and the Diacrisia virginica granulosis viruses

The biochemical and biophysical characteristics of the closely related Diacrisia virginica and Hyphantria cunea granulosis virus isolates were examined. Sucrose gradient sedimentation patterns of alkali-solubilized DGV and HcGV capsules were identical. The top, middle, and bottom fractions from either viral isolate were infectious when injected into susceptible host larvae. Electrophoretic analysis of alkaline-solubilized granulin extracts demonstrated that both viruses contain alkaline proteolytic activity. The major granulin protein (～28,000 daltons) of both isolates comigrated in a SDS-PAGE. Electrophoretic separation of the virus proteins demonstrated some quantitative differences between the two granulosis viruses. The enveloped nucleocapsids and the nucleocapsids of the two viruses were morphologically indistinguishable.     

Isolation of ribosomal RNA precursors from Physarum polycephalum

Ribosomal RNA synthesis in Physarum polycephalum was studied by labeling intact microplasmodia with [³H]uridine. Labeled, high-molecular-weight RNA species were found in a 30,000 S structure released by phenol extraction at room temperature. RNA was released from the structure by further phenol extraction at 65–70 °C. If the labeling period was 15 min or longer, the labeled RNA was seen by polyacrylamide gel electrophoresis to be of two major types, a heterodisperse collection of 45-35 S molecules and a 26 S species. If the labeling was carried out for 30 min in the presence of cycloheximide, the major labeled species had an electrophoretic mobility corresponding to 40 S. Studies of the labeling kinetics, methylation, and base composition of these RNA molecules indicate that they are precursors to ribosomal RNA. The molecular weights of the homogeneous 40 and 26 S precursors are 3.0 × 10⁶ and 1.45 × 10⁶ daltons, respectively, in comparison with molecular weights of 1.29 × 10⁶ and 0.68 × 10⁶ daltons for the completed ribosomal RNA's.     

Transcription and processing of rRNA in higher plant cells (Daucus carota,Petroselinum crispum,Acer pseudoplatanus)

Actively dividing cells from parsley (Petroselinum crispum) and carrot (Daucus carota) (bothApiaceae) andAcer pseudoplatanus (Aceraceae) were used to detect the primary gene product of the rDNA in plant cells. Parsley and carrot cells were labelled with [³²P] orthophosphate. In both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels under non-denaturing conditions. Its molecular weight did not exceed 2.5 × 10⁶ daltons. The component emerged from the heterogenous material after a labelling period of 5–10 min. In parsley cells 45 min after onset of incubation labelled mature rRNA (25S and 18S) arrived in the cytoplasm. InAcer pseudoplatanus (incubation period 60 min) two rapidly labelled components did emerge from polyacrylamide gels; their molecular weights were 2.3 and 3.2^? 3.4 × 10⁶ daltons. After electrophoresis under denaturing conditions the larger component was no longer present, thus indicating that it was an aggregate of different RNA molecules. The molecular weights of the rRNA precursors ofD. carota andP. crispum determined after electrophoresis in formamide gels were about 2.1 × 10⁶ daltons. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 × 10⁶ daltons.     

Growth of Pseudomonas chlororaphis on apolyester–polyurethane and the purification andcharacterization of a polyurethanase–esterase enzyme

A Pseudomonas chlororaphis was found to degrade and utilize apolyester polyurethane as a sole carbon and energy source. Polyurethane utilization by P.chlororaphis followed simple Michaelis–Menten kinetics. The K_s and μ_max values were 0.802 mg·ml⁻¹ and 1.316 doublings·h⁻¹, respectively. The enzymes from P. chlororaphis responsible for polyurethanedegradation were found to be extracellular. Analysis of the polyurethane degrading proteins, usingnon-denaturing polyacrylamide gel electrophoresis, revealed three active protein bands with R_f values of 0.25, 0.417 and 0.917. A polyurethane degrading enzyme was purifiedand displayed esterase activity. This enzyme was inhibited by phenylmethylsulfonyl fluoride andhad a molecular weight of 27,000 daltons.     

Repair Replication in Escherichia coli As Measured by the Photolysis of Bromodeoxyuridine

The ability to selectively photolyze bromouracil-(BrUra-)containing repaired regions in cellular DNA has allowed us to estimate the average size of repaired regions in ultraviolet (UV) light-irradiated Escherichia coli. Cells were labeled with thymidine-³H, irradiated at 254 nm, and incubated in nonradioactive bromodeoxyuridine (BrdUrd). After incubation the cells were exposed to 10⁶ ergs·mm^-2 at 313 nm, lysed, and sedimented in alkaline sucrose gradients so as to measure the average molecular weight of single DNA strands. In strains that had excised ~45 cyclobutane pyrimidine dimers/10⁸ daltons, the 313 nm treatment resulted in ~6 single-strand breaks/10⁸ daltons. In an excisionless strain, the same treatment resulted in only 1.5 breaks/10⁸ daltons. From the determination of the sensitivities of fully substituted DNAs to 313 nm light, we calculate that the repaired regions in excising strains of E. coli contain an average of 4-6 BrUra residues. Photoreactivation experiments indicate that the excision of pyrimidine dimers in the presence of BrdUrd is the primary source of repaired regions selectively photolyzed by 313 nm radiation.     

Virus particle packaging in baculovirus and cytoplasmic polyhedrosis virus inclusion bodies

The structure of the inclusion bodies (IBs) of three multiply enveloped nuclear polyhedrosis viruses (MNPVs), one singly enveloped NPV (SNPV), two granulosis viruses (GVs) and one cytoplasmic polyhedrosis virus (CPV) were compared. A method was devised to calculate the numbers of virus particles and nucleocapsids in IBs using data from light microscopy and thin sections. The three MNPVs, from Agrotis segetum (English and Polish virus isolates) and Mamestra brassicae had similar concentrations of virus particles ranging from 17.3 to 19.6 per μm³ of IB. Plusia gamma SNPV had a higher density of 59.6 virus particles per μm³ of IB, which partly compensated for its having smaller IBs (mean volume 0.65 μm³) than the MNPVs (2.60–9.71 μm³). The English A. segetum MNPV isolate had the most nucleocapsids in each virus particle (mean, 4.04) and the largest IBs (mean volume, 9.71 μm³), giving 674 nucleocapsids per IB on average. The GVs, from A. segetum and Pieris brassicae, mainly contained one nucleocapsid per IB. P. gamma CPV IBs had a much higher density of virus particles than the baculoviruses (260 per μm³ compared with 17–60 per μm³). These data are discussed in relation to the biological properties of these viruses, and possible adaptational advantages of alternative IB designs are considered.     

An electron microscope heteroduplex study of the ribosomal DNAs of Xenopus laevis and Xenopus mulleri

The arrangement of the genes and spacers has been analyzed in ribosomal DNA of Xenopus laevis and Xenopus mulleri by heteroduplex mapping and visualization of ribosomal RNA-DNA hybrids. Heterologous reassoeiated molecules show a characteristic pattern in which two perfectly duplexed regions, whose lengths are those predicted by the known lengths of the 18 S and 28 S genes, are separated by a small substitution loop of about 0.23 × 10⁶ daltons and a large region of partial homology which averages 3.24 × 10⁶ daltons. These mismatched regions are entirely consistent with the known sequence divergence previously described (Brown et al., 1972) for the transcribed and non-transcribed spacer regions of the two rDNAs, respectively. Hybrids of X. laevis rDNA with 18 S and 28 S rRNA contain two duplex regions of the expected lengths for the 18 S and 28 S genes separated by 0.49 × 10⁶ daltons of single-strand DNA. This latter value is the length of the transcribed spacer region between the 18 S and 28 S RNAs that has been measured within the 40 S RNA precursor molecule by secondary structure mapping (Wellauer &; Dawid, personal communication). There is also a longer single-strand region separating one 18 S + 28 S gene set from the next; this is considered to be mainly non-transcribed spacer.We conclude that the 18 S and 28 S genes are separated by about 0.5 × 10⁶ daltons of DNA of which about half is homologous in the two Xenopus species. This region is part of the transcribed spacer. In addition, the longer non-transcribed spacer can be seen to have some homology between the two species; the location of this homology is fairly reproducible between molecules and has been carefully documented by contour length measurements.