Physiological and biochemical contributions to the taxonomy of the genus Prototheca

16 strains of the genus Prototheca do not produce extracellular amylolytic enzymes. The base composition of their DNA shows rather continuous values from 62% to 78% GC (guanine + cytosine). Their assignment to four species and their possible relationship with Chlorella protothecoides are discussed.Abbreviations Used GC guanine + cytosine - SSC saline sodium citrate     

DNA methylase from HeLa cell nuclei.

A DNA methylase has been purified 270-fold from HeLa cell nuclei by chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite. The enzyme transfers methyl groups from S-adenosyl-L-methionine to cytosine residues in DNA. The sole product of the reaction has been identified as 5-methylcytosine. The enzyme is able to methylate homologous (HeLa) DNA, although to a lesser extent than heterologous DNA. This may be due to incomplete methylation of HeLa DNA synthesized in vivo. The HeLa enzyme can methylate single-stranded DNA, and does so to an extent three times greater than that of the corresponding double-stranded DNA. In single-stranded M. luteus DNA, at least 2.4% of the cytosine residues can be methylated in vitro by the enzyme. The enzyme also can methylate poly (dG-dC-dG-dC) and poly (dG, dC). Bilateral nearest neighbors to the 5-methylcytosine have been determined with M. luteus DNA in vitro and HeLa DNA in vivo. The 5' neighbor can be either G or C while the 3' neighbor is always G and this sequence is, thus, p(G/C)pmCpG.     

Methanogenium,a new genus of marine methanogenic bacteria,and characterization ofMethanogenium cariaci sp. nov. andMethanogenium marisnigri sp. nov.

A new genus of marine methanogenic bacteria and two species within this genus are described.Methanogenium is the proposed genus andMethanogenium cariaci the type species. Cells of the type species are Gram-negative, peritrichously flagellated, irregular cocci with a periodic wall surface pattern. Colonies formed by these bacteria are yellow, circular and umbonate with entire edges. The DNA base composition is 52 mol% guanine plus cytosine. Formate or hydrogen and carbon dioxide serve as substrates for growth. Cells ofMethanogenium marisnigri are of similar shape but smaller diameter thanM. cariaci. The colonies ofM. marisnigri are convex, and the DNA base composition is 61 mol % G+C. Formate or hydrogen and carbon dioxide are growth substrates. Sodium chloride is required for growth of both methanogens.Abbreviations SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2 ethanesulfonic acid) - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

A Mutant of Uracil DNA Glycosylase That Distinguishes between Cytosine and 5-Methylcytosine

We demonstrate that a mutant of uracil DNA glycosylase (N123D:L191A) distinguishes between cytosine and methylcytosine. Uracil DNA glycosylase (UDG) efficiently removes uracil from DNA in a reaction in which the base is flipped into the enzyme’s active site. Uracil is selected over cytosine by a pattern of specific hydrogen bonds, and thymine is excluded by steric clash of its 5-methyl group with Y66. The N123D mutation generates an enzyme that excises cytosine. This N123D:L191A mutant excises C when it is mispaired with A or opposite an abasic site, but not when it is paired with G. In contrast no cleavage is observed with any substrates that contain 5-methylcytosine. This enzyme may offer a new approach for discriminating between cytosine and 5-methylcytosine.     

Ballistosporous yeasts found on the surface of plant materials collected in New Zealand

Six new species of ballistosporous yeast, the genusSporobolomyces, were isolated from dead leaves and fruit of plants collected in New Zealand;Sp. novazealandicus, Sp. dimmenae, Sp. coprosmicola, Sp. coprosmae, Sp. dracophyllus, andSp. taupoensis. These species differ from any hitherto known species ofSporobolomyces based on chemotaxonomic characteristics.Abbreviations B. Bullera - Ben. Bensingtonia - Sp. Sporobolomyces - Sporid. Sporidiobolus - T. Tilletiopsis - U. Udeniomyces - G + C guanine plus cytosine     

An extremely thermophilic Methanococcus from a deep sea hydrothermal vent and its plasmid

An extremely thermophilic methanogen was isolated from a hydrothermal vent core sample from Guaymas Basin, Gulf of California, at a depth of 2003 m. The isolate, designated strain AG86, was a coccoid autotroph using H₂-CO₂ as energy and carbon source with a growth temperature range of 48 to 92°C, optimum, 85°C. AG86 required NaCl and Mg²⁺ and trace amounts of selenite and tungstate. Vitamins were not required. However, yeast extract, Casamino acids and Trypticase stimulated growth significantly. When grown in the presence of these stimulants and at the optimal growth temperature and pH 6.5, the minimum doubling time was 20 min. Cells were fragile and readily lysed by detergents. The mol% G+C was 33%. These results and partial 16S rRNA sequencing indicated that AG86 belonged to the genus Methanococcus and closely resembled Methanococcus jannaschii. Tests for extrachromosomal DNA revealed a plasmid in AG86 and two plasmids in M. jannaschii. Different patterns were obtained from restriction endonuclease digestion of the three plasmids, and no homology was observed with DNA-DNA hybridization.Abbreviations CCC DNA covalently close circular DNA - DM defined marine medium - G+C Guanine plus cytosine - MPN most probable number     

A mutant of Mycoplasma mycoides subsp. mycoides lacking the H2O2-producing enzyme l-α-glycerophosphate oxidase

A universal rapid procedure to determine the DNA base composition (mol% guanine + cytosine) of Gram-positive bacteria is described. Cells of Gram-positive bacteria were lysed with achromo-peptidase and the mol% G + C of their DNAs were determined by using high performance liquid chromatography. One ml of a Gram-positive bacterial suspension which matched MacFarland No. 3 standard turbidity was sufficient to determine the mol% G + C within 3 h.     

Fermentation of glutamate by Selenomonas acidaminophila sp. nov.

From an anaerobic digester a novel type of strictly anaerobic, Gram-negative, non-sporeforming mesophilic bacterium was isolated. The cells were curved rods, motile by means of lateral flagella and contained b- and c-type cytochromes. The G+C content of the DNA was 48.0±1.0 mol%. The isolate was able to ferment only glutamate, aspartate, lactate and pyruvate. Organic fermentation products were acetate, propionate and succinate. Propionate was probably formed via a reductive succinate pathway. Strain DKglu16 is described as the type strain of a new species, Selenomonas acidaminophila sp. nov., in the family Bacteroidaceae.Abbreviations atm atmosphere - Pa Pascal - SSC standard saline citrate - G+C guanine+cytosine - _max maximum specific growth rate     

Determination of the replication time of nucleolar organizer DNA after 5-azacytidine treatment for restricted parts of the S period

Summary In vivo exposure to 5-azacytidine (10^–6M) depressed the incorporation of methyl groups to GC rich regions ofAllium cepa L. DNA. Nearly 22% of its 5-methylcytosine residues were under-methylated. The treatment stimulated 1.8 times the rate of [³H]uridine incorporation, as measured in meristems proliferating under steady state kinetics. Nucleologenesis was shortened from 2.7 to 1.6 h in synchronous binucleate cells after 5-azacytidine treatment lasting the whole S period of their previous interphase. By hypomethylating DNA sequences replicated at different times during the S period, it could be inferred that the cistron replication took place in early S. Thus, nucleogenesis was shortened to only 0.6 h after such treatment. Sequential short treatment periods using [³H]thymidine confirmed that the nucleolar organizer regions of the chromosomes replicate in early S.Abbreviations A adenine - AZA 5-azacytidine - C cytosine - EDTA ethylenediamine tetraacetic acid - mC 5-methylcytosine - T thymine - TE tris-HCl EDTA buffer - HPLC high pressure liquid chromatography - NOR nucleolar organizer region - PPO 2,5-diphenyloxazole - POPOP 1,4-di[2(5-phenyloxazolyl) benzene - TCA trichloroacetic acid - tris tris (hydroxy-methyl) aminomethane     

High diversity in DNA of soil bacteria.

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caenorhabditis elegans DNA does not contain 5-methylcytosine at any time during development or aging.

DNA, isolated from age-synchronous senescent populations of Caenorhabditis elegans has been quantitatively and qualitatively analyzed for the presence of 5-methylcytosine. High performance liquid chromatography on two wild-type and several mutant strains of C. elegans failed to detect any 5-methylcytosine. The restriction endonuclease isoschizomers, HpaII and MspI, were used to digest genomic DNA after CsCl purification and failed to detect any 5' cytosine methylation at any age. We conclude that C. elegans does not contain detectable (0.01 mole percent) levels of 5-methylcytosine.     

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (^m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of ^m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung ⁺ host proficient in uracil excision repair.     

High diversity in DNA of soil bacteria

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clostridium halophilium sp. nov. and C. litorale sp. nov., an obligate halophilic and a marine species degrading betaine in the Stickland reaction

Two new mesophilic, sporeforming, gram-positive, strictly anaerobic, rod-shaped bacteria were isolated which utilized betaine in the Stickland reaction. Strain M1 was obtained from pasteurized hypersaline sediments. Cells were motile rods and formed spherical terminal spores. Betaine was used with hydrogen and several amino acids as electron donors. In addition, several carbohydrates served as substrates. Growth required 1.5% NaCl with an optimum at 6.0% NaCl. The guanine plus cytosine content of the DNA was 26.9%. This strain is described as a new species, Clostridium halophilum.Strain W6 was isolated from marine sediments. Cells were motile rods and formed ovoid, subterminal spores. Betaine was used with hydrogen and several amino acids as electron donors. Carbohydrates were not fermented. Growth optimum was at 1.0% NaCl. The guanine plus cytosine content of the DNA was 26.1%. This strain is described as a new species, Clostridium litorale.Non standard abbreviations DMG N,N-dimethylglycine - TMA trimethylamine - PY peptone-yeast extract - PYG peptone-yeast extract-glucose     

The distribution of 5-methylcytosine in the nuclear genome of plants.

We have determined the 5-methylcytosine (5mC) content in high molecular weight DNA, from two dicot (tobacco and pea) and two monocot (wheat and maize) plant species, fractionated according to base composition. The results show that the proportion of 5mC in the genomic fractions increases linearly with their guanine + cytosine (G + C) content while the proportion of non-methylated cytosine remains almost constant. This can be interpreted as a consequence of a difference in mutation pressure related to spontaneous deamination of 5mC to thymine between the different compartments of plant genomes.     

The mealybug chromosome system I: Unusual methylated bases and dinucleotides in DNA of aPlanococcus species

The methylation status of the nuclear DNA from a mealybug, aPlanococcus species, has been studied. Analysis of this DNA by High Performance Liquid Chromatography and Thin Layer Chromatography revealed the presence of significant amounts of 5-—methylcytosine. Since analysis of DNA methylation using the Msp I/Hpa II system showed only minor differences in susceptibility of the DNA to the two enzymes, it seemed possible that 5-methylcytosine (5mC) occurred adjacent to other nucleotides in addition to its usual position, next to guanosine. This was verified by dinucleotide analysis of DNA labelledin vitro by nick translation. These data show that the total amount of 5-methylcytosine in this DNA is slightly over 2.3 mol %, of which 0.61% occurs as the dinucleotide 5mCpG, 0.68% as 5mCpA, 0.59% as 5mCpT and 0.45% as 5mCpC. 5mCpG represents approximately 3.3% of all CpG dinucleotides. The experimental procedure would not have permitted the detection of 5mCp5mC, if it occurs in this system. Unusually high amounts of 6-methyladenine (approximately 4 mol %) and 7-methylguanine (approximately 2 mol %) were also detected, 6-methyladenine and 7-methylguanine occurred adjacent to all four nucleotides. The total G+C content was 33.7% as calculated from dinucleotide data and 32.9% as determined from melting profiles.     

A density functional theory study on the kinetics and thermodynamics of N-glycosidic bond cleavage in 5-substituted 2'-deoxycytidines

B3LYP/6-311+G(2d,p)//B3LYP/6-31+G(d) density functional theory calculations were employed to explore the kinetics and thermodynamics of gas-phase N-glycosidic bond cleavage induced by nucleophilic attack of C1' with a hydroxide ion in 5-substituted 2'-deoxycytidines. The results showed that, among the 5-substituted 2'-deoxycytidine derivatives examined [XdC, where X = H (dC), CH(3) (medC), CH(2)OH (hmdC), CHO (fmdC), COOH (cadC), F (FdC), or Br (BrdC)], fmdC and cadC exhibited the lowest energy barrier and largest exothermicity for N-glycosidic bond cleavage. These results paralleled previously reported nucleobase excision activities of human thymine DNA glycosylase (hTDG) toward duplex DNA substrates harboring a thymine and 5-substituted cytosine derivatives when paired with a guanine. Our study suggests that the inherent chemistry associated with the nucleophilic cleavage of N-glycosidic bond constitutes a major factor contributing to the selectivity of hTDG toward 5-substituted dC derivatives. These findings provided novel insights into the role of TDG in active cytosine demethylation.     

Unusual properties of the DNA from Xanthomonas phage XP-12 in which 5-methylcytosine completely replaces cytosine.

Xanthomonas phage XP-12 contains 5-methylcytosine completely replacing cytosine. This substitution confers several unusual properties upon XP-12 DNA. The buoyant density of XP-12 DNA in CsCl gradients is 1.710 g/cm-3, 0.16 g/cm-3 lower than that expected for a normal DNA with the same percentage of adenine plus thymine. The melting temperature for XP-12 DNA in 0.012 M Na+ is the highest reported for any naturally occurring DNA, 83.2 degrees C, 6.1 degrees C higher than that of normal DNAs with the same percentage of adenine plus thymine. Unlike the minor amounts of 5-methylcytosine found in most plant and animal DNAs, the 5-methylcytosine residues of XP-12 derive their methyl group from the 3-carbon of serine instead of from the thiomethyl carbon of methionine. .     

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine.

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.