The mitochondrial genome of wild-type yeast cells. IV. Genes and spacers

The organization of the mitochondrial genome of wild-type Saccharomyces cerevisiae cells has been investigated further, by degrading mitochondrial DNA with micrococcal nuclease. Under the conditions used, this enzyme very strongly degrades the A + T-rich stretches (spacers) whereas it only inflicts a limited number of breaks into the G + C-rich stretches (genes). The macromolecular fragments derived from the “genes” have been separated from the oligonucleotides originating from the “spacers” by gel filtration, and both sorts of products have been investigated. It has been shown (a) that the spacers are very homogeneous in base composition and have a G + C content lower than 5% (mitochondrial DNA has a G + C content of 18%); (b) that the genes are very heterogeneous in base composition, the G + C content ranging from about 25% to 50%, when the average size of the fragments is 1·2 × 10⁵; smaller fragments, molecular weight 4 × 10⁴, having a G + C level as high as 65%, have been isolated in a yield of 10%; the average G + C content of genes is about 32%; (c) that genes and spacers are present in about equal amounts in the mitochondrial genome and that they have comparable average sizes.     

DNA of some anaerobic rumen fungi: G + C content determination

The nuclear DNAs from five species of anaerobic rumen fungi have been isolated and purified by means of two extraction methods (with and without 8 M urea). Their G + C contents have been characterized by the thermal denaturation procedure of Marmur and Doty. As has already been shown in Neocallimastix frontalis, the results obtained by the two techniques demonstrated a very low G + C content (less than 20%) and the constant presence of satellite DNA.     

Heterogeneous base distribution in mitochondrial DNA of Neurospora crassa.

The mitochondrial DNA of Neurospora crassa has a heterogeneous intramolecular base distribution. A contiguous piece, representing at least 30% of the total genome, has a G+C content that is 6% lower than the overall G+C content of the DNA. The genes for both ribosomal RNAs are contained in the remaining, relatively G+C rich, part of the genome.     

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.     

Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen

Summary A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacteriumMethanobacterium thermoautotrophicum H has been cloned and sequenced. This DNA complements mutations in both the purE₁ and purE₂ loci ofEscherichia coli. The sequence of theM. thermoautotrophicum DNA predicts that complementation inE. coli results from the synthesis of a polypeptide with a molecular weight of 36,249. A polypeptide apparently of this molecular weight is synthesized inE. coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA. We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogenMethanobrevibacter smithii. The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology. The genome ofM. thermoautotrophicum has a molar G+C content of 49.7%, whereas the genome ofM. smithii is 30.6% G+C. Conservation of encoded amino acids while accommodating the very different G+C contents is accomplished by use of different codons that encode the same amino acid. The majority of base changes occur at the third codon position. The intergenic regions of the clonedM. thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters. Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene fromM. smithii maintains a codon usage that conforms to the RNY rule.     

Sequence homologies of (guanosine + cytidine)-rich regions of mitochondrial DNA of Saccharomyces cerevisiae.

The nucleotide sequences of two distinct regions of mitochrondrial DNA of Saccharomyces cerevisiae are reported. The regions studied have a high content of G + C (45%) and contain closely spaced Hpa II and Hae III restriction sites. Both regions have sequences that are homologous over a lenght of 47 base-pairs. In addition, the two regions are highly palindromic. These data support certain aspects of the organization of mitochondrial DNA proposed by Prunell and Bernardi (Prunell, A., and Bernardi, G. (1977) J. Mol. Biol. 110, 53--74).     

The relationship of DNA base composition and individual protein composition in micro-organisms

Summary To investigate the dependence of protein composition on DNA base composition, a set of data on individual proteins with known amino acid compositions from a spectrum of bacterial species has been compiled. It is found that similar relationships of amino acid frequency to G + C content exist for these proteins as for the bulk proteins studied by Sueoka (1961). The data are analysed by linear and cubic regression, and a measure of the proportions of A + T-rich and G + C-rich codons in the underlying messenger RNAs is put forward. The theoretical limits on the G + C content of coding DNA are discussed, and inference are made about the various selective forces acting on DNAs of different G + C contents.     

Physical characterization of a plasmid (pTT1) isolated from Thermus thermophilus

A small circular supercoiled DNA molecule species with a molecular weight of about 5.4 × 10⁶ has been isolated from the extreme thermophile Thermus thermophilus HB8. This plasmid (pTT1) has a G plus C content of 68%, similar to that of the host chromosome. The superhelix density is the same as that of bacteriophage PM2 DNA. A physical map of the plasmid has been obtained using restriction endonucleases.     

Physical properties of a plasmid-like DNA from Euglena gracilis

A small circular extrachromosomal DNA of the flagellate protozoan Euglena gracilis has been characterized as having a contour length of 11.3 kb, with a consistent restriction map. The buoyant density (rho = 1.717) and melting temperature (tm = 89 degrees C) both indicate a base content of 59% G + C. The DNA is found in both wild-type cells and those lacking plastids. The copy number is estimated to be about 1000.     

Purification and Properties of a Thermostable Pullulanase from a Newly Isolated Thermophilic Anaerobic Bacterium, Fervidobacterium pennavorans Ven5

Extremely thermophilic anaerobic fermentative bacteria growing at temperatures between 50 and 80(deg)C (optimum, 65 to 70(deg)C) were isolated from mud samples collected at Abano Terme spa (Italy). The cells were gram-negative motile rods, about 1.8 (mu)m in length and 0.6 (mu)m in width, occurring singly and in pairs. Cells commonly formed spheroids at one end similar to Fervidobacterium islandicum and Fervidobacterium nodosum. The new isolate differs from F. nodosum by the 7% higher G+C content of its DNA (40.6 mol%) but is similar to Fervidobacterium pennavorans and F. islandicum in its G+C content and phenotypic properties. The phylogenetic dendrogram indicates that strain Ven5 belongs to the order Thermotogales and shows the highest 16S ribosomal DNA sequence similarity to F. pennavorans, F. islandicum, and F. nodosum, with similarities of 99.0, 98.6, and 96.0%, respectively. During growth on starch the strain produced a thermostable pullulanase of type I which preferentially hydrolyzed (alpha)-1,6 glucosidic linkages. The enzyme was purified 65-fold by anion-exchange, gel permeation, and hydrophobic chromatography. The native pullulanase has a molecular mass of 240,000 Da and is composed of three subunits, each with a molecular mass of 77,600 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimal conditions for the activity and stability of the purified pullulanase were pH 6.0 and 85(deg)C. At pH 6.0, the half-life of the enzyme was over 2 h at 80(deg)C and 5 min at 90(deg)C. This is the first report on the presence of pullulanase type I in an anaerobic bacterium.     

A Francisella tularensis pathogenicity island required for intramacrophage growth

Francisella tularensis is a gram-negative, facultative intracellular pathogen that causes the highly infectious zoonotic disease tularemia. We have discovered a ca. 30-kb pathogenicity island of F. tularensis (FPI) that includes four large open reading frames (ORFs) of 2.5 to 3.9 kb and 13 ORFs of 1.5 kb or smaller. Previously, two small genes located near the center of the FPI were shown to be needed for intramacrophage growth. In this work we show that two of the large ORFs, located toward the ends of the FPI, are needed for virulence. Although most genes in the FPI encode proteins with amino acid sequences that are highly conserved between high- and low-virulence strains, one of the FPI genes is present in highly virulent type A F. tularensis, absent in moderately virulent type B F. tularensis, and altered in F. tularensis subsp. novicida, which is highly virulent for mice but avirulent for humans. The G+C content of a 17.7-kb stretch of the FPI is 26.6%, which is 6.6% below the average G+C content of the F. tularensis genome. This extremely low G+C content suggests that the DNA was imported from a microbe with a very low G+C-containing chromosome.     

A microanalytical procedure for determination of the base composition of DNA

A new procedure for the determination of the percentage guanine plus cytosine (% G+C; mol/100 mol) values of microquantities of DNA is described. Its principle is a DNA-polymerase-I-directed nick translation of DNA in the presence of dGTP, dTTP, [3H]dCTP, and [alpha-32P]dATP. Kinetics experiments indicate that the plateau value is reached in about 20 min of incubation under our experimental conditions. Percentage G+C is obtained from the linear relation 1/(% G+C) = 0.01 K [32P]/[3H] + 0.01, where the ratio of trichloroacetic-acid-precipitable radioactivity is taken into account, the K value being determined for each experiment by using a few reference DNAs of known composition. This procedure has proven suitable for analysis of plasmidic, viral and cellular DNAs of different base composition (25-75% G+C), shape (linear and circular double-stranded DNA) and size (100-150 000 base pairs). Usual methods for % G+C analysis (buoyant density and melting temperature determinations) yield unreliable results in the presence of either modified or unusual bases: the double-labeling procedure is still valid under these conditions. The latter is, therefore, the method of choice for analysis or rare DNA species which are available in very small quantities (it requires amounts of DNA as low as 1 ng, i.e. several order of magnitude lower than those used for chromatographic analysis of DNA hydrolysates). Since the obtention of highly purified DNA is an essential prerequisite for the double-labeling procedure, a method for purification of bacterial DNA is detailed in the present work.     

Clostridium alkalicellum sp. nov., an obligately alkaliphilic cellulolytic bacterium from a soda lake in the Baikal region

The first anaerobic alkaliphilic cellulolytic microorganism has been isolated from the Verkhnee Beloe soda lake (Buryatiya, Russia) with pH 10.2 and a salt content of up to 24 g/l. Five strains were characterized. Strain Z-7026 was chosen as the type strain. The cells of the isolate are gram-positive spore-forming rods. A mucous external capsule is produced. The microorganism is obligately alkaliphilic, growing in a pH range of 8.0-10.2, with an optimum at pH 9.0. Sodium ions and, in carbonate-buffered media, sodium chloride are obligately required. The microorganism is slightly halophilic; it grows at 0.017-0.4 M Na+ with an optimum at 0.15-0.3 M Na+. The metabolism is fermentative and strictly anaerobic. Cellulose, cellobiose, and xylan can be used as growth substrates. Plant and algal debris can be fermented. Lactate, ethanol, acetate, hydrogen, and traces of formate are produced during cellulose or cellobiose fermentation. Yeast extract or vitamins are required for anabolic purposes. The microorganism fixes dinitrogen and is nitrogenase-positive. It is tolerant to up to 48 mM Na2S. Growth is not inhibited by kanamycin or neomycin. Chloramphenicol, streptomycin, penicillin, ampicillin, ampiox, bacillin, novobiocin, and bacitracin suppress growth. The DNA G+C content is 29.9 mol %. According to the nucleotide sequence of its 16S rRNA gene, strain Z-7026 is phylogenetically close to the neutrophilic cellulolytic bacteria Clostridium thermocellum (95.5%), C. aldrichii (94.9%), and Acetivibrio cellulolyticus (94.8%). It is proposed as a new species: Clostridium alkalicellum sp. nov.     

Dehalobacter restrictus gen. nov. and sp. nov., a strictly anaerobic bacterium that reductively dechlorinates tetra- and trichloroethene in an anaerobic respiration

The highly enriched anaerobic bacterium that couples the reductive dechlorination of tetrachloroethene to growth, previously referred to as PER-K23, was obtained in pure culture and characterized. The bacterium, which does not form spores, is a small, gram-negative rod with one lateral flagellum. It utilized only H₂ as an electron donor and tetrachloroethene and trichloroethene as electron acceptors in an anaerobic respiration process; it could not grow fermentatively. Acetate served as a carbon source in a defined medium containing iron as the sole trace element, the two vitamins thiamine and cyanocobalamin, and the three amino acids arginine, histidine, and threonine. The cells contained menaquinones and b-type cytochromes. The G+C content of the DNA was 45.3 ± 0.3 mol%. The cell wall consisted of type-A3γ peptidoglycan with ll-diaminopimelic acid and one glycine as an interpeptide bridge. The cells are surrounded by an S-layer; an outer membrane was absent. Comparative sequence analysis of the 16S rRNA sequence showed that PER-K23 is related to gram-positive bacteria with a low G+C content of the DNA. Based on the cytological, physiological, and phylogenetic characterization, it is proposed to affiliate the isolate to a new genus, Dehalobacter, with PER-K23 as the type strain of the new species Dehalobacter restrictus. Received: 14 July 1997 / Accepted: 27 November 1997     

Correlation of the DNA nucleotide makeup with the physiological and cytological characteristics of spore-forming anaerobic bacteria]

The nucleotide composition of DNA from 12 studied species of anaerobic bacteria belongs to AT type, with G+C varying from 28.4 to 36.8 mole%. In the anaerobic group of Clostridium bifermentans, a correlation has been established between the nucleotide composition of DNA, the type of appendages on spores, and some physiologo-biochemical characteristics. The nucleotide composition of DNA in the spores of four anaerobic species is shifted toward GC type as compared to DNA in the vegetative cells. Data on the content of GC pairs in DNA of the spores may sometimes be of a higher taxonomic value than the corresponding evidence on DNA of the vegetative cells.     

Image and flow cytometric analysis of DNA content in breast cancer. Relation to estrogen receptor content and lymph node involvement

A comparative flow-cytometric and image-cytometric study was performed on 166 human breast cancers. Parallel measurements of 67 cases showed a good correlation between the DNA indices measured with each of the techniques. However, minor ploidy abnormalities were detected with flow cytometry. Only with this technique about 70% of the tumors appeared to be aneuploid. DNA profiles obtained with image cytometry frequently contained DNA values above the modal G2 value of the tumor. At least part of these broadly scattered DNA profiles resulted from multiploid tumors, as was found with flow cytometry. The mean percentage of cells with DNA contents exceeding the modal G0G1 value in image-cytometric DNA profiles (non- G0G1 cells) appeared to be highest in aneuploid lymph-node-positive (N+) tumors and lowest in near-diploid lymph-node-negative (N-) tumors. Near-diploid tumors were more frequently (70%) estrogen receptor positive (ER+) than were aneuploid tumors (50%), whereas highly aneuploid tumors with low or negative ER contents tended to be N+. Tumors with a high percentage of non- G0G1 cells value were predominantly estrogen-receptor negative (ER-).     

Sequence analysis of two yeast mitochondrial DNA fragments containing the genes for tRNA Ser UCR and tRNA Phe UUY.

Two restriction enzyme fragments containing yeast mitochondrial tRNA genes have been characterized by DNA sequence analysis. One of these fragments is 320 base pairs long and contains a tRNA Ser gene. The corresponding tRNA SER was isolated from yeast mitochondria and its nucleotide sequence also was determined. This mitochondrial tRNA is 90 nucleotides in length, has a G + C content of 38%, and has UGA as the anticodon. A portion of a 680-base-pair DNA fragment containing a tRNA Phe gene was also sequenced. The portion of this gene which codes for the mature tRNA is 75 base pairs in length, has a G + C content of 33%, and contains the anticodon GAA. Neither gene contains an intervening sequence or codes for the 3' CCA terminus. Both are surrounded by regions of more than 90% A + T. The significance of these sequences is discussed.     

Spirochaeta bajacaliforniensis sp. n. from a microbial mat community at Laguna Figueroa,Baja California Norte,Mexico

A new anaerobic spirochete was isolated from anaerobic muds beneath the laminated sediment in the evaporite flat at Laguna Figueroa, Baja California Norte, Mexico. The organism is a member of the stratified microbial community involved in the deposition of the laminated sediments in the lagoon. The size of the spirochete is 0.3 by 30 m, with a wave amplitude of 0.5 m and a wavelength of 1.25 m. The periplasmic flagella have a 1-2-1 arrangement. The outer membrane of the modified Gramnegative cell wall (the sheath) is irregularly crenulated and has a sillon. The growth medium contained yeast extract, trypticase, cellobiose, sodium thioglycolate and at least 20% natural seawater. Chemically defined artificial seawater media did not support growth. Optimal growth occurred with a seawater concentration of 80% at 36° C and a pH of 7.5. Glucose was fermented to acetate, ethanol, carbon dioxide and hydrogen. The guanine+cytosine content of the DNA was 50 mol %. the spirochete body reacts positively to antibodies raised against eukaryotic brain tubulin protein. On the basis of its free-living anaerobic habitat, its unique morphological and physiological characteristics and G+C ratio, it is proposed that this isolate be considered a new species and named Spirochaeta bajacaliforniensis.     

Substrate preferences of human placental DNA methyltransferase investigated with synthetic polydeoxynucleotides

A DNA methyltransferase partly purified from human placenta has been tested on a variety of synthetic polydeoxynucleotides. The results showed that: the enzyme is most active as a 'maintenance' or 'hemi-' methylase but also has some de novo methylating activity; the presence or absence of A or T in the substrate strand has little influence on maintenance or de novo activity, while polymers containing C but not G in the same strand are poor de novo substrates and bind poorly to the enzyme; single-stranded polymers are about as good substrates as double-stranded ones, and the effects of nucleotide composition (particularly G and mC content) on enzyme activity with single strands are similar to those with double-stranded polymers; strands in which all the cytosines are methylated bind the enzyme well. A mechanism is suggested involving two different sites on the enzyme that recognize CG and mCG, and which rationalizes the activity of eukaryotic DNA methyltransferases towards single-stranded DNA.