Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.     

Three-Piece-Ligation PCR and Application in Disruption of Chlorophyll Synthesis Genes in Synechocystis sp. PCC 6803

Linear DNA, consisting of a drug-resistance marker and long flanking sequences, was synthesized by one-step polymerase chain reaction after a three-piece ligating reaction. Chlorophyll synthesis genes, chlH and chlL in Synechocystis sp. PCC 6803, were replaced by a kanamycin-resistance marker through double recombinations with flanking homology regions. Under LAHG conditions, the chlL but not chlH mutant stopped chlorophyll synthesis, while both synthesized chlorophyll in the light. Received: 20 June 2001 / Accepted: 30 July 2001     

<Emphasis Type="Italic">Campylobacter coli</Emphasis> Pulsed Field Gel Electrophoresis Genotypic Diversity Among Sows and Piglets in a Farrowing Barn

Genotypes of Campylobacter coli isolates from feces of three sows and rectal swabs of 17 piglets were examined by pulsed field gel electrophoresis (PFGE). All of the animals originated from a single farrowing barn of a farrow-to-finish swine operation. Five Campylobacter colonies were picked from a single agar plate for each sample after broth enrichment and growth on Campy-Cefex agar. Genotypes were examined by PFGE after genomic DNA digestion with SmaI and SacII restriction endonucleases. Twenty SmaI genotypes and 12 SacII genotypes were detected among 99 Campylobacter coli isolates. There was no pattern of shared genotypes between sows and their respective piglets, nor between littermates. Results indicate that a high number of Campylobacter genotypes may coexist in related pigs from a single housing facility. Received: 12 October 2001 / Accepted: 7 December 2001     

A genome-specific repetitive DNA sequence from Oryza eichingeri: characterization,localization, and introgression to O. sativa

In the course of transferring the brown planthopper resistance from a diploid, CC-genome wild rice species, Oryza eichingeri (IRGC acc. 105159 and 105163), to the cultivated rice variety 02428, we have isolated many alien addition and introgression lines. The O. eichingeri chromatin in some of these lines has previously been identified using genomic in situ hybridization and molecular-marker analysis. Here we cloned a tandemly repetitive DNA sequence from O. eichingeri IRGC acc105163, and detected it in 25 introgression lines. This repetitive DNA sequence showed high specificity to the rice CC genome, but was absent from all the four tetraploid species with BBCC or CCDD genomes. The monomer in this repetitive DNA sequence is 325–366-bp long, with a copy number of about 5,000 per 1 C of the O. eichingeri genome, showing 88% homology to a repetitive DNA sequence isolated from Oryza officinalis (2n=2x=24, CC). Fluorescent in situ hybridization revealed 11 signals distributed over eight O. eichingeri chromosomes, mostly in terminal or subterminal regions. Received: 28 November 2000 / Accepted: 3 April 2001     

Global DNA methylation profiling in peripheral blood cells of South African women with gestational diabetes mellitus

Background/Objective: Recently, several studies have reported that DNA methylation changes in tissue are reflected in blood, sparking interest in the potential use of global DNA methylation as a biomarker for gestational diabetes mellitus (GDM). This study investigated whether global DNA methylation is associated with GDM in South African women.

Methods: Global DNA methylation was quantified in peripheral blood cells of women with (n?=?63) or without (n?=?138) GDM using the MDQ1 Imprint® DNA Quantification Kit.

Results: Global DNA methylation levels were not different between women with or without GDM and were not associated with fasting glucose or insulin concentrations. However, levels were 18% (p?=?0.012) higher in obese compared to non-obese pregnant women and inversely correlated with serum adiponectin concentrations (p?=?0.005).

Discussion: Contrary to our hypothesis, global DNA methylation was not associated with GDM in our population. These preliminary findings suggest that despite being a robust marker of overall genomic methylation that offers opportunities as a biomarker, global DNA methylation profiling may not offer the resolution required to detect methylation differences in the peripheral blood cells of women with GDM. Moreover, global DNA methylation in peripheral blood cells may not reflect changes in placental tissue. Further studies in a larger sample are required to explore the candidacy of a more targeted approach using gene-specific methylation as a biomarker for GDM in our population.     

MGMT hypomethylation is associated with DNA damage in workers exposed to low-dose benzene

Objective: This study aims to assess the effects of low-dose benzene on DNA damage and O⁶-methylguanine-DNA methyltransferase (MGMT) methylation in occupational workers.

Materials and methods: We recruited 96 nonsmoking male petrochemical industry workers exposed to low-dose benzene and 100 matched control workers. Urinary S-phenylmercapturic acid (SPMA) and S-benzylmercapturic acid (SBMA) were measured for indicating internal exposure of benzene and toluene. The degree of DNA damage was determined by the Comet assay. The levels of MGMT methylation were detected quantitatively by bisulphite-PCR pyrosequencing assay.

Results: The benzene-exposed workers had significantly higher levels of urinary SPMA, degree of DNA damage but decreased MGMT methylation than the controls (all p?<?0.05). In contrast, the level of urinary SBMA does not differ between benzene-exposed workers and the controls. In all participants, MGMT methylation was negatively associated with the urinary SPMA and the degree of DNA damage, indicating that epigenetic regulation might be involved in response to low-dose benzene exposure-induced genetic damage.

Discussion and conclusion: MGMT methylation could be a potent biomarker associated with low-dose benzene exposure and benzene-induced DNA damage.     

Trials of direct detection and identification of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG 1 and AG 2 subgroups using specifically primed PCR analysis

Specifically primed polymerase chain reaction (PCR) analysis was used for direct detection and identification of Rhizoctonia solani isolates belonging to AG 1 subgroups (IA, IB, and IC) and AG 2 subgroups (2-1 and 2-2). A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract PCR templates. PCR-specific primer sets for each group were designed from sequences in the regions of the 28S ribosomal DNA of this fungus. The results of specifically primed PCR analysis showed that AG 1-IA, AG 1-IB, AG 1-IC, AG 2-1, and AG 2-2 primers sets contributed detection from the same AG isolates and could escape detection from different AG isolates at a high level of frequency. In this experiment, we suggested that our synthesized primer sets might provide a method for the direct detection and identification of AGs of R. solani. Received: June 28, 2001 / Accepted: November 14, 2001     

Phylogeny of Saururaceae based on mitochondrial <Emphasis Type="Italic">matR</Emphasis> gene sequence data

DNA sequences of matR gene from three species of Saururaceae and the selected outgroups, Chloranthus holostegius and Zippelia begoniaefolia, are reported. All DNA sequences of six species in four genera of Saururaceae and the two outgroups are analyzed on PAUP 4.0 8b to reconstruct the phylogeny. A single matR gene tree is generated from parsimony, distance, and likelihood analyses, respectively. The three trees with the same topology are slightly different in bootstrapping support for some clades. The result indicates that Saururaceae is monophyletic. Anemopsis is sister to Houttuynia, and the two genera form the first diverging lineage of the family. The sister group relationship between Saururus and Gymnotheca is also supported by a relatively high bootstrap value. The result is different from all the former phylogenetic opinions on Saururaceae based on morphology, but it is supported by the evolution of flower-bract stalk in Saururaceae. In addition, some characteristics of the matR gene are analyzed. The MatR gene is a relatively better tool to reconstruct the molecular clock because the base substitution bias greatly decreases in the gene. Received: January 19, 2001 / Accepted: October 22, 2001     

Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes

This study was aimed at determining whether gene cassettes (promoter-coding sequence-terminator) can be efficiently used in microprojectile acceleration-mediated co-transformation of rice in the place of whole plasmids, and to what extent their use influences the integration and expression of the co-transferred gene of interest. Two non-linked marker genes (yfp and hph) were co-introduced by microprojectile bombardment into cells of embryogenic calli in three separate experiments. Three different DNA structures were compared for their ability to transiently and stably transform rice cells: supercoiled or linearized whole-plasmid DNA, gene cassette DNA and single-stranded gene cassette DNA coated with Escherichia coli single-stranded binding (SSB) proteins. Our results demonstrate that microprojectile bombardment-mediated transformation of rice using gene cassettes is possible without significantly reducing transformation efficiency in comparison to the use of whole-plasmid DNA. Furthermore, no obvious difference in transgene integration pattern and inheritance was observed among plants transformed with gene cassettes compared to those transformed with the whole plasmid, except that concatemerization of molecules prior to integration was rarely observed in gene cassette transformants. Received: 4 April 2001 / Accepted: 13 August 2001     

Multilocus Hybridization Typing in Pediococcus acidilactici Strains

In previous a study we demonstrated the presence of several genomic subpopulations within a collection of Pediococcus acidilactici strains isolated from different environments, through a multilocus typing analysis taking into consideration housekeeping conserved loci and protein coding genes of the primary metabolism. In this study, representative strains of five genomic subpopulations previously described (I, II, III, V, VII) were analyzed by restriction analysis of chromosomal DNA and subsequent hybridization assays using as probes amplified fragments obtained from five housekeeping genes (16S rDNA, rpoC, ldhD, ldhL, and metS). A computer similarity and clustering analysis of hybridization data showed the subdivision of P. acidilactici strains in five distinct genotypes according to the grouping previously obtained confirming that pediocin AcH/PA-1 producer strains represent one genomic lineage within the species P. acidilactici. Received: 30 January 2001 / Accepted: 16 May 2001     

PCR detection of Hordeum bulbosum introgressions in an H. vulgare background using a retrotransposon-like sequence

Retrotransposon-like sequences are ideal tools for initial screening assays to distinguish between closely related species because of their ubiquitous presence, high copy number, chromosome coverage and rapid sequence evolution. A retrotransposon-like sequence, pSc119.1, cloned from Secale cereale (rye) has been used to obtain PCR primers that are capable of detecting small introgressions of Hordeum bulbosum (bulbous barley grass) chromatin in a Hordeum vulgare (cultivated barley) background. Combining this PCR-based assay with a crude but effective high-throughput DNA extraction has enabled the rapid identification of plants possessing H. bulbosum introgressions from large numbers of progeny from H. vulgare×H. bulbosum crosses. These plants are then further characterized by more-refined cytological, molecular and pathological techniques to locate and map the introgressed chromatin and to evaluate their disease resistance. Received: 18 April 2001 / Accepted: 23 August 2001     

Identification of a Broad-Specificity Xylosidase/Arabinosidase Important for Xylooligosaccharide Fermentation by the Ruminal Anaerobe Selenomonas ruminantium GA192

Strains of Selenomonas ruminantium vary considerably in their capacity to ferment xylooligosaccharides. This ability ranges from strain GA192, which completely utilized xylose through xylotetraose and was able to ferment considerable quantities of larger oligosaccharides, to strain HD4, which used only the simple sugars present in the hydrolysate. The ability of S. ruminantium GA192 to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidase activities. The production of these activities appears to be regulated in response to carbon source used for growth. Both arabinosidase and xylosidase were induced by growth on xylose or xylooligosaccharides, but no activity was detected in glucose-or arabinose-grown cultures. A genetic locus from S. ruminantium GA192 was cloned into Escherichia coli JM83 that produced both xylosidase and arabinosidase activities. Analyses of crude extracts from the E. coli clone and S. ruminantium GA192 by using native polyacrylamide gel electrophoresis and methylumbelliferyl substrates indicated that a single protein was responsible for both activities. The enzyme expressed in E. coli was capable of degrading xylooligosaccharides derived from xylan. DNA sequencing of the locus demonstrated the presence of an open reading frame that encodes for a protein of 61,174 molecular weight. Received: 12 January 2001 / Accepted: 5 March 2001     

Stable Integration and Functional Expression of Flounder Growth Hormone Gene in Transformed Microalga, Chlorella ellipsoidea

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 μg of fGH protein expression per one liter culture containing 1 × 10⁸ cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding. Received March 26, 2001; accepted July 10, 2001.     

Genus- and Isolate-Specific Real-Time PCR Quantification of <Emphasis Type="Italic">Erwinia</Emphasis> on Leaf Surfaces of English Oaks (<Emphasis Type="Italic">Quercus robur</Emphasis> L.)

In order to quantify pathogenic epiphytic bacteria on leaf surfaces of the important European forest tree Quercus robur without time-intensive cultivation and separation of microorganisms, methods were developed to selectively quantify DNA copy numbers of the genus Erwinia in DNA isolated from the leaf surface. By using the combination of the two different real-time PCR techniques SYBR®-Green and TaqMan®, methods were developed not only to allow quantification of the total DNA copy number of Erwinia on the oak leaf surface, but also to distinguish between two significantly different groups of Erwinia strains. In the present work, these techniques were successfully applied to quantify the copy number of the genus Erwinia and its subgroups compared with the total bacteria number in DNA samples extracted from the upper leaf surface of English oaks collected on the 4^th of June 2001 (Julian day 155). Received: 24 June 2002 / Accepted: 25 October 2002     

Detection and Phylogenetic Analysis of Novel Crenarchaeote and Euryarchaeote 16S Ribosomal RNA Gene Sequences from a Great Barrier Reef Sponge

The presence of Archaea in the Great Barrier Reef marine sponge Rhopaloeides odorabile was investigated by 16S ribosomal RNA community analysis of total DNA extracted from the sponge tissue. The 16S rRNA gene sequences corresponding to group I crenarchaeotes and group II euryarchaeotes were recovered from R. odorabile tissue. The location of archaeal cells within the sponge tissue was investigated using fluorescently labeled oligonucleotide probes. The presence of Archaea was confirmed within all regions of the sponge tissue from R. odorabile, with a significantly higher number of archaeal cells located in the pinacoderm than the mesohyl region. This is the first report of euryarchaeaotes associated with marine sponges. Received April 16, 2001; accepted June 27, 2001     

New mitochondrial genome organization in three interspecific somatic hybrids of Medicago sativa including the parent-specific amplification of substoichiometric mitochondrial DNA units

Three somatic hybrid plants produced by protoplast fusion between Medicago sativa and each of the three species Medicago coerulea, Medicago falcata and Medicago arborea have been analysed for the composition of their mitochondrial DNA. Restriction fragment length polymorphism (RFLP) analysis of mitochondrial genes in somatic hybrids and their parental lines showed various degrees of rearrangement. The M. sativa+M. coerulea hybrid retained all of the M. coerulea-specific bands but lost all the major M. sativa- specific bands. The M. sativa+M. falcata hybrid showed only M. sativa-specific bands together with non-parental bands, and the M. sativa+M. arborea hybrid showed a partial incorporation of bands from both parents together with non-parental bands. The three different outcomes were attributed mainly to differences in the genetic distance between the parents of each hybrid. Analysis of the sexual progeny of the M. sativa+M. coerulea hybrid showed that a residual mitochondrial DNA subunit of M. sativa was retained in the hybrid cytoplasm. This subunit was amplified and inherited in a mutually exclusive, allelic-like fashion with its M. coerulea homologous counterpart in the sexual progeny of the hybrid. Possible mechanisms for the partitioning of mitochondrial DNA in the generative lineage of the somatic hybrids are discussed in relation to the creation of new nucleus-cytoplasm assortments otherwise impossible to obtain by a sexual cross in Medicago. Received: 5 January 2001 / Accepted: 23 March 2001     

Exact sampling formulas for multi-type Galton-Watson processes

 Exact formulas for the mean and variance of the proportion of different types in a fixed generation of a multi-type Galton-Watson process are derived. The formulas are given in terms of iterates of the probability generating function of the offspring distribution. It is also shown that the sequence of types backwards from a randomly sampled particle in a fixed generation is a non-homogeneous Markov chain where the transition probabilities can be given explicitly, again in terms of probability generating functions. Two biological applications are considered: mutations in mitochondrial DNA and the polymerase chain reaction. Received: 10 June 2001 / Revised version: 21 November 2001 / Published online: 23 August 2002 Mathematics Subject Classification (2000): Primary 60J80, Secondary 92D10, 92D25 Key words or phrases: Multi-type Galton-Watson process – sampling formula – PCR – mitochondrial DNA     

Phylogeography of a freshwater sculpin, Cottus nozawae, from the northeastern part of Honshu Island, Japan

 The fluvial sculpin, Cottus nozawae, is a coldwater-adapted fish distributed in Hokkaido Island and the northeastern part of Honshu Island (Tohoku District), Japan. Mitochondrial DNA (mtDNA) control region sequencing was used to investigate the geographic distribution of genetic variation and phylogeography of C. nozawae. Most populations possessed unique haplotypes, few being shared across river systems. Phylogenetic analysis of the sequences of the mtDNA control region and adjacent regions of C. nozawae revealed three distinct phylogenetic groups that differed by 3.05% to 3.11%, corresponding to distinct geographic regions, Hokkaido Island, northern Tohoku District, and Yamagata Prefecture (southwestern Tohoku District), respectively. The divergence times of three groups were estimated to be about 1.5 million years ago by applying a general rate for mtDNA, suggesting that the divergence among them might have occurred in the early Pleistocene. Divergence among the haplotypes within the group from the northern Tohoku District was also high (1.84%), no haplotypes being shared by local populations in different river systems in this region. Local populations from a single river system in this region comprise a distinct lineage that differed from other river systems. Such genetically divergent population structures among the different regions and river systems are considered to have resulted mainly from long-term isolation and restricted gene flow among river systems, probably promoted by the fluvial benthic life history and low dispersal ability of this species. Received: April 12, 2001 / Revised: December 1, 2001 / Accepted: December 19, 2001     

MutS2 Family Protein from Pyrococcus furiosus

MutS2 protein of Pyrococcus furiosus has been cloned and over-expressed. Initial characterization reveals that PfuMutS2 possesses a thermostable ATPase activity and a thermostable, nonspecific DNA binding activity. However, PfuMutS2 does not have any detectable mismatch-specific DNA binding activity. It is the first in vitro characterization of an MutS2 family protein. Received: 23 April 2001 / Accepted: 27 August 2001     

Efficient Electrotransformation of <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> with a Mini-Replicon Derived from the <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> Plasmid pGA1

Efficient transformation of the human pathogen Corynebacterium diphtheriae was achieved with novel cloning vectors consisting of a mini-replicon from the cryptic C. glutamicum plasmid pGA1 as well as of the aph(3′)-IIa or tetA(Z) antibiotic resistance genes. Plasmid-containing transformants of C. diphtheriae were recovered at frequencies ranging from 1.3 × 10⁵ to 4.8 × 10⁶ colony forming units (cfu)/μg of plasmid DNA. Vector DNA was directly transferred from Escherichia coli into C. diphtheriae with frequencies up to 5.6 × 10⁵ cfu/μg of plasmid DNA. On the basis of the pGA1 mini-replicon, an expression vector system was established for C. diphtheriae by means of the P _tac promoter and the green fluorescent reporter protein. In addition, other commonly used vector systems from C. glutamicum, including the pBL1 and pHM1519 replicons, and the sacB conditionally lethal selection marker from Bacillus subtilis, were shown to be functional in C. diphtheriae. Thus, the ability to apply the standard methods of C. glutamicum recombinant DNA technology will greatly facilitate the functional analysis of the recently completed C. diphtheriae genome sequence. Received: 26 November 2001 / Accepted: 15 February 2002