Fractionation of plant Polyadenylated RNA on the basis of poly(A) size

Polyadenylated RNA from Vicia faba meristematic root cells was fractionated on the basis of mean poly(A) size by a thermal stepwise elution from poly(U) Sepharose. Such a procedure allowed the elimination of contaminating RNA at 30° and the collection of two populations of purified polyadenylated RNA at 40° and 50°, respectively. RNA eluting at the higher temperature carried a poly(A) segment (mean size of 100 nucleotides), twice as large as the RNA eluting at the lower temperature.     

Human liver nuclear transcortin its postulated role in glucocorticoid regulation of genetic activity

Highly purified human transcortin was injected into rabbits, and the antibody subsequently obtained was employed for the demonstration of transcortin-like molecules within various subcellular fractions of the human liver cell. Results obtained via quantitative double diffusion ouchterlony procedures indicate that proteins extracted from the nucleus or from chromatin form continuous precipitin lines of identity with those of transcortin. Fluorescein-tagged anti-transcortin permitted the visual localization of this molecule within isolated nuclei. Cortisol binding studies of all the subcellular fractions, particularly that extracted from the chromatin, suggest that such proteins do indeed bind cortisol specifically, as well as responding to exogeneous additions to the buffer (sulfhydryl reagents) as does purified transcortin. Purified transcortin when dialyzed exhaustively loses its cortisol binding ability, although the latter can be restored after its incubation with chromatin at 4°C. The restoration of such activity is dependent upon a dialyzable, heat-resistant chromatin component which itself lacks cortisol binding activity and which increases the sedimentation characteristics of dialyzed transcortin. The effect of transcortin on the in vitro synthesis of RNA in an Escherichia coli RNA polymerase human liver chromatin system is also presented. All of the above results are interpreted to indicate that transcortin is involved directly in the regulation of that genetic activity observed subsequent to the administration of cortisol.     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24°C are transferred rapidly to 32°C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)⁺ RNA is 24°C and 35% at 32°C. Kinetic analysis of cDNA-poly(A)⁺ RNA hybridizations reveals three abundance classes of poly(A)⁺ RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24°C and 13000 at 32°C. The intermediately abundant and highly abundant classes are represented by 100–200 and 1–3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)⁺ RNA with increase in temperature.     

Evidence for a role of RNA in eukaryotic chromosome structure. Metabolically stable, small nuclear RNA species are covalently linked to chromosomal DNA in HeLa cells

An investigation of metabolically stable, chromatin-associated RNA in HeLa cells has revealed that three small RNA species, 193, 171 and 127 nucleotides in length, are covalently linked to double-stranded chromosomal DNA through phosphodiester bonds. These DNA-linked RNAs appear to be members of the small nuclear RNA species that have been identified in a wide variety of eukaryotic cells, and they are tentatively identified as species C, D and G′, in the nomenclature system currently employed for HeLa cell small nuclear RNAs. These DNA-linked RNAs do not appear to be involved in priming DNA replication, since they are of relatively high metabolic stability ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 19}$ hours in HeLa cells with a 21·5-hour cell generation time) and since their covalently contiguous DNA stretches are not enriched in newly replicated material. They lack saturated pyrimidine bases (level of detection = 0·15 mol %) and are therefore not “chromosomal RNA”, as defined by its proponents. The covalent linkage of these small RNA species with chromosomal DNA was discovered by virtue of the fact that when highly purified HeLa cell chromatin is dissociated by chaotropic solutes, these RNAs are released in association with small pieces of double-stranded DNA (approx. 475 nucleotide pairs). These DNA-RNA complexes can then be purified by removing the bulk, high molecular weight DNA by ultra-centrifugation. The resulting DNA-RNA complexes are shown to be covalently joined by several criteria, including equilibrium density-gradient centrifugation in either Cs₂SO₄/dimethylsulfoxide or aqueous Cs₂SO₄/formaldehyde after thermal denaturation (90 °C in 50% formamide, which is 55 deg. C above the melting temperature of this DNA), by the chromat ographicbehavior of the complexes on hydroxylapatite before and after thermal denaturation, and by the demonstration of alkali-resistant ribonucleotides flanking the 3′ hydroxyl termini of the DNA, the latter criterion providing evidence for 3′ to 5′ DNA-RNA phosphodiester bonds. Reconstruction experiments involving addition of the purified RNAs to nuclei or chromatin demonstrate that the covalent DNA-RNA linkages do not arise by ligation events during cell fractionation. Further experiments indicate the existence of a dynamic equilibrium of these small nuclear RNA species between chromosomal and nucleoplasmic loci in vivo, and other considerations suggest that this equilibrium may be cell cycle-dependent. The DNA adjacent to these covalently linked RNAs has the same melting temperature as total HeLa chromosomal DNA and its reassociation kinetics reveal the presence of both repeated and non-repeated sequences, implying that the DNA-linked RNAs are widely distributed throughout the HeLa cell genome. It is proposed that these DNA-linked RNAs are involved in the tertiary structure of chromatin, particularly in relation to the cell cycle.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

Human bone marrow colony formation in diffusion chambers. Normal values.

In situ hybridization techniques have previously employed a series of manipulations to effect denaturation of chromosomal DNA and reannealing of DNA-RNA hybrids. This report presents a new protocol which combines the denaturation and reannealing processes. DNA is heated in a solution of 50% formamide, 50% 4 × SSC containing the RNA to be hybridized. After l h at 70 °C the preparation is slowly cooled to 37 °C over a period of 6 h and incubated at 37 °C for an additional 10 h. This technique eliminates the possibility of premature reannealing of the DNA while employing hybridization conditions which, in vitro, lead to accurate base pairing.     

Themoanaerobacterium calidifontis sp. nov., a novel anaerobic, thermophilic, ethanol-producing bacterium from hot springs in China

A novel thermophilic Gram staining positive strain Rx1 was isolated from hot springs in Baoshan of Yunnan Province, China. The strain was characterized as a hemicellulose-decomposing obligate anaerobe bacterium that is rod-shaped (diameter: 0.5–0.7 μm; length: 2.0–6.7 μm), spore-forming, and motile. Its growth temperature range is 38–68 °C (optimum 50–55 °C) and pH range is 4.5–8.0 (optimum 7.0). The maximum tolerance concentration of NaCl was 3 %. Rx1 converted thiosulfate to elemental sulfur and reduced sulfite to hydrogen sulfide. The bacterium grew by utilizing xylan and starch, as well as a wide range of monosaccharide and polysaccharides, including glucose and xylose. The main products of fermentation were ethanol, lactate, acetate, CO₂, and H₂. The maximum xylanase activity in the culture supernatant after 30 h of incubation at 55 °C was 16.2 U/ml. Rx1 DNA G + C content was 36 mol %. 16S rRNA gene sequence analysis indicated that strain Rx1 belonged to the genus Thermoanaerobacterium of the family ‘Thermoanaerobacteriaceae’ (Firmicutes), with Thermoanaerobacterium aciditolerans 761–119 (99.2 % 16S rRNA gene sequence similarity) being its closest relative. DNA–DNA hybridization between Rx1 and T. aciditolerans 761–119 showed 36 % relatedness. Based on its physiological and biochemical tests and DNA–DNA hybridization analyses, the isolate is considered to represent a novel species in the genus Thermoanaerobacterium, for which the name Thermoanaerobacterium calidifontis sp. nov. is proposed, with the type strain is Rx1 (=JCM 18270 = CCTCC M 2011109).     

Thermal stability of carbonic anhydrase immobilized within polyurethane foam

Thermal stability of carbonic anhydrase (CA) immobilized within polyurethane (PU) foam was investigated. The catalytic activity of the enzyme was estimated by using p‐nitrophenyl acetate (p‐NPA) as the substrate in tris buffer containing 10% acetonitrile. The immobilized CA was stable during the repeatable washings and stability tests over 45 days stored in tris buffer at ambient conditions indicating that the CA was covalently attached to the polyurethane (PU) foam by crosslinking. The immobilized CA was found to be 98% stable below 50°C, whereas a drastic decrease was seen at temperatures between 50 and 60°C. The optimum temperature for the immobilized CA was found to be 45°C and it lost its activity completely at 60°C. Thermal deactivation energies for the free and immobilized CA were estimated to be 29 and 86 kcal/mol, respectively. The association of unfolded CA with the polymeric backbone chains of the PU foam was also addressed. It was concluded that the immobilized CA was highly stable at temperatures less than 50°C and could be used in biomimetic CO₂ sequestration processes. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

RNA from callus cultures and leaves of Nicotiana tabacum

MAK column chromatography has been used to analyse RNA from normal and crown gall callus cultures and leaves of Nicotiana tabacum. To determine the elution behaviour of well-defined DNA-like RNAs with different GC content, complementary RNAs (c-RNA) synthesized on Agrobacterium tumefaciens DNA and crown gall DNA were used. The elution profile of the RNA from all three tissues followed a similar pattern. By salt gradient elution the RNA in the tRNA region showed a remarkably high CMP content which was significantly higher for the normal tissues than for crown gall tissue. RNA from the callus cultures contained more DNA-like RNA (D-RNA) with a higher turnover rate than RNA from leaves. Because of its relatively low poly A content, measured as RNase A + T₁ resistance, as well as its high turnover rate, the salt-eluted D-RNA is thought to be heterogeneous nuclear RNA (Hn-RNA) and not mRNA. RNA molecules that might represent the mRNA population, having intramolecular poly A tracts, were subsequently eluted by a salt gradient, a low salt buffer and with the chaotropic agent guanidine thiocyanate, which removed tenaciously bound (TB-RNA) in two fractions, α and β. Crown gall RNA showed both a different labelling behaviour and a higher poly A content in the α and β fractions compared to the normal tissues. c-RNAs may be eluted at different salt concentrations because of their different GC content. They give rise to a considerable fraction of TB-RNA which in the presence of tobacco leaf RNA was split into fractions similar to α and β. No fraction was found amongst these RNAs which did have intramolecular poly A tracts.     

Transcriptional activity and chromatin structural changes in a temperature-sensitive mutant of BHK cells blocked in early G1.

AF8, a temperature-sensitive mutant of BHK $\text{21}\text{13}$ cells, has been shown to arrest at the non-permissive temperature in the G1 phase of the cell cycle. When AF8 cells are released from density-dependent arrest of growth by trypsinization and replating at lower density, 60–80% of the cells enter DNA synthesis and divide at the permissive temperature (33 °C), while only 20% enter DNA synthesis at the non-permissive temperature (39.5 °C). The temperature-sensitive block has been localized 4 to 8 h before the onset of DNA synthesis which begins at 12 h after stimulation. Two biochemical events of the prereplicative phase have been temporally related to this temperature-sensitive block. RNA synthesis as measured in isolated nuclei increases initially at both temperatures, then levels off and declines to control levels at 39.5 °C while continuing to increase at 33 °C. Parallel changes are found in circular dichroism spectra and ethidium bromide binding capacity of isolated chromatin. The results suggest that these biochemical changes are involved in the regulation of the prereplicative phase and the subsequent entrance of cells into DNA synthesis.     

Liposomes from the thermophilic eubacterium Thermus SP. fluorescence polarization and permeability properties

• 1.1. The physical properties of liposomes prepared from total and polar lipid extracts of Thermus SPS 11 and SPS 17 grown at 50°C and at the optimal growth temperature (73–75°C) have been studied using 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization (P) and correlated to the permeability properties of the bilayers.
• 2.2. P data show the presence of a broad phase transition from a gel to a liquid-crystal phase for all liposomes studied. Liposomes from lipid fractions of Thermus SPS 11 and SPS 17 grown at 50°C were in a less fluid state than those of bacteria grown at the optimal growth temperature.
• 3.3. Comparing the permeabilities to ethyleneglycol, glycerol and erythritol of liposomes from lipid extracts of Thermus SPS 11 and SPS 17 grown at the two temperatures already mentioned, liposomes from lipid extracts of bacteria grown at 50°C were, in general, more permeable to ethyleneglycol and glycerol and less permeable to erythritol.
• 4.4. Liposomes from lipid fractions with higher carotenoid (CA) content were in a more rigid state within the whole range of temperatures studied, and their permeabilities to the three polyols were significantly lower than in the low-CA samples.

     

Hybridization properties of nucleolar ribonucleic acid.

Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.     

Pressure effects on thermal preference behaviour in gammarid amphipods from 600–1000m in Lake Baikal

Temperature preference behaviour of gammarid crustaceans from depths between 600–2000 m in Lake Baikal was studied in a system which provided a stable temperature gradient at pressures ranging from 50–150 atm. At the pressure of their habitat, these animals show well-defined modal distributions of sojourn temperatures around mean values from 3.0–5.5°C, av. 3.9 ± 0.3°C; mean modal T_p is estimated at 3.5°C. Year-round habitat temperatures are 3.0–3.6°C. The effect of changing pressure upon sojourn temperatures was explored over the range 50–150 atm. The slope of the mean sojourn temperature/pressure curves was 2.1°C/100 atm, significantly greater than 0. Mean nodal temperature estimates indicate that the corresponding slope in the range of 50–100 atm is 3°C/100 atm, and in the range of 100–150 atm, is likely to exceed 5°C/100 atm.