The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

Location of Satellite and Homogeneous DNA Sequences on Human Chromosomes

HUMAN DNA^1,2 contains at least two satellite fractions—satellite I (0.5% of the genome) which bands at a density of 1.687 g/cm³ in neutral CsCl and satellite II (2% of the genome) which bands at 1.693 g/cm³. The main band DNA has an average buoyant density between 1.670 and 1.720 g/cm³ and a light shoulder (constituting 15% of the genome) with a buoyant density of 1.696 g/cm³, referred to as homogeneous mainband. Satellite DNA has been observed in many higher organisms³, usually with an unknown function, notable exceptions being cistrons coding for ribosomal RNA⁴ and the DNA coding for histone messenger RNA⁵. To investigate possible functions of human repetitive DNA we have studied the annealing of complementary RNA fractions to chromosomes using in situ hybridization^6,7. We describe here preliminary observations using human satellite II and homogeneous mainband fractions.     

Phylogenetic Origin of the Chloroplast*†

SYNOPSIS. The 16S ribosomal RNA of the chloroplast of Euglena gracilis strain Z has been characterized in terms of its 2-dimensional electrophoretic “fingerprint” (T1 ribonuclease). Over 100 spots were resolved on the “fingerprint” and each spot was characterized as to which RNA oligonucleotide fragment(s) it contained. When compared to similar analyses of prokaryotic 16S rRNAs and eukaryotic cytoplasmic 18S rRNAs, the chloroplast 16S rRNA was a typically prokaryotic RNA, but bore little if any relationship to eukaryotic 18S rRNAs. Therefore, the cistrons for chloroplast 16S rRNA are related to the equivalent prokaryotic cistrons, but, apparently, are not related to the equivalent eukaryotic cistrons. Among the organisms available for comparison, the Euglena chloroplast 16S rRNA appears most closely related to the 16S rRNA of the eukaryote, Porphyridium cruentum (a red alga), and at least distantly related to the 16S rRNAs of the blue-green algae and perhaps also to the bacilli.     

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

Melon rugose mosaic virus, the cause of a disease of watermelon and sweet melon

A mechanically transmissible virus with isometric particles c. 32 nm in diameter, was isolated from infected watermelons and sweet melons in the People's Democratic Republic of Yemen. Purified virus preparations contained two major sedimenting components with sedimentation coefficients of 61S and 117S. In isopycn ic centrifugation in CsCl the particles formed a single band of buoyant density 1.39 g cm^-3. Preparations of virus particles comprised of a single polypeptide of mol. wt c. 22 000 and ssRNA of mol. wt 2.1 × 10⁶. The virus was serologically related to three of six subgroups of tymoviruses tested. The name melon rugose mosaic virus is proposed for this newly described virus.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10⁶ daltons (native) DNA.In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA^Val, tRNA₁^Met and tRNA₂^Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm^?3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.     

Rottboellia yellow mottle virus,a new sobemovirus affecting Rottboellia cochinchinensis (Itch grass) in Nigeria1

A hitherto undescribed virus, termed rottboellia yellow mottle virus (RoYMV), affecting Rottboellia cochinchinensis (syn. R. exaltata, itch grass) at Ibadan, Nigeria, was investigated. RoYMV virions are isometric, c. 29 nm in diameter, and sediment homogeneously at 114s. In isopycnic CsCl gradients, RoYMV virions band sharply at a buoyant density of 1.379 g cm^-3, but in Cs₂SO₄ gradients, virions band at two zones, at the densities of 1.300 g cm^-3 and 1.325 g cm^-3. Treatment with EDTA at pH 8.0 reduced the sedimentation value of RoYMV to c. 87s and rendered it susceptible to proteinase-K, SDS and NaCl. The apparent molecular weight of RoYMV coat protein was c. 27 000. Virions encapsidate a single-stranded RNA of mol. wt 1.4 × 10⁶ Da. Besides R. cochinchinensis, RoYMV was mechanically transmissible only to maize (Zea mays). No reaction occurred when RoYMV was tested against antisera to 44 isometric plant viruses (belonging to 12 groups), including several that affect Gramineae. RoYMV exhibits striking similarities with other viruses belonging to the sobemovirus group, and it is tentatively designated as a new member of the sobemovirus group.     

Partial Characterization of Artichoke Virus M

A virus with filamentous particles 697 nm in length was isolated from artichoke plants in Southern Italy and identified as a new possible member of Carlavirus group, for which the name artichoke virus M (AVM) is suggested. AVM could not be transmitted by sap inoculation to herbaceous hosts and was always present in artichoke in mixed infections with other viruses. Virus particles had a buoyant density in CsCl of 1.31 g × cm^?3 and contained a single species of nucleic acid with an apparent size of 7.5 Kb and a single coat protein species with a mol. wt of 31,000. The virus was distantly related serologically to carnation latent and poplar mosaic carlaviruses but not to other members of the group including the recently described artichoke latent S carlavirus. Cytological alterations consisted of complex cytoplasmic inclusions composed of deranged organelles, lipid droplets and accumulations of membranes.     

Olive latent ringspot virus, a newly recognised virus infecting olive in Italy

A manually transmissible virus, for which the name olive latent ringspot virus (OLRV) is proposed, was isolated from a symptomless olive tree. The virus was mechanically transmitted to test plants. Purified preparations of OLRV contained three classes of isometric particle, c. 28 nm in diameter, with sedimentation coefficients of 525 (T), 975 (M) and 1325 (B) and containing 0, 30 and 43% nucleic acid respectively. At equilibrium in CsCl gradients, the buoyant densities of T and M components were 1–29 and 1–43 g/cm³ respectively, whereas B component separated into two sub-components with buoyant densities of 1–51 g/cm³ (BJ and 1–52 g/cm³ (B₂). Particle preparations contained two species of single-stranded RNA with mol. wt 1–40 times 10⁶ and 2–65 times 10⁶, both necessary for infectivity. The coat protein of OLRV, dissociated under strong denaturing conditions, separated into four components in polyacrylamide gel electrophoresis. Over 75% of the protein was found in a band with mol. wt 57 600, but all four components were recognised as oligomers of a monomeric form with mol. wt 14 300. OLRV was serologically unrelated to 26 different isometric plant viruses including 17 recognised nepoviruses. Its properties strongly indicate that it is a hitherto undescribed member of the nepovirus group.     

Precursors of chloroplast ribosomal RNA in Euglena gracilis

Incorporation of ³²P into mature chloroplast rRNA species of MW 1.1 × 101 and 0.56 × 10⁶ has been followed in Euglena gracilis by pulse and pulse chase experiments. Mature rRNA species have precursors of MW 1.16 × 10⁶ ± 0.01 × 10⁶ and 0.64 × 106 ± 0.01 × 10⁶ resp. These precursors have base composition and hydridization properties similar to those of the mature, rRNA species. No evidence of a single common precursor to these molecules was found. Rifampicin did not affect the synthesis of chloroplast rRNA.     

The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. III. Ribosomal RNA cistrons of the nucleus and leucoplast

The colorless alga Polytoma obtusum has been found to possess leucoplasts, and two kinds of ribosomes with sedimentation values of 73S and 79S. The ribosomal RNA (rRNA) of the 73S but not the 79S ribosomes was shown to hybridize with the leucoplast DNA (rho - 1.682 g/ml). Nuclear DNA of Polytoma (rho = 1.711) showed specific hybridization with rRNA from the 79S ribosomes. Saturation hybridization indicated that only one copy of the rRNA cistrons was present per leucoplast genome, with an average buoyant density of rho = 1.700. On the other hand, about 750 copies of the cytoplasmic rRNA cistrons were present per nuclear genome with a density of rho = 1.709. Heterologous hybridization studies with Chlamydomonas reinhardtii rRNAs showed an estimated 80% homology between the two cytoplasmic rRNAs, but only a 50% homology between chloroplast and leucoplast rRNAs of the two species. We conclude that the leucoplasts of Polytoma derive from chloroplasts of a Chlamydomonas-like ancestor, but that the leucoplast rRNA cistrons have diverged in evolution more extensively than the cistrons for cytoplasmic rRNA.     

Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA

Polyadenylated RNA, isolated from total cellular RNA of photoautotrophically grown Euglena gracilis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with ¹²⁵I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA.     

The chromosomal distribution of satellite DNA in the germ-line and somatic tissues of the gall midge,Heteropeza pygmaea

Somatic DNA from Heteropeza pygmaea separated in CsCl gradients into a main band DNA (?=1.685 g/cm³) and a satellite band (?=1.716 g/cm³) comprising 15% of the total DNA. The satellite melted sharply at 93.0°C in SSC, 10.4°C higher than the main band DNA. Satellite DNA reassociated rapidly, banding in CsCl heavier than native satellite but lighter than denatured satellite. The complementary strands of the satellite formed a single band in alkaline gradients and hence are apparently similar in G+T composition. — Filter hybridization experiments with Xenopus ribosomal RNA showed that the satellite band does not contain ribosomal cistrons. — Complementary RNA (cRNA) transcribed in vitro from isolated satellite bound extensively to satellite DNA but not to main band DNA. — The strain of Heteropeza used here contained about 58 chromosomes in germ-line cells and reproduced only paedogenetically. During early cleavage, the presumptive somatic nuclei eliminate most of their chromosomes (E-chromosomes) and retain only ten (S-chromosomes). In situ hybridizations with satellite-cRNA showed satellite DNA (prepared from predominately somatic tissues) to be localized in the centromeric heterochromatin of S- and E-chromosomes. Silver grain comparisons suggested that the amount of satellite is equivalent in both types of chromosomes. — Lastly we found that both diploid and polyploid cells contain similar amounts of satellite DNA. We interpreted this to mean that during polyploidization, as has been demonstrated during polytenization, satellite DNA does not replicate or replicates only slightly while other DNA fractions increase.     

Purification and characterization of two tyrosyl-tRNA synthetase activities from soybean cotyledons

The tyrosyl-tRNA synthetases located in cytoplasm and chloroplasts of soybean cotyledons were purified to near homogeneity by ammonium sulfate precipitation, DEAE-cellulose chromatography, hydroxylapatite chromatography, and DEAE-Sephadex A-25 chromatography. Purified cytoplasmic tyrosyl-tRNA synthetase shows only a single band in acrylamide gel electrophoresis which corresponds to a MW of 126000. In SDS-acrylamide gel electrophoresis the enzyme again shows only a single band which corresponds to a MW of 61 000. Chloroplast tyrosyl-tRNA synthetase shows only one band in both acrylamide and SDS-acrylamide gel electrophoresis with MWs being 98 000 and 43 000, respectively. For cytoplasmic tyrosyl-tRNA synthetase the apparent K_ms determined are 6.8 μM L-tyrosine, 49 μM ATP, and 8.9 × 10^?8 M tRNA (as total tRNA). Apparent K_ms for chloroplast tyrosyl-tRNA synthetase are 4.9 μM L-tyrosine, 214 μM ATP and 2.2 × 10^?8 M tRNA (as BDC-ethanol fraction tRNA). Fractionation of soybean cotyledon-tRNA on RPC-5 columns gives 4 tyrosyl-tRNA species, the first two species (tRNA_{1 and 2}^Tyr) are acylated only by cytoplasmic tyrosyl-tRNA synthetase while the last two species (tRNA_{3 and 4}^Tyr) are acylated only by chloroplast tyrosyl-tRNA synthetase.     

The molecular integrity of chloroplast ribosomal ribonucleic acid

Instability of chloroplast rRNA has been observed with essentially all chloroplast RNA preparations. This paper describes experiments that show that, under normal conditions of preparation and fractionation, only the heavy chloroplast component (mol.wt. 1.1x10(6)) is unstable, the light chloroplast rRNA (mol.wt. 0.56x10(6)) and the cytoplasmic rRNA species (mol.wt. 1.3x10(6) and 0.70x10(6)) being stable. The stability of the 1.1x10(6)-mol. wt. molecule varies with different plant species, as also does the size and the number of fragments produced. Cleavages in three particular regions of the molecule are very frequent within the range of tissues studied. The 1.1x10(6)-mol.wt. rRNA is, however, stabilized by the presence of Mg(2+) during the preparation and fractionation of the RNA.     

Further properties of wineberry latent virus and evidence for its possible involvement in calico disease

The flexuous filamentous particles of wineberry latent virus (WLV) were found to measure 620. 12 nm and not 510. 12 nm as previously reported. Analysis of dsRNA from infected plants detected a major species of c. 5.7. 10⁶ mol. wt and minor species of lower mol. wt. Purified virus particles formed a major and a minor buoyant density component in solutions of caesium salts with densities of 1.26 and 1.25 g cm^-3 in Cs₂SO₄ and 1.30 and 1.29 g cm^-3 in CsCl. The particles contained a single nucleic acid species, presumably single stranded RNA, and a single polypeptide of estimated mol. wt 2.78. 10⁶ and 31 000 respectively. In indirect ELISA, purified particles of WLV and particles in plant sap failed to react specifically with antiserum to nine carlaviruses, 12 potexviruses, three capilloviruses or apple chlorotic leafspot closterovirus, nor was WLV found to react with several of these antisera in immunosorbent electron microscopy or immunoblots. In Marion and Olallie blackberry, WLV in mixture with raspberry bushy dwarf virus (RBDV), but not RBDV alone, induced veinal line-pattern symptoms resembling those of calico disease reported from the USA.