Physicochemical characterization of the ribosomal RNA species of the Mollusca. Molecular weight, integrity and secondary-structure features of the RNA of the large and small ribosomal subunits.

1. The rRNA species of the Cephalopoda Octopus vulgaris and Loligo vulgaris were found to have unexpectedly high sedimentation coefficients and molecular weights. In 0.1 M-NaCl the L-rRNA (RNA from large ribosomal subunit) has the same s20 value as the L-rRNA of the mammals (30.7S), whereas the S-rRNA (RNA from small ribosomal subunit) sediments at a faster rate (20.1S) than the S-rRNA of both the mammals and the fungi (Neurospora crassa) (17.5S). The molecular weights of the L-rRNA were determined by gel electrophoresis in formamide and found to be 1.66 X 10(6) (Octupus) and 1.89 X 10(6) (Loligo); the mol.wt. of the S-rRNA of both species is 0.96 X 10(6), i.e. much larger than that of the mammals (0.65 X 10(6)) and almost coincident with that of the '23S' RNA of the prokaryotes. 2. By contrast, the less evolved Gastropoda and Lamellibranchiata (Murex trunculus and Macrocallista chione) have S-rRNA and L-rRNA species with mol.wts. of 0.65 X 10(6) and approx. 1.40 X 10(6).3. All the mature L-rRNA molecules of the cephalopoda are composed of two unequal fragments held together by regions of hydrogen-bonding having a similar, low, thermal stability in the two species; the molecular weights of the two fragments composing the L-rRNA are estimated to be 0.96 X 10(6) and 0.88 X 10(6) (Loligo) and 0.96 X 10(6) and 0.65 X 10(6) (Octupus). THe S-rRNA of both species is a continuous chain with exactly the same molecular weight (0.96 X 10(6)) as the heavier of the two fragments of the L-rRNA. 4. The secondary-structure features of the L-rRNA and S-rRNA species of the Caphalopoda were investigated by thermal 'melting' analysis in 4.0 M-guanidinium chloride; 60-70% of the residues are estimated to form short, independently 'melting' bihelical segments not more than 10 base-pairs in length. 5. Bases are unevenly distributed between non-helical and bihelical portions of the rRNA molecules, G and C residues being preferentially concentrated in bihelical comains. 6. The secondary-structure regions of the L-rRNA species of Octopus and Loligo are heterogenous, including two discrete fractions of independently 'melting' species that give rise to biphasic 'melting' profiles: a fraction consisting of shorter (G + C)-poorer segments (60-68% G + C, not more than 5 base-pairs in length) and a fraction consisting of longer (G + C)-richer segments (80-88% G + C, 5-10 base-pairs in length). No evidence for heterogeneity has been detected in the S-rRNa.     

Dinoflagellate ribosomal RNA; an evolutionary relic?

The apparent molecular weights of the rRNA subunits of the dinoflagellate Peridinium cinctum fa. westii were determined by polyacrylamide gel electrophoresis to obtain an indication of its evolutionary status. The apparent molecular weights of the Peridinium light and heavy rRNA subunits were 0.7 and 1.23 X 10(6) respectively. The light rRNA is therefore typically eukaryotic but the heavy rRNA has a significantly lower MW than previously reported for higher eukaryotes which range from 1.3 to 1.75 X 10(6) depending on their evolutionary position. Thus by this criterion Peridinium is similar to red algae and may represent a eukaryotic stage, lower than fungi or green plants.     

Molecular weight distribution of ribosomal proteins from several vertebrate species.

Two-dimensional polyacrylamide gel electrophoresis of proteins from the separated ribosomal subunits of rabbit reticulocytes, rabbit liver, mouse liver, rat liver, chicken liver, and toad liver was performed using the "pH 4.5/SDS" system previously described (Martini and Gould, 1975), with internal standards to measure the molecular weight distributions. With few exceptions, the patterns were remarkably similar, indicating a high degree of conservation during evolution of both net charge (largely determining mobility in the first dimension) and size (determining mobility in the second dimension). The aggregate mass (sum of molecular weights) of both small and large subunit proteins, about 0.65 X 10(6) and 0.95 X 10(6) daltons respectively, were invariant. These figures are significantly smaller than the hydrodynamically determined mass of protein in the subunits. The implications of this discrepancy, which is opposite that found in the prokaryotes, is discussed.     

Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila.

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6 X 10(6) Da, compared with 20 proteins totalling 0.35 X 10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15 X 10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87 X 10(6) Da mass); a smaller difference. 0.15 X 10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8 X 10(6) and 1.65 X 10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.     

Structure, subunit composition, and molecular weight of RD-114 RNA.

The properties and subunit composition of the RNA extracted from RD-114 virions have been studied. The RNA extracted from the virion has a sedimentation coefficient of 52S in a nondenaturing aqueous electrolyte. The estimated molecular weight by sedimentation in nondenaturing and weakly denaturing media is in the range 5.7 X 10(6) to 7.0 X 10(6). By electron microscopy, under moderately denaturing conditions, the 52S molecule is seen to be an extended single strand with a contour length of about 4.0 mum corresponding to a molecular weight of 5.74 X 10(6). It contains two characteristic secondary structure features: (i) a central Y- or T-shaped structure (the rabbit ears) with a molecular weight of 0.3 X 10(6), (ii) two symmetreically disposed loops on each side of and at equal distance from the center. The 52S molecule consists of two half-size molecules, with molecular weight 2.8 X 10(6), joined together within the central rabbit ears feature. Melting of the rabbit ears with concomitant dissociation of the 52S molecule into subunits, has been caused by either one of two strongly denaturing treatments: incubation in a mixture of CH3HgOH and glyoxal at room temperature, or thermal dissociation in a urea-formamide solvent. When half-size molecules are quenched from denaturing temperatures, a new off-center secondary structure feature termed the branch-like structure is seen. The dissociation behavior of the 52S complex and the molecular weight of the subunits have been confirmed by gel electrophoresis studies. The loop structures melt at fairly low temperatures; the dissociation of the 52S molecule into its two subunits occurs at a higher temperature corresponding to a base composition of about 63% guanosine plus cytosine. Polyadenylic acid mapping by electron microscopy shows that the 52S molecule contains two polyadenylic acid segments, one at each end. It thus appears that 52S RD-114 RNA consists of two 2.8 X 10(6) dalton subunits, each with a characteristic secondary structure loop, and joined at the 5' ends to form the rabbit ears secondary structure feature. The observations are consistent with but do not require the conclusion that the two 2.8 X 10(6) dalton subunits of 52S RD-114 RNA are identical.     

Characteristics of Pseudomonas putida plasmid DNAs

Physico-chemical characteristics of plasmid DNAs isoalted from Pseudomonas putida G7 were studied as well as the behavior of these DNAs in th eourse of chromatography on columns with Sepharose 4B and kieselguhr with methylated albumin (MAC). This strain was found to contain several plasmid DNAs having molecular weights of 33-36X10(6), 15-18X10(6), and 3-5X10(6) dalton. The plasmid DNAs of biodegradation are supposed to be located in the vicinity of chromosomes, and only a small part of them is characterized by extrachromosomal localization.     

Mitochondrial gene sequences show fungal homology for Pneumocystis carinii

A 6.8 kilobase fragment of mitochondrial DNA from Pneumocystis carinii encodes for apocytochrome b, NADH dehydrogenase subunits 1, 2, 3, and 6, cytochrome oxidase subunit II, and the small subunit of ribosomal RNA. Comparative sequence analysis with a series of organisms representative of the fungal and protozoan groups shows that P. carinii has, consistently, an average similarity of 60% with the fungi but only 20% with the protozoa. The data indicate homology with the fungi for this opportunistic pathogen.     

The ribosomes of Plasmodium berghei: isolation and ribosomal ribonucleic acid analysis.

Ribosomes and high molecular weight ribosomal ribonucleic acid (rRNA) from the blood stages of Plasmodium berghei parasites were studied in preparations free from host ribosome contamination. Purified malarial ribosomes were isolated in high yield from a population of ultrastructurally intact, viable parasites by hypertonic lysis with Triton X-100 and differential centrifugation. These ribosomes were shown to be derived from active polysomes and could be dissociated into subunits by puromycin-0.5 M KCl treatment. Malarial rRNA extracted from purified 40S and 60S ribosomal subunits was characterized by electrophoretic, sedimentation and base ratio analyses. Like certain other protozoa, the P. berghei 40S ribosomal subunit possessed an exceptionally large RNA species (mol. wt 0.9 X 10(6), while RNA isolated from the parasite's 60S subunit (mol. wt 1.5 X 10(6)) was specifically 'nicked' to produce one large component (mol.wt 1.2 X 10(6)) and one small component (mol.wt 0.3 X 10(6)) in equimolar quantities. These rRNA's migrate identically on polyacrylamide gels after heating to 63 degrees C for 5 min or under denaturing conditions in the presence of formamide, indicating an absence of aggregation and non-specific degradation of the rRNA species. Base composition studies showed P. berghei rRNA to be low in guanosine and cytosine content, as is the case for protozoa generally.     

The ribosomal RNA of the trypanosomatid protozoan Crithidia fasciculata: physical characteristics and methylated sequences.

When extracted and analyzed under conditions which maintain noncovalently associated RNA-RNA complexes, the bulk cellular RNA of Crithidia fasciculata contains species of apparent molecular weights 1.3, 0.825, 0.08, 0.065, and 0.045 x 10(6) in addition to 5S rRNA and tRNA. Heat denaturation results in the disappearance of the 1.3 x 10(6) dalton RNA and the appearance of three new species having molecular weights of 0.67, 0.575, and 0.059 x 10(6). In addition, the apparent molecular weight of the 0.825 x 10(6) dalton component is reproducibly lowered to 0.81 x 10(6) after heat treatment. With the exception of tRNA, all of the RNA species are present in close to equimolar amounts in either undenatured or heat-denatured C. fasciculata bulk cellular RNA. On the basis of previous observations on the ribosomal RNA of the closely related organism, Crithidia oncopelti (Spencer, R. & Cross, G.A.M. (1976) J. Gen. Microbiol. 93, 82-88), the 1.3 and 0.825 x 10(6) dalton RNA's are considered to be components of the large and small subunits, respectively, of C. fasciculata ribosomes, but the subunit localization of the other RNA's described here has not yet been determined. O2'-Methylnucleosides account for about 1.4 mol% of the total nucleoside constituents of unfractionated C. fasciculata rRNA. Quantitative analysis suggests that the rRNA molecules in a C. fasciculata ribosome contain a total of 95-100 O2'-methyl groups, distributed in 80-85 Nm-Np sequences (including four 'hypermodified' Nm-Np, each containing a modification of a base or base-sugar linkage in addition to sugar methylation), six different Nm-Nm-Np sequences, and one Nm-Nm-Nm-Np sequence. While the specific pattern of O2'-methylation in the rRNA of C. fasciculata is distinct, both qualitatively and quantitatively, from the pattern observed in other organisms, Crithidia rRNA does contain certain 'universal' O2'-methylated sequences which appear to have been extensively conserved in evolution. The base-methylated nucleoside, N6,N6-dimethyladenosine (m26A), has been isolated from both C. fasciculata and wheat embryo rRNA in the form of the alkali-resistant dinucleotide, m26A-m26Ap. This dinucleotide and its enzymatic degradation products have been characterized by examination of their ultraviolet absorption spectra and electrophoretic and chromatographic properties.     

Butyrate-binding protein from rat and mouse liver

A novel component which specifically binds butyrate was found in rat and mouse liver. This component, termed butyrate binding protein (BBP), is localized in the cytosolic fraction and exhibits protein characteristics, such as heat- and protease-sensitivity. The size of BBP was found to be 7.6S, while it was converted to subunits of 45,000--48,000 dalton by treatment with sodium dodecyl sulfate. The dissociation constant of the binding of BBP with butyrate was 2.22 X 10(-6) M in the standard assay. 30-Fold purification of BBP was achieved by batch-wise adsorption and elution from CM-cellulose and hydroxylapatite column chromatography. BBP is clearly distinguishable from the fatty acid-binding protein found previously on the basis of its size and binding specificity.     

Light-scattering investigation of the subunit structure and dissociation of octopoda hemocyanins

The molecular weights, subunit dissociation, and conformation in solution of the hemocyanins of three species of octopi were investigated by light-scattering, ultracentrifugation, absorbance, and circular dichroism methods. The molecular weights of the hemocyanins of Octopus bimaculoides, Octopus bimaculatus, and Octopus rubescens obtained by light scattering were 3.3 X 10(6), 3.4 X 10(6), and 3.5 (+/- 0.3) X 10(6), respectively. The average molecular weights of the fully dissociated hemocyanins of the same octopi, investigated at alkaline pH and in the presence of 8 M urea and 6 M guanidinium chloride (GdmCl), were found to be close to one-tenth of those of the parent proteins, with average molecular masses of 3.4 X 10(5), 3.3 X 10(5), and 3.3 (+/- 0.3) X 10(5). These findings confirm the earlier observations of van Holde and co-workers with other cephalopod hemocyanins that the basic cylindrical assembly of molluscan hemocyanins consists of 10 subunits. Circular dichroism and absorbance measurements suggest that the dissociated subunits at alkaline pH and in concentrated urea solutions retain their native, multidomain folding. Fairly concentrated GdmCl above 3-4 M is necessary to unfold fully the dissociated hemocyanin chains. Molecular weight measurements studied as a function of reagent concentration with the urea and Hofmeister salt series as dissociating agents show that the ureas are very effective dissociating agents, while the salts are ineffective to moderately effective reagents for octopus hemocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ecology of gonococcal plasmids.

Of 261 strains of Neisseria gonorrhoeae examined for plasmids, 6 were plasmid-free, 217 contained only a small multicopy 2.6 X 10(6) dalton plasmid and 38 carried a large 24.5 X 10(6) dalton plasmid. Restriction enzyme digests and DNA-DNA hybridization studies revealed that the large plasmids isolated between 1940 and 1978 share a common core of DNA sequences (70 to 100%) and represent a group of closely related molecules.     

Multiple loop purification method for selective cultivation of Pentatrichomonas hominis

It is always troublesome having protozoan cultures contaminated with other organisms in the laboratory. The method described here produces high efficiencies of purification for fast moving flagellate protozoa. A human strain Pentatrichomonas hominis was employed in the study to examine the effects of multiple loop tubes on the purification of flagellates. Trichomonads were harvested from a trypticase yeast extract iron-serum-33 (TYI-S-33) medium, adjusted to 2 X 10(5) organisms/ml, and mixed with an equal volume of 2 X 10(6) organisms/ml of bacteria. The isolation was performed at 37 degrees C in TYI-S-33 medium containing a suitable amount of antibiotics (1000 U/ml of penicillin, 1000 micrograms/ml of streptomycin, and 4 micrograms/ml of fungizone). Four days later, 10(6) organisms/ml of protozoa, free of bacteria, were observed at the other end of the single loop and the double loop tubes. About the same amount of flagellates could be found at the other end of the triple loop tube six days after incubation. The traditional U-shaped tubes were used as controls and 10(5) cells/ml of flagellates were recovered in the presence of bacteria two days after incubation. An axenic culture of P. hominis was successfully isolated from the feces of a Formosan rock-monkey, Macaca cyclopsis, by this method. Purified trichomonads were recovered from a double loop purification tube five days after incubation.     

Detection and characterization of plasmids in Pseudomonas glycinea.

Pathogenic strains of Pseudomonas glycinea were shown to possess plasmid deoxyribonucleic acid by dye-buoyant density gradient centrifugation. The size and number of plasmids of four different isolates were determined by neutral sucrose gradient centrifugation. Two isolates were found to harbor a single plasmid; however, they differed in size, having molecular weights of 43 X 10(6) and 54 X 10(6). Two other isolates each contained two different plasmids. Plasmids with molecular weights of 43 X 10(6) and 73 X 10(6) were observed in one isolate, and the other carried plasmids with molecular weights of 25 X 10(6) and 87 X 10(6). An auxotrophic mutant derived from the latter strain was found to contain plasmids of identical size. The plasmids were found to be under stringent control of replication, having plasmid copies of 1.0 to 2.7 per chromosome equivalent. By the dye-cesium chloride technique, the mutant showed twice as much covalently closed circular deoxyribonucleic acid as did the parental strain.     

Further studies on the size and composition of the chick embryo fibroblast cytosolic DNA complex

The chick embryo fibroblast cytosolic DNA complex shows anomalous elution behaviour on agarose gel column chromatography. The indicated molecular size varies between 5 X 10(5) dalton (higher exclusion limit gels) and 1.4 X 10(6) dalton (lower exclusion limit gels). Chromatography on lower exclusion limit gels shows the [3H]thymidine labelled (DNA) complex as a sharp peak, coincident with a peak of [3H]uridine and [3H]lysine labelling and similar pulse labelling patterns for the three precursors but with DNA labelling lagging behind RNA and protein. Both cultured and uncultured cell cytosols show an A260 peak coincident with the [3H]precursor labelling peaks.     

The Mitochondrial Genomes of Two Nematodes, Caenorhabditis Elegans and Ascaris Suum

The nucleotide sequences of the mitochondrial DNA (mtDNA) molecules of two nematodes, Caenorhabditis elegans [13,794 nucleotide pairs (ntp)], and Ascaris suum (14,284 ntp) are presented and compared. Each molecule contains the genes for two ribosomal RNAs (s-rRNA and l-rRNA), 22 transfer RNAs (tRNAs) and 12 proteins, all of which are transcribed in the same direction. The protein genes are the same as 12 of the 13 protein genes found in other metazoan mtDNAs: Cyt b, cytochrome b; COI-III, cytochrome c oxidase subunits I-III; ATPase6, Fo ATPase subunit 6; ND1-6 and 4L, NADH dehydrogenase subunits 1-6 and 4L: a gene for ATPase subunit 8, common to other metazoan mtDNAs, has not been identified in nematode mtDNAs. The C. elegans and A. suum mtDNA molecules both include an apparently noncoding sequence that contains runs of AT dinucleotides, and direct and inverted repeats (the AT region: 466 and 886 ntp, respectively). A second, apparently noncoding sequence in the C. elegans and A. suum mtDNA molecules (109 and 117 ntp, respectively) includes a single, hairpin-forming structure. There are only 38 and 89 other intergenic nucleotides in the C. elegans and A. suum mtDNAs, and no introns. Gene arrangements are identical in the C. elegans and A. suum mtDNA molecules except that the AT regions have different relative locations. However, the arrangement of genes in the two nematode mtDNAs differs extensively from gene arrangements in all other sequenced metazoan mtDNAs. Unusual features regarding nematode mitochondrial tRNA genes and mitochondrial protein gene initiation codons, previously described by us, are reviewed. In the C. elegans and A. suum mt-genetic codes, AGA and AGG specify serine, TGA specifies tryptophan and ATA specifies methionine. From considerations of amino acid and nucleotide sequence similarities it appears likely that the C. elegans and A. suum ancestral lines diverged close to the time of divergence of the cow and human ancestral lines, about 80 million years ago.     

烟青虫质型多角体病毒的形态及某些理化特性

本文对烟青虫(Heliothis assulta)质型多角体病毒(CPV)的形态大小以及某些理化特性进行了研究。烟青虫CPV多角体为正五角形的十二面体,大小为0.8-4.6μm;CPV 粒子为外形呈六角形或球形的廿面体,大小为62nm。CPV多角体蛋白的主要多肽为一种,分子量23000,为非糖蛋白。 用SDS-酚法提取的CPV基因,经Rnase I和Dnase I处理后在1%琼脂糖凝胶上电泳,结果表明 其基因是双链RNA,并由10个基因片段组成。各片段大小为0.3-2.68X10⁶,总分子量为15.85x10⁶。本文所报道的烟青虫质型多角体病毒在国内外尚属首次。     

Mutagenesis and cloning of hly determinants of the paP20 plasmid

The data obtained indicate that pAP20 plasmid determinants for the synthesis and excretion of Escherichia coli haemolysin are located on the plasmid SalI-fragment f3. The size of f3 fragment is 12 X 10(6) dalton.     

Characterisation of ribosomal proteins from HeLa and Krebs II mouse ascites tumor cells by different two-dimensional polyacrylamide gel electrophoresis techniques

Summary Electrophoresis of ribosomal proteins according to Kaltschmidt and Wittmann, 1970a, b (pH 8.6/pH 4.5 urea system) yielded 29 proteins for the small subunits and 35 and 37 proteins for the large subunits of Krebs II ascites and HeLa ribosomes, respectively. Analysis of the proteins according to a modified technique by Mets and Bogorad (1974) (pH 4.5/pH 8.6 SDS system) revealed 28 and 29 proteins in the small subunits and 37 and 38 proteins in the large subunits of Krebs II ascites and HeLa ribosomes.The molecular weights of the individual proteins were determined by: 1. three-dimensional gel electrophoresis; 2. two-dimensional gel electrophoresis at pH 4.5/pH 8.6 in SDS. The molecular weights for 40S proteins ranged from 10,000 to 39,000 dalton (number average molecular weight: 21,000). The molecular weights for the 60S proteins ranged from 14,000 to 44,000 dalton (number average molecular weight: 23,000) using the three-dimensional technique. A molecular weight range from 10,000 to 38,000 dalton (number average molecular weight: 21,000) was obtained for the 40S subunits, whereas the molecular weights for the 60S ribosomal proteins (average molecular weight: 26,000) ranged from 12,000 to 69,000 dalton using the pH 4.5/pH 8.6 SDS system.The molecular weights of Krebs II ascites and HeLa ribosomal proteins are compared with those obtained by other authors for different mammlian species.     

Isolation and characterization of canine distemper virus-specific RNA

Ten species of virus-specific RNA were detected in Vero cells infected with the FXNO strain of canine distemper virus (CDV). The largest RNA was the genome-sized RNA and the nine smaller species were polyadenylated RNAs. Similar results were obtained for nine other strains of CDV. The molecular weights of these ten RNAs were determined to be 4.61 X 10(6), 2.46 X 10(6), 1.52 X 10(6), 1.32 X 10(6), 1.19 X 10(6), 1.07 X 10(6), 0.77 X 10(6), 0.65 X 10(6), 0.58 X 10(6), and 0.48 X 10(6). By in vitro translation of the polyadenylated RNAs in a rabbit reticulocyte lysate system, three different proteins which probably correspond to H, NP, and M were synthesized from the fraction containing RNAs 7, 8, 9, and 10.