Enzymic glycosphingolipid synthesis on polymer supports.

Two synthetic approaches leading toN-4-carboxymethyl-2-nitrobenzyloxycarbonyl derivatives of model compounds and of glycosylsphingosines are described. The carboxymethyl group can be converted into a hydrazido derivative and the resulting products react with an active ester polymer yielding light-sensitive polymeric products that may subsequently serve as acceptors in glycosyltransferase reactions.Author for correspondence. Address until September 1990; Graduate Department of Biochemistry, Brandeis University, 415 South Street, Waltham, MA 02254, USA     

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200–300 μg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-l-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) · poly(dG-dC) and poly(dA-dT) · poly(dA-dT), but not poly(dI-dC) · poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the ‘de novo’ activity of the enzyme.     

Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Zinc-finger–FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP–scFokI). DNA cleavage assays revealed that the AZP–scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP–FokI dimer. The DNA cleavage pattern of AZP–scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP–scFokI. Furthermore, we demonstrated that AZP–scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP–scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.     

DNA analysis of tropical plant species: An assessment of different drying methods

Use of molecular techniques to study plant systematics has been restricted to either largely temperate groups or groups from which seed is readily obtained. Dried material has advantages over other methods of preservation for molecular analyses; it is cheap, easily undertaken, overcomes the difficulties associated with seed recalcitrance and the absence of seeding plants in the field, and most taxonomists are familiar with the method. Three drying techniques were assessed using material collected in the tropics. No differences in either the quantity or the quality of DNA extracted from material dried by these methods was detected. The implications of using dried material in molecular analyses are discussed.     

Simple and rapid preparation of gapped plasmid DNA for incorporation of oligomers containing specific DNA lesions

The cytotoxicity, mutagenicity, and carcinogenicity of DNA base lesions are largely determined by the responses of cellular DNA repair proteins, DNA polymerases, and signaling pathways. Elucidation of these processes is thus of high biochemical interest. Such studies increasingly rely on DNA substrates containing specific lesions at defined locations. Although short synthetic DNA oligomers have frequently proved useful, circular plasmid substrates are preferable for much biochemical work, and essential for in vivo studies. However, the complexity of current approaches for preparing such substrates and limitations inherent in the procedures have posed problems. We present here a simple, highly versatile procedure for preparing gapped duplex plasmids, into which oligomers incorporating specific lesions can easily be inserted. Endonuclease N.BstNBI was used to nick twice the same strand of a pUC19-derived plasmid (pUC19HBDa), at two GAGTCNNNN sequences separated by 22 bases. Removal of the 22-nt oligomer and further purification produced a highly pure gapped plasmid. To illustrate application of this procedure, 22-nt oligonucleotides containing a single uracil residue were ligated into the gapped molecules. The pUC19HB(Da) plasmid can be modified to accept almost any DNA-lesion-containing oligomer. Using this new approach to incorporate specific DNA lesions into popular reporter genes will facilitate in vivo study of cellular responses to DNA damage.     

Non-equivalency of genera inAngiospermae: Evidence from DNA hybridization studies

The problem of taxa equivalency in phylogenetically distant groups can hardly be solved by comparing morphological differences alone. An attempt is made to approach the problem by means of DNA comparisons, e.g., DNA hybridization. Data obtained forCompositae, Umbelliferae andIridaceae indicate that both unique and repetitive DNA sequence comparisons lead to the conclusions that genera within these families are not equivalent, e.g., the differences in the DNA among the species ofIris are much more pronounced than among those ofAchillea; some genera ofUmbelliferae occupy an intermediate position.     

Identification of the G-genome donor to Triticum timopheevii by DNA:DNA hybridizations

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid Triticum species ancestral to the G genome of Triticum timopheevii. Total genomic, unique-sequence, and repeated-sequence fractions of ³H-T. timopheevii DNA were hybridized to the corresponding fractions of unlabeled DNAs of T. searsii, T. speltoides, T. sharonensis, T. longissimum, and T. bicorne. The heteroduplex thermal stabilities indicated that, of the five species examined, T. speltoides was the most closely related to the G genome of T. timopheevii. Thus, T. spelotides appears to be the G-genome donor to T. timopheevii. The thermal stability profiles further indicated that the repeated DNA fractions from the five diploid species and the tetraploid T. timopheevii are more similar than the unique DNA fractions. This indicates that all of these species are closely related and that the sequences which comprise the current repeated fractions in the various species have not undergone any significant change since the formation of various species.Published with the approval of the Director of the West Virginia Agriculture and Forestry Experiment Station as Scientific Paper No. 1850.     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of steam-pretreated spruce using crude Trichoderma reesei and Trichoderma atroviride enzymes

The aim of this study was to compare the performance of the enzymes produced by Trichoderma reesei Rut C30 and the good extracellular β-glucosidase-producing mutant Trichoderma atroviride TUB F-1663 to that of commercial preparations in the enzymatic hydrolysis and the simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce (SPS).The concentrated TUB F-1663 enzyme was found to be the most efficient in the hydrolysis of washed SPS at 50 g/L water-insoluble solids (WIS) in terms of the glucose produced (18.5 g/L), even in comparison with commercial cellulases (14.1–16.7 g/L). The enzyme preparations were studied at low enzyme loadings (5 FPU/g WIS) in SSF to produce ethanol from SPS. The enzyme supernatant and whole fermentation broth of T. atroviride as well as the whole broth of T. reesei proved to be as efficient in SSF as the commercial cellulase mixtures (ethanol yields of 61–76% of the theoretical were achieved), while low ethanol yields (<40%) were obtained with the β-glucosidase-deficient T. reesei supernatant.Therefore, it seems, that instead of using commercial cellulases, the TUB F-1663 enzymes and the whole broth of Rut C30 may be produced on-site, using a process stream as carbon source, and employed directly in the biomass-to-bioethanol process.     

Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis

Mitochondrial DNA (mtDNA) analysis with Hae III, Hind. III and Msp I was performed in 45 Exophiala jeanselmei strains (30 Phialophora jeanselmei and 15 Phialophora gougerotii strains) and 31 Exophiala dermatitidis strains. The results were as follows, 1) P. jeanselmei and P. gougerotii are identical, 2) E. jeanselmei is classified into 18 types based on restriction profiles, 3) two strains of E. jeanselmei CBS 577.76 and CBS 578.76 are identified as E. dermatitidis, 4) E. dermatitidis has no intraspecific variation and is definitely distinct from E. jeanselmei, 5) E. jeanselmei is suggested to be a complex organism because of extensive mtDNA polymorphism.     

Two translesion synthesis DNA polymerase genes, AtPOLH and AtREV1, are involved in development and UV light resistance in Arabidopsis

Plants are continually exposed to external and internal DNA-damaging agents. Although lesions can be removed by different repair processes, damages often remain in the DNA during replication. Synthesis of template damages requires the replacement of replicative enzymes by translesion synthesis polymerases, which are able to perform DNA synthesis opposite specific lesions. These proteins, in contrast to replicative polymerases, operate at low processivity and fidelity. DNA polymerase η and Rev 1 are two proteins found in eukaryotes that are involved in translesion DNA synthesis. In Arabidopsis, DNA polymerase η and Rev 1 are encoded by AtPOLH and AtREV1 genes, respectively. Transgenic plants over-expressing AtPOLH showed increased resistance to ultraviolet light. Only plants with moderate AtREV1 over-expression were obtained, indicating that this enzyme could be toxic at high levels. Transgenic plants that over-expressed or disrupted AtREV1 showed reduced germination percentage, but the former exhibited a higher stem growth rate than the wild type during development.     

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.     

Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair

Summary A new recombination gene called recR has been identified and located near dnaZ at minute 11 on the current linkage map of Escherichia coli. The gene was detected after transposon mutagenesis of a recB sbcB sbcC strain and screening for insertion mutants that had a reduced efficiency of recombination in Hfr crosses. The recR insertions obtained conferred a recombination deficient and extremely UV sensitive phenotype in both recB recC sbcA and recB recC sbcB sbcC genetic backgrounds. recR derivatives of recBC⁺sbc⁺ strains were proficient in conjugational and transductional recombination but deficient in plasmid recombination and sensitive to UV light. Strains carrying recR insertions combined with mutations uvrA and other rec genes revealed that the gene is involved in a recombinational process of DNA repair that relies also on recF and recO, and possibly recJ, but which is independent of recB, recC and recD. The properties of two other insertions, one located near pyrE and the other near guaA, are discussed in relation to their proximity to recG and xse (the gene for exonuclease VII), respectively.     

Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependent on specific combinations of amino acids in the primary structure of the expressed protein.

Summary In this study we present the characterization of the temperature-sensitive mutant allele cdc9-1 encoding DNA ligase, of Saccharomyces cerevisiae strain A364A by DNA sequencing. Comparison with the published wild-type sequence from strain SKI revealed 13 nucleotide exchanges between these two sequences, which are derived from non-isogenic genetic backgrounds. Only four of these changes, distributed over the whole coding region, lead to amino acid exchanges in the protein chain. Our analysis of the sequence of the wild-type CDC9 allele from strain A364A revealed differences from the isogenic cdc9-1 allele in only two nucleotides: one silent change and one leading to a single amino acid exchange. The latter is therefore responsible for the temperature-sensitive phenotype. A mosaic protein, in which a region carrying this amino acid exchange has been inserted in place of the corresponding part of CDC9 from the non-isogenic strain SKI, is not temperature sensitive. The exchange of a longer stretch of DNA leading to atteration of three amino acids of the protein compared with the original sequence of SKI is required to obtain a temperature-sensitive DNA ligase in this strain, while in strain A364A a single amino acid change is sufficient for expression of a temperature-sensitive protein.     

Isolation and characterization of two middle repetitive DNA sequences of nuclear tobacco genome

Summary Two DNA sequences, R8.1 and R8.3, representing two distinct classes of tobacco genomic repeated DNA, were cloned and characterized by Southern blot analysis. Both R8.1 and R8.3 were found to be homologous to the Nicotiana tomentosiformis component of the allotetraploid Nicotiana tabacum genome, and each of them represents about 0.3% of nuclear DNA. The R8.1 and R8.3 differ in the mode of distribution in chromosomes, as revealed by in situ DNA/DNA hybridization.     

DNA homology between Candida albicans strains: Evidence to justify the synonymous status of C. stellatoidea

Genetic relatedness between strains of C. albicans and C. stellatoidea was studied by measuring G + C content and overall sequence homology. G + C contents determined by high-performance liquid chromatography were 32.6 to 34.2% for 26 strains of C. albicans and 33.0 to 33.9% for eight strains of C. stellatoidea. DNA-DNA hybridization with two C. albicans and two C. stellatoidea probes revealed that all 34 test strains formed a single cluster in which the extents of hybridization with the heterologous probes ranged between 77.9 and 105.6% of those with the homologous probes. These results give support to the unification of C. albicans and C. stellatoidea into a single species.     

Phylogenetic relationships of the genera Arthroderma and Nannizzia inferred from mitochondrial DNA analysis

The phylogenetic relationship of the genera Arthroderma and Nannizzia, was investigated by mitochondrial DNA analysis based on the restriction-fragment-length polymorphisms. Phylogenetic trees made on ten species. A. benhamiae, A. insingulare, A. quadrifidum, A. simii, A. vanbreuseghemii, N. fulva, N. grubyia, N. gypsea, N. incurvata and N. otae showed no definite distinctions between the genera Arthroderma and Nannizzia. These results support the conclusion of Weitzman et al. that the genera Arthroderma and Nannizzia are congeneric.     

Enzymatic isolation of living embryo sacs of Petunia

Summary A 20%–25% yield of isolated and living embryo sacs of Petunia hybrida L. was obtained using an enzymatic maceration mixture containing 3% driselase (soluble fraction only), 0.1% MES buffer, pH 5.5, and 8% mannitol. For each maceration ± 450 ovules were incubated in 1 ml enzyme solution for 2 h at 30° C in a shaking waterbath (150 rpm). Subsequently, the enzyme solution was replaced by Brewbaker and Kwack's medium, pH 6.5, supplemented with 10% mannitol (BKM). Gentle agitation of the suspension resulted in the liberation of embryo sacs, which were then collected with a micropipette using a dissecting microscope and transferred to fresh BKM. The embryo sacs isolated are intact and living, and have maintained their original shape and organization When stored in BKM at room temperature the isolated embryo sacs remain alive for 8 h. Storage at 4° C results in a prolongation of viability of up to 80 h. Prolonged incubation of ovules or reincubation of isolated embryo sacs in the maceration mixture results in the liberation of the gametophytic cells as individual, living protoplasts.     

Phylogeny of Nannizzia incurvata,N. gypsea,N. fulva and N. otae by restriction enzyme analysis of mitochondrial DNA

Sequence divergence among Nannizzia incurvata, N. gypsea, N. fulva and N. otae was studied genetically using digestion profiles of mitochondrial DNA with restriction enzymes, Hae III, Hha I, Hind III and Xba I. Only N. fulva was subdivided into two types on restriction profiles. Phylogenetic relationships suggest that 1) N. gypsea is more closely related to N. fulva than N. incurvata, 2) the phylogenetic distance between N. otae and the other three species is larger than the distances between the other three species.     

Chloroplast DNA restriction site mapping and the phylogeny ofRanunculus (Ranunculaceae)

A chloroplast DNA restriction site map forRanunculus sceleratus (Ranunculaceae) was constructed using 14 restriction endonucleases. The total size of the chloroplast genome is 152.4kb. No inversions were detected relative to the tobacco chloroplast DNA. Cladistic analyses of chloroplast DNA restriction site polymorphism were employed in order to elucidate the phylogeny among 76 species of the genusRanunculus in a wide sense and one species ofTrautvetteria. A total of 341 informative restriction site changes were detected. Parsimony jackknifing, bootstrapping and decay analysis were undertaken in order to evaluate the amount of support for the monophyletic groups. The results suggest that the analysed species ofRanunculus are divisible into two main clades. Only few of the traditional sections and subgenera ofRanunculus are monophyletic. The genusTrautvetteria is nested within a clade comprising, e.g.Ranunculus cymbalaria, R. andersonii, R. lapponicus andR. ficaria. SubgenusBatrachium lies within a larger clade containing, e.g.R. sceleratus andR. hyperboreus. Contractions of the inverted repeat due to parallel deletions of 200–300 bp close to the J_SB have occurred in many clades and the phylogenetic distribution of this size reduction was mapped among the species.