DNA base composition of planktonic species of Anabaena (Cyanobacteria) and its taxonomic value

Fifty axenic strains of planktonic Anabaena, including 24 strains of the straight form and 26 strains of the coiled form, were examined for their DNA base composition (GC content). The taxonomic value of their GC content at species level was evaluated by comparing their morphological, physiological and biochemical properties. The DNA base composition determined for all fifty strains ranged from 35.9 to 56.4 mol% GC. The straight-form strains were in the range of 35.9-56.4 mol% GC, while coiled forms were in the range of 38.1-50.3 mol% GC. In general, strains assigned to the same species showed similar DNA base composition. However, of three strains of A. affinis Lemmermann that were separated into two categories, two had 40.6-40.9 mol% GC, and the third strain 45.6 mol% GC. It is noteworthy that the DNA base composition of the newly established species A. eucompacta Li et Watanabe was 45.5 mol% GC, which differed from 39.5 mol% GC of the morphologically close species, A. compacta (Nygarrd) Hickel.     

Kinetics of methylation in Escherichia coli K-12.

Newly synthesized DNA is undermethylated in E. coli K-12. The amount of N6-methyl deoxyadenylic acid in labeled DNA varied from 0.3 mol% of total adenine for a 2-min pulse to 1.7 mol% for DNA that was labeled for more than two generations.     

Taxonomy of alkaliphilic Bacillus strains

The DNA base compositions of 78 alkaliphilic Bacillus strains were determined. These strains were grouped as follows: DNA group A, guanine-plus-cytosine (G+C) content of 34.0 to 37.5 mol% (17 strains); DNA group B, G+C content of 38.2 to 40.8 mol% (33 strains); and DNA group C, G+C content of 42.1 to 43.9 mol% (28 strains). DNA group A includes the type strain of Bacillus alcalophilus Vedder 1934. DNA-DNA hybridization studies with DNA group A strains revealed that only one strain, strain DSM 2526, exhibited a high level of DNA homology with B. alcalophilus DSM 485T (T = type strain). Neither strain DSM 485T nor any other DNA group A strain is homologous to any of the Bacillus type strains with comparable base compositions. Six strains formed a distinct group containing three highly homologous strains and three strains exhibiting greater than 50% DNA homology.     

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10?mol%, 50?mol% and 90?mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90?mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.     

Rapid GC mol% screening of primary culture lysates using horizontal slab gel electrophoresis

An electrophoretic technique for the rapid screening of GC mol% of bacterial DNA was modified and evaluated. Modifications of the technique included its adaptation to horizontal slab electrophoresis. Primary culture lysates (one per gel) of bacterial strains with unknown ratios of G+C/A+T+G+C (GC mol%), and reference strains whose GC mol% had been determined by thermal denaturation, were simultaneously electrophoresed for 2 h in polyacrylamide-agarose gels and mobilites of the chromosomal DNA bands were compared with the GC mol% values obtained from thermal denaturation curves. Results indicated a positive correlation (r = 0.80, df = 23) between GC mol% and electrophoretic mobility. The procedure, as modified, requires a minimum of equipment and resources and allows for the determination of GC mol% values with sufficient accuracy to serve as a means for inexpensive and routine screening of bacterial isolates.     

Base composition heterogeneity of Euglena gracilis chloroplast DNA.

Euglena gracilis chloroplast DNA has an average buoyant density of 1.685 gm/cm3, corresponding to 25 mol% G . C base pairs. To test for base compositional heterogeneity within this 130 kilobase pairs (kbp) genome, previously mapped restriction endonuclease fragments were isolated, and characterized by equilibrium buoyant density centrifugation. The chloroplast DNA can be characterized as containing two major buoyant density components. A segment of 17 kbp, representing 13% of the genome and containing the rRNA genes is 43--44 mol% G . C. The remaining 113 kbp, accounting for 87% of the genome, has an average 20--21 mol% G . C content.     

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.     

Influence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA

To investigate the possibility of producing charge-neutral gene delivery complexes with extended, non-particulate structures, DNA was allowed to self-assemble with a series of hydrophilic cationic polymers containing quaternary charged trimethylammonio ethylmethacrylate (TMAEM, 5, 15, 50, 100 mol%) copolymerised with hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA, 95, 85, 50, 0 mol%, respectively). Copolymers were all able to bind DNA, assessed using ethidium bromide fluorescence, although copolymers with low TMAEM content did not expel ethidium bromide. Increasing TMAEM content of the copolymers changed the morphology of the complexes from extended (5-15 mol% TMAEM), through partially condensed particles (50 mol%) to discrete nanoparticles (100 mol% TMAEM). Complexes based on copolymers with low TMAEM content (5-50 mol%) showed less resistance to degradation by nucleases and lower surface charge (21.2+/-5.9-45.1+/-3.9 mV) than those formed using 100 mol% TMAEM (57.8+/-8.2 mV). They also showed significantly less association with phagocytic cells in vitro (human leucocytes, uptake decreased by up to 92.3%; murine peritoneal macrophages, uptake decreased by up to 69.6%), although in vivo their hepatic accumulation was only slightly decreased (maximum decrease 27.6%). Finding the appropriate balance of hydrophilicity and stability is key to development of effective vectors for gene delivery.     

Physico-chemical characteristics of the DNA of the nuclear polyhedrosis virus of the moth Porthetria dispar L

DNA preparations were obtained after dissolving the inclusion bodies, polyhedra virus particles, from the purified bundle virus of Porthetria dispar L. nuclear polyhedrosis. The DNA molecules in the preparations obtained are of different conformation and separate within the CsCl density gradient in the presence of ethidium bromide into supercoiled catenated and relaxed circular molecules (with the admixture of linear molecules). The circular DNA was studied by electron microscopy. The size of virus genome according to the data of reassociation kinetics of DNA is about 100 MD. Estimated on the basis of the values of buoyant density (p) and the melting temperature (Tmelt.) the content of guanine-cytosine pairs (GC pairs) in the viral DNA varies from 61 up to 65 mol%, and in the insect cell DNA--from 38 up to 40 mol%. The viral and cellular DNA are distinctly separated by centrifugation within the CsCl density gradient.     

QUANTUM AND CONTINUOUS EVOLUTION OF DNA BASE COMPOSITION IN THE YEAST GENUS PICHIA

This paper investigates the noncontinuous nature and evolution of the base composition of nuclear DNA (expressed as mol% guanine + cytosine) in species of the yeast genus Pichia (sensu Kurtzman, 1984b). The pattern of change in the G + C contents in species of this genus, which range from about 27 to 52 mol%, was evaluated. When specifically those species of Pichia were analyzed that have evolved in necroses of cactus species and associated Drosophila, a periodic change in the G + C contents of approximately 3.0–3.2 mol% was detected by a “bootstrapping” method, Fourier analysis, and a nonlinear trigonometric model. Pichia species occurring in exudates of broad-leaved deciduous trees or associated Drosophila and substrates such as soil and water (“other”) showed a periodicity of 2.5–2.6 mol%, whereas species associated with conifers and associated bark beetles showed no significant periodicity. Periodicity in the most recent association (cactus and resident Drosophila) as compared to the lack of periodicity in the oldest association (conifer-beetle) may indicate mixed evolutionary processes. Low mol% G + C values appear more frequently in the relatively recent cactus and Drosophila-associated yeast species. In addition, low mol% G + C species do not display the ancestral bud-meiosis mode of sexual reproduction which occurs frequently in medium to high mol% G + C yeasts. It was found that the mol% G + C content of the Drosophila- and cactus-associated Pichia species is positively correlated with the number of compounds fermented or respired by these yeast species. Possible reasons for the periodic changes in mol% G + C content accompanying speciation include aneuploidy, allopolyploidy, the presence of nuclear plasmids, and regular differences in moderately repetitive portions of DNA. Since significant DNA complementarity is virtually limited to species within a relatively narrow G + C group, this suggests that there are at least two processes which alter the G + C content between species, one saltational and one continuous.     

DNA base composition of Rickettsia tsutsugamushi determined by reversed-phase high-performance liquid chromatography

The DNA base composition of Rickettsia tsutsugamushi was determined by reversed-phase high-performance liquid chromatography and compared with that of Rickettsia rickettsii. The G+C contents were 28.1 to 30.5 mol% for R. tsutsugamushi and 32.1 mol% for R. rickettsii.     

Myxozyma neglecta sp. nov. (Candidaceae) a new yeast species from South Africa

Three strains of Myxozyma mucilagina including the type strain were reexamined. Based on differences in their carbon utilization pattern, mobility of isoenzymes, mol% G + C of their DNA and extent of DNA complementary the new species Myxozyma neglecta is proposed.     

Differentiation of Gluconacetobacter liquefaciens and Gluconacetobacter xylinus on the basis of DNA base composition,DNA relatedness,and oxidation products from glucose

Gluconacetobacter liquefaciens and Gluconacetobacter xylinus share very similar phenotypic characteristics. They are differentiated by the production of a reddish-brown water-soluble pigment of the former and cellulose production of the latter. However, the loss of the two distinguishing features questions the separate standings of the two species. The DNA base composition and the DNA relatedness of strains of the two species, including other established species of acetic acid bacteria, were determined. G. liquefaciens strains had the higher guanine-plus-cytosine content (G+C content) in DNA, ranging from 63.5 to 66.9 mol%, and G. xylinus had the lower range, from 59.4 to 63.2 mol%. DNA hybridization revealed a low level of DNA similarity between the two species. G. liquefaciens strains produced 2,5-diketogluconic acid and pyrones from glucose, and G. xylinus strains produced 5-ketogluconic acid. From these results, it is unequivocal that G. liquefaciens is a distinct species from G. xylinus.     

A Contrast of the Plasma Membrane Lipid Composition of Oat and Rye Leaves in Relation to Freezing Tolerance

The lipid composition of the plasma membrane isolated from leaves of spring oat (Avena sativa L. cv Ogle) was vastly different from that of winter rye (Secale cereale L. cv Puma). The plasma membrane of spring oat contained large proportions of phospholipids (28.8 mol% of the total lipids), cerebrosides (27.2 mol%), and acylated sterylglucosides (27.3 mol%) with lesser proportions of free sterols (8.4 mol%) and sterylglucosides (5.6 mol%). In contrast, the plasma membrane of winter rye contained a greater proportion of phospholipids (36.6 mol%), and there was a lower proportion of cerebrosides (16.4 mol%); free sterols (38.1 mol%) were the predominant sterols, with lesser proportions of sterylglucosides (5.6 mol%) and acylated sterylglucosides (2.9 mol%). Although the relative proportions of individual phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine, and the molecular species of these two phospholipids were similar in oat and rye, the relative proportions of di-unsaturated species of these two phospholipids were substantially lower in oat than in rye. The relative proportions of sterol species in oat were different from those in rye; the molecular species of cerebrosides were similar in oat and rye, with only slight differences in the proportions of the individual species. After 4 weeks of cold acclimation, the proportion of phospholipids increased significantly in both oat (from 28.8 to 36.8 mol%) and rye (from 36.6 to 43.3 mol%) as a result of increases in the proportions of phosphatidylcholine and phosphatidylethanolamine. For both oat and rye, the relative proportions of di-unsaturated species increased after cold acclimation, but the increase was greater in rye than in oat. In both oat and rye, this increase occurred largely during the first week of cold acclimation. During the 4 weeks of cold acclimation, there was a progressive decrease in the proportion of cerebrosides in the plasma membrane of rye (from 16.4 to 10.5 mol%), but there was only a small decrease in oat (from 27.2 to 24.2 mol%). In both oat and rye, there were only small changes in the proportions of free sterols and sterol derivatives during cold acclimation. Consequently, the proportions of both acylated sterylglucosides and cerebrosides remained substantially higher in oat than in rye after cold acclimation. The relationship between these differences in the plasma membrane lipid composition of oat and rye and their freezing tolerance is presented.     

Separation of Mycobacterium gadium from other rapidly growing mycobacteria on the basis of DNA homology and restriction endonuclease analysis

DNA was isolated from Mycobacterium gadium with high purity. Its G + C content was between 64 and 67 mol%. The homology of M. gadium DNA with DNA from three other rapidly growing mycobacteria was less than 22%, which indicates that M. gadium is a discrete genomic species. Analysis of the DNAs with restriction endonucleases supported this finding.     

Unique modification of adenine in genomic DNA of the marine cyanobacterium Trichodesmium sp. strain NIBB 1067.

The genomic DNA of the marine nonheterocystous nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067 was found to be highly resistant to DNA restriction endonucleases. The DNA was digested extensively by the restriction enzyme DpnI, which requires adenine methylation for activity. The DNA composition, determined by high-performance liquid chromatography (HPLC), was found to be 69% AT. Surprisingly, it was found that a modified adenine which was not methylated at the usual N6 position was present and made up 4.7 mol% of the nucleosides in Trichodesmium DNA (15 mol% of deoxyadenosine). In order for adenine residues to be modified at this many positions, there must be many modifying enzymes or at least one of the modifying enzymes must have a degenerate recognition site. The reason(s) for this extensive methylation has not yet been determined but may have implications for the ecological success of this microorganism in nature.     

超慢生大豆根瘤菌DNA G+C含量和DNA同源性测定

用热变性温度法和液相复性速率法分别测定了超慢生大豆根瘤菌(ESG,extra-slow-growing soybean rhizobia)DNA G+C mol%及与其它根瘤菌间的DNA同源性.结果表明,ESG的DNA G+C mol含量在59.2—63.5%之间,且不同地区不同血清型的ESG代表菌株DNA同源率在70%以上,说明它们是遗传型一致的类群.ESG与在大豆上结瘤的快生大豆根瘤菌(Rhizobium fredii USDA205)同源率为14.8%,与慢生大豆根瘤菌(Bradyrhizobiumjaponicum)三个DNA同源组的同源率分别为20.5%,30.0%,19.4%.测定结果还表明,ESG与其它根瘤菌遗传学的亲缘关系也很远.