Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Summary Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 ) were present; instead, smaller circles characteristic for each mutant sudied were found. Eco Rl digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type.The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 . These circles ranged in size from 0.89 to greater than 20 ; only one molecule out of some 200 molecules was thought to be of full length (31 ). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm³) and a second at 1.699 g/cm³. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23×10⁶ daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20×10⁶ daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length.These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho ^- mutation in the yeast, S. cerevisiae.     

Germ line-restricted,highly repeated DNA sequences and their chromosomal localization in a Japanese hagfish (Eptatretus okinoseanus)

The various species of Japanese hagfish, namely, Eptatretus okinoseanus (types A and B), Eptatretus burgeri and Myxine garmani, are known to eliminate a fraction of their chromosomes during early embryogenesis. High molecular weight DNA from germ line cells and somatic cells of these hagfish species was isolated and digested with different restriction enzymes. The DNA fragments were separated by agarose gel electrophoresis. Digestion with BamHI and DraI generated two weak bands and one weak band, respectively, that were estimated to be about 90, and 180 bp and about 90 bp long and were limited to the germ line DNA in both types of E. okinoseanus. DNA filter hybridization experiments showed that the two BamHI fragments and the one DraI fragment were present almost exclusively in the germ line DNA of E. okinoseanus. Thus, these DNA fragments appear to be eliminated during embryogenesis. Moreover, evidence was obtained that these fragments are highly and tandemly repeated. Molecular cloning and sequence analysis revealed that the BamHI fragments are mainly composed of a family of closely related sequences that are 95 bp long (EEEo1, for Eliminated Element of E. okinoseanus 1), and the DraI fragment is composed of another family of closely related sequences that are 85 bp long (EEEo2). The two DNA families account for about 19% of the total eliminated DNA in E. okinoseanus type A. Fluorescence in situ hybridization experiments demonstrated that the two families of DNA are located on several C-band-positive, small chromosomes that are limited to germ cells in both types of E. okinoseanus.by W. Hennig     

Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I,from Bacillus subtilis – Bsu121I,a type II restriction endonuclease from Bacillus subtilis

A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified. The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease. The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp. The endonuclease activity required Mg⁺² as cofactor like other type II endonucleases.     

Isolation and Characterization of Transposon-Insertional Mutants from <Emphasis Type="Italic">Paenibacillus </Emphasis><Emphasis Type="Italic">polymyxa</Emphasis> E681 Altering the Biosynthesis of Indole-3-Acetic Acid

We screened a mini-Tn10 insertional mutant library of the spore-forming bacterium Paenibacillus polymyxa E681 with variable indole-3-acetic acid (IAA) productivity. Four mutants, of which two showed a decrease in IAA production and the other two showed an increase in IAA production, were finally selected. Further analyses demonstrated different levels of IAA intermediates from culture supernatant of wild-type strain and mutants. In addition, mutants showed different promotions on the early growth of 10-day-old maize in terms of the increase in shoot and root weights. DNA fragments flanking the transposon insertion in four mutants were cloned and sequenced. The target sites of insertion were gene gpr1, disrupted at two sites, 49 bp downstream of the spo0F gene, and relA/spoT homologue, which codes for GPR1/FUN34/YaaH family protein, stage 0 sporulation protein F, and RelA/SpoT domain protein, respectively. This evidence suggests that there may be a number of genes involved in the regulation of IAA biosynthesis of P. polymyxa.     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.     

Detection of Deletion Mutants in Rice Via Overgo Hybridization Onto Membrane Spotted Arrays

Overlapping nucleotides or overgos were designed and used to detect deletions in rice mutants that had been generated by treatment with a chemical (diepoxybutane, DB) or irradiation (gamma ray, GR). As a proof of concept, DNA of three spotted leaf 11 (spl11) deletion mutants, GR5612, GR5717 and DB2487, and the wild-type DNA (IR64) were spotted onto nylon membranes in different concentrations and probed with radiolabeled overgos designed from different regions within the Spl11 gene. At least 3 μg per spot of DNA was required to show unambiguous and visible signals. Overgos designed from regions putatively deleted in GR5612 consistently did not hybridize to the GR5612 genomic DNA, whereas DNA from wild-type IR64 and from mutants in other regions consistently hybridized to the overgos. DNA of mutant DB2487 hybridized to all the overgos tested, suggesting the deletion is small and undetectable by the overgos. Overall, this study demonstrates that overgos can be employed to detect, verify, and characterize deletions, particularly in single copy genes, in mutant genomes. However, the technique requires careful adjustment of DNA concentration before spotting and the spotting of relatively high DNA concentrations onto the membranes.     

Frameshift and double-amber mutations in the bacteriophage T4 uvsX gene: analysis of mutant UvsX proteins from infected cells

Summary The bacteriophage T4 uvsX gene encodes a 43 kDa, single-stranded DNA-dependent ATPase, double-stranded DNA-binding protein involved in DNA recombination, repair and mutagenesis. Mutants of uvsX have a DNA-arrest phenotype and reduced burst size. Western blot immunoassay of UvsX peptides made by a number of amber mutants revealed amber peptides ranging from 25–32 kDa. Wild-type UvsX protein was also detected in lysates of cells infected with uvsX amber mutants, suggesting that their mutations are suppressed by translational ambiguity. We investigated the effects of mutations near the 5 end of uvsX. A frameshift mutation was engineered at codon 33. Western immunoblots for UvsX protein demonstrated that the frameshift mutant expresses no detectable wild-type UvsX; instead, a 37 kDa reactive peptide was detected. In order to determine if this peptide represents truncated UvsX protein, the mutation was regenerated in the cloned uvsX gene and expressed in transformed Escherichia coli. Endopeptidase digestion of the 37 kDa protein from the cloned gene generated peptide fragments indistinguishable from those obtained from wild-type UvsX. A double-amber mutant of uvsX was also generated by oligonucleotide site-directed mutagenesis. No UvsX protein was detected in lysates of cells infected with the uvsX-am64am67 double mutant. Plaque size and sensitivity to UV inactivation for both the double-amber and the frame-shift mutants were indistinguishable from those of other uvsX mutants. Mutations in uvsY had no demonstrable effect on efficiency of plating or UV sensitivity of uvsX mutants. Thus, null mutants of uvsX are viable.     

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Summary Fructose was shown to be phosphorylated by a specific phosphoenolpyruvatc-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose Ell. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

Cloning of the GSH1 and GSH2 Genes Complementing the Defective Biosynthesis of Glutathione in the Methylotrophic Yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh⁺ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the -glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh^– phenotype of the gsh2 mutant. The Gsh⁺ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24, having the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme -glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Cloning of seven differently complementing DNA fragments with chl functions from Escherichia coli K12

Summary Seven genomic libraries of chromosomal Escherichia coli K12 wild-type DNA were constructed in plasmid vectors. These were used to transform chl insertion mutants. Selection for growth on nitrate under anaerobic conditions yielded four plasmids which complemented mutants of the chlA, B, E and G types. The chromosomal fragments were mapped with restriction enzymes and subcloned. Three complementation groups were observed among the chlA mutants and two among the chlE mutants. The established complementation groups plus mutants of the chlD type represent eight distinct functions, which are all believed to be required for the molybdenum cofactor activity in the reduction of nitrate to nitrite by E. coli.     

Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: genome size, fragment identification, and gene localization.

Four restriction endonucleases, AseI (5'-ATTAAT), SpeI (5'-ACTAGT), DraI (5'-TTTAAA), and SnaBI (5'-TACGTA), generated DNA fragments of suitable size distributions for mapping the genome of Rhodobacter sphaeroides by transverse alternating field electrophoresis. AseI produced 17 fragments, ranging in size from 3 to 1,105 kilobases (kb), SpeI yielded 16 fragments (12 to 1,645 kb), DraI yielded at least 25 fragments (6 to 800 kb), and SnaBI generated 10 fragments (12 to 1,225 kb). A total genome size of approximately 4,400 +/- 112 kb was determined by summing the fragment lengths in each of the digests generated by using the different restriction endonucleases. The total genomic DNA consisted of chromosomal DNA (3,960 +/- 112 kb) and the five endogenous plasmids (approximately 450 kb total) whose cognate DNA fragments have been unambiguously identified. A number of genes have been physically mapped to the AseI-generated restriction endonuclease fragments of total genomic DNA by Southern hybridization analysis with either homologous or heterologous specific gene probes or, in the case of several auxotrophic and pigment-biosynthetic mutants apparently generated by Tn5, a Tn5-specific probe. Other genes have been mapped by a comparison with wild-type patterns of the electrophoretic banding patterns of the AseI-digested genomic DNA derived from mutants generated by the insertion of either kanamycin or spectinomycin-streptomycin resistance cartridges. The relative orientations, distance, and location of the pufBALMX, puhA, cycA, and pucBA operons have also been determined, as have been the relative orientations between prkB and hemT and between prkA and the fbc operon.     

茄科作物青枯菌NADH脱氢酶F亚基在细胞运动和致病性中的功能研究

[目的]劳尔氏菌（Ralstonia solanacearum）在茄科作物上引起严重的细菌性青枯病,本研究旨在发掘青枯劳尔氏菌与致病相关的基因。[方法]利用Tn5转座子构建随机插入突变体,分析生物膜形成、细胞运动和致病性;对有表型变化的突变体,运用TAIL-PCR方法鉴定Tn5插入位点,确定所突变的基因。[结果]以模式菌株GMI000为出发菌,总共获得了400个突变体,其中2个突变体不能形成生物膜,在软琼脂平板上的运动能力下降;接种感病番茄植物,这2个突变体都不能引起萎焉症状。TAIL-PCR结果显示,2个突变体的Tn5插入位点都在NADH脱氢酶F亚基（nuoF）中,距离翻译起始位点分别为103-bp和225-bp。ripAY基因启动子推动的nuoF基因互补载体,完全恢复了2个突变体的表型。[结论]NADH脱氢酶复合物是微生物呼吸电子传递链中的第一步催化酶。我们的结果表明,NADH脱氢酶复合物对R.solanacearum生物膜形成、细胞运动和致病性也有重要作用。     

Saccharomyces species assignment by long range ribotyping

Type strains of 10 genotypically distinctSaccharomyces species are differentiated by ribosomal DNA restriction fragment analysis (ribotyping). The full length of the chromosomal ribosomal repeat was amplified in two parts, the 18SrDNA including both ITS regions (2600 bp) and the 25SrDNA (3300 bp). Restriction fragments generated by 9 enzymes from these two products yield characteristic patterns, by which unknownSaccharomyces isolates are assigned to the type strains. For convenient separation and detection only fragments longer than 200 bp were monitored. In contrast to molecular differentiation methods of highest resolution as RAPD-PCR or fingerprinting, the results from ribotyping are absolutely reproducible and thereby suitable for databases. The phylogeny computed from the discrete character matrix for presence/absence of fragments by the PHYLIP program package is in complete accordance to the phylogeny derived from ribosomal RNA sequence analysis. By this the field of application of the long range ribotyping can be regarded basically as equal to DNA sequence analysis of the same locus. Because distant relationships are recognized, missidentified genera were detected upon the species assignment. This cannot be done by methods of higher resolution like RAPD-PCR or fingerprinting.     

Nuclear tRNATyr genes are highly amplified at a single chromosomal site in the genome of Arabidopsis thaliana

Summary We have examined the organization of tRNA^Tyr genes in three ecotypes of Arabidopsis thaliana, a plant with an extremely small genome of 7 × 107 bp. Three tRNA^Tyr gene-containing EcoRI fragments of 1.5 kb and four fragments of 0.6, 1.7, 2.5 and 3.7 kb were cloned from A. thaliana cv. Columbia (Col-O) DNA and sequenced. All EcoRl fragments except those of 0.6 and 2.5 kb comprise an identical arrangement of two tRNA^Tyr genes flanked by a tRNA^Ser gene. The three tRNA genes have the same polarity and are separated by 250 and 370 bp, respectively. The tRNA^Tyr genes encode the known cytoplasmic tRNA_GA ^Tyr. Both genes contain a 12 by long intervening sequence. Densitometric evaluation of the genomic blot reveals the presence of at least 20 copies, including a few multimers, of the 1.5 kb fragment in Col-O DNA, indicating a multiple amplification of this unit. Southern blots of EcoRl-digested DNA from the other two ecotypes, cv. Landsberg (La-O) and cv. Niederzenz (Nd-O) also show 1.5 kb units as the major hybridizing bands. Several lines of evidence support the idea of a strict tandem arrangement of this 1.5 kb unit: (i) Sequence analysis of the EcoRI inserts of 2.5 and 0.6 kb reveals the loss of an EcoRI site between 1.5 kb units and the introduction of a new EcoRI site in a 1.5 kb dimer. (ii) Complete digestion of Col-O DNA with restriction enzymes which cleave only once within the 1.5 kb unit also produces predominantly 1.5 kb fragments. (iii) Partial digestion with EcoRI shows that the 1.5 kb fragments indeed arise from the regular spacing of the restriction sites. The high degree of sequence homology among the 1.5 kb units, ranging from 92% to 99%, suggests that the tRNA^Ser/tRNA^Tyr cluster evolved 1–5 million years ago, after the Brassicaceae diverged from the other flowering plants about 5–10 million years ago.     

Nucleotide Sequence of Polymerase Chain Reaction Product Amplified from Rickettsia japonica DNA Using Rickettsia rickettsii 190-Kilodalton Surface Antigen Gene Primers

The PCR product amplified from Rickettsia japonica with the primer pair Rr 190.70p and Rr 190.602n of R. rickettsii 190-kDa antigen gene was cloned into M13mp19 RF DNA at the EcoRI site and sequenced by chemiluminescent DNA sequencing. The sequence revealed a molecular size of 533 base pairs (bp). The primer-flanking region of 491 bp, an open reading frame, was compared with the corresponding region of R. rickettsii, demonstrating 35 nucleotide substitutions in R. japonica. The sequence of primer portions in R. japonica DNA was also analyzed, revealing one nucleotide substitution in the Rr 190.70p and two in the Rr 190.602n portion. The homology in the overall sequence of PCR-amplified regions between R. japonica and R. rickettsii was 93% in nucleotide and 85% in putative amino acid structure. The sequence contains no cleavage site for the restriction endonuclease AfaI but two PstI sites giving three fragments of 121, 159, and 253 bp, which differentiated R. japonica from other spotted fever group rickettsiae in addition to R. rickettsii. The cleavage sites for endonucleases AluI, HinfI, and MunI that disappeared or appeared in the sequence by nucleotide substitution differentiated R. japonica from others, as did PstI. The estimation of molecular size of DNA fragments on polyacrylamide gel electrophoresis is discussed.     

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation

Summary Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod^-) or nitrogen fixation (Fix^-). Seven Nod^- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod^- mutants and 14 Fix^- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His^- Nod^- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix^- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod^- mutants and one His^-Nod^- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod^- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.