Restriction endonuclease mapping of a plasmid that confers oncogenicity upon Agrobacterium tumefaciens strain B6-806

The 26 SmaI digest fragments of pTi-B6-806 plasmid have a total molecular weight (121 × 10⁶) which accounts for the size of the plasmid as determined by contour length measurements. We have determined the physical arrangement of all SmaI digest fragments with reference to HpaI digest fragments. Hybridization of individual labeled SmaI digest fragments to HpaI digest fragments (cellulose nitrate transfers) allowed the latter to be ordered and located the SmaI boundary fragments. Recleavage of isolated HpaI fragments with SmaI revealed the SmaI fragments located within each HpaI fragment. The order of these internal SmaI fragments within a given HpaI fragment was determined by partial digestion of the latter with SmaI and hybridization of the resulting fragments with SmaI boundary fragments. From the sizes of partial digest fragments containing each boundary, the order of occurrence of SmaI fragments from each end was deduced. The complete map of the SmaI digest fragments is presented. The map of the HpaI digest fragments is presented with the following ambiguity: The order of fragments 12, 15, and 16, which map within SmaI fragment 1, was not determined. The SmaI digest fragments that contain DNA sequences transferred to plant cells during tumor induction, fragments 3b and 10c, were found to be contiguous on the physical map.     

Restriction map of CELO virus DNA

A restriction map of chicken embryo lethal orphan (CELO) virus DNA was reported with ten restriction endonucleases (XbaI, XhoI, SalI, HindIII, EcoRI, BglI, KpnI, BamHI, PstI and SstI). CELO virus DNA was estimated by comparing CELO virus DNA fragments with marker DNA fragments to have a molecular weight of 29.3·10⁶.     

A physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome

A combined physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleasesSwaI (5′-ATTTAAAT-3′),PacI (5′-TTAATTAA-3′), andPmeI (5′-GTTTAAAC-3′) yielding 26, 27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacentSwaI fragments, a genomic cosmid library ofC. glutamicum was screened for chromosomal inserts containingSwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect theSwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blottedSwaI digests using as probesPacI andPmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partialSwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes toSwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.     

A site-specific endonuclease from Caulobacter crescentus CB-13: restriction endonuclease CcrI

A site-specific restriction endonuclease (CcrI) has been identified from Caulobacter crescentus CB-13. This enzyme has been purified to homogeneity and the cleavage patterns with various DNAs and sequence data show that CcrI recognizes the same sequence as the XhoI restriction endonuclease (5′-C^↓-T-C-G-A-G-3′). Ccr has an absolute requirement for magnesium ions with an optimum concentration of 4 mM. The enzyme is optimally active at pH 8.0 and is stable up to 70°C. CcrI has a molecular weight of 65300 and exists as a monomer in its native state. Most of the physical characteristics observed for CcrI were similar to those observed for XhoI. Kinetic studies on CcrI and XhoI suggest that the enzymes interact with λ DNA in the same manner; however, with ?X-174 R.F. DNA, CcrI has a greater affinity for the supercoiled molecule than XhoI.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.?coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.     

Metabolic classification of South American Ilex species by NMR-based metabolomics

The genus Ilex to which mate (Ilex paraguariensis) belongs, consists of more than 500 species. A wide range of metabolites including saponins and phenylpropanoids has been reported from Ilex species. However, despite the previous works on the Ilex metabolites, the metabolic similarities between species which can be used for chemotaxonomy of the species are not clear yet. In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomics was applied to the classification of 11 South American Ilex species, namely, Ilex argentina, Ilex brasiliensis, Ilex brevicuspis, Ilex dumosa var. dumosa, I. dumosa var. guaranina, Ilex integerrima, Ilex microdonta, I. paraguariensis var. paraguariensis, Ilex pseudobuxus, Ilex taubertiana, and Ilex theezans. ¹H NMR combined with principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) showed a clear separation between species and resulted in four groups based on metabolomic similarities. The signal congestion of ¹H NMR spectra was overcome by the implementation of two-dimensional (2D)-J-resolved and heteronuclear single quantum coherence (HSQC). From the results obtained by 1D- and 2D-NMR-based metabolomics it was concluded that species included in group A (I. paraguariensis) were metabolically characterized by a higher amount of xanthines, and phenolics including phenylpropanoids and flavonoids; group B (I. dumosa var. dumosa and I. dumosa var. guaranina) with oleanane type saponins; group C (I. brasiliensis, I. integerrima, I. pseudobuxus and I. theezans) with arbutin and dicaffeoylquinic acids, and group D (I. argentina, I. brevicuspis, I. microdonta and I. taubertiana) with the highest level of ursane-type saponins. Clear metabolomic discrimination of Ilex species and varieties in this study makes the chemotaxonomic classification of Ilex species possible.     

Accurate identification of three cryptic species of rodents of the genus Calomys using RAPD-PCR and mitDNA RFLP markers

Calomys musculinus, Calomys laucha and Calomys venustus are cryptic species with overlapping distribution ranges. C. musculinus is the natural reservoir of Junin virus (Arenaviridae), the etiological agent of Argentine Hemorrhagic Fever. In epidemiological studies it is very important to unequivocally identify the species of individuals collected in the field in order to test virus infection. The purpose of this work was to describe molecular markers allowing a prompt and clear characterization of individuals of the three species. We studied the D-loop region of mitochondrial DNA by restriction fragment length polymorphism (RFLP). This region was amplified by PCR and the product was digested with nine restriction endonucleases (RE). Eight recognize 4 bp restriction sites (Taqα I, Tsp509 I, Aci I, Rsa I, Alu I, Nla III, Hae III and Mse I) and one recognized a 6 bp sequence (Ase I). Two of them (Aci I and Hae III) did not distinguish any of the three species. Alu I did not discriminate between C. musculinus and C. laucha, but clearly distinguished both from C. venustus. Taq I did not distinguish C. laucha from C. venustus, but differentiated both species from C. musculinus. Mse I distinguished the three species, but some of the polymorphisms of C. musculinus are very similar to C. laucha's restriction pattern. The enzyme Nla III distinguished the three species, but it is highly polymorphic within species. The enzymes Tsp509 I, Rsa I and Ase I clearly discriminated the three species, and patterns obtained with the three of them are recommended for reliable identification of individuals collected in the field. The same DNA samples were used to obtain Random Amplified Polymorphic DNAs (RAPDs) patterns. Several bands produced with primers A02, A06, A08, A09, B09 and OPA02 are species specific and could also be used for identification.     

Sse9I restriction-modification system: Organization of genes and structural comparison of proteins

The nucleotide sequence was established for the operon of the Sse9I type II restriction-modification system of Sporosarcina species 9D. The enzymes of the Sse9I system recognize the 5′-AATT-3′ tetranucleotide. The operon includes three genes, sse9IC-sse9IR-sse9IM, which are transcribed unidirectionally and code, respectively, for the controller protein (C.Sse9I), restriction endonuclease (R.Sse9I), and DNA methyltransferase (M.Sse9I). The region immediately upstream of sse9IC was found to contain a conserved nucleotide sequence (C box) providing a binding site for C. Sse9I. The amino acid sequences of C.Sse9I and R.Sse9I were compared with those of related proteins. In the case of R.Sse9I, the highest homology was observed with the R.MunI (5′-CAATTG-3′) and R.EcoRI (5′-GAATTC-3′) regions that harbor the amino acid residues involved in recognizing the AATT inner tetranucleotide. The sse9IR gene was cloned in an expression vector, and recombinant R.Sse 9I was isolated.     

Molecular cloning and physical map of bacteriophage PM2 DNA

The entire genome and the DNA fragments of the lipid-containing bacteriophage pM2 were cloned in the pBR322 plasmid vector. A physical map including the sites for the following restriction enzymes was obtained: HpaII, HaeIII, TthI, Sau96I, AvaII, PstI, BstNI, AccI, HincII, HpaI and HindIII. No restriction sites on PM2 DNA were found for BalI, BamHI, BclI, BglI, BglII, BstEII, KpnI, PvuII, SacI, SalI, Sau3A, XbaI and XhoI.     

Novel reports of glands in Neotropical species of Indigofera L. (Leguminosae,Papilionoideae)

By considering controversial discussions in the literature with regard to gland denomination in Indigofera species, as well as the taxonomic value of secretory structures in Leguminosae, we aim to morphologically detail glands that had been previously observed in I. microcarpa and I. sabulicola, and to investigate the occurrence of glands in vegetative and reproductive organs of other six Neotropical species that belong to the genus. Glands analyzed through scanning electronic microscopy (SEM) in combination with anatomic analyses correspond to secretory trichomes that are classified into seven types. Main variations in relation to types occurred with regard to head shape and peduncle size. Trichome heads were multicellular, with a thin cuticle. Hollow heads with conspicuous inner space characterized only one type (type 1); the other trichome types had massive heads. Peduncles, which varied from biseriate to multiseriate, had thick, pecto-cellulosic cell walls. Trichomes were found on stems, stipules, petioles, rachis, petiolules, leaflets, bracteoles, sepals, standards and fruits, more commonly along the margins. Each of the eight Indigofera species analyzed had at least two different trichome types out of the seven types that occurred in reproductive and vegetative organs of these taxa. Various types of secretory trichomes were found in I. campestris, I. lespedezioides, I. microcarpa, I. spicata, I. suffruticosa and I. truxillensis. Stems and rachis were the vegetative organs in which a greater variety of trichomes occurred, and sepals were parts of reproductive organs with the same status. Five out of the seven secretory trichome types occurred on both vegetative and reproductive organs. Distribution and gland types differed between species and these gland distribution patterns can be used as diagnostic characters. Reports of glands in Indigofera campestris, I. hirsuta, I. lepedezioides, I. suffruticosa, I. spicata and I. truxillensis, their recognition as secretory trichomes, and the morphological variety of types found for such trichomes are novel data for Indigofera.     

Inonotus tenuicontextus sp. nov. (Hymenochaetaceae) from Guizhou, southwest China with a preliminary discussion on the phylogeny of its kin

Inonotus tenuicontextus collected from Guizhou, southwest China was described and illustrated as a new species based on a combination of phylogenetic and morphological evidence. It is characterized by perennial and effused-reflexed to pileate basidiocarps; duplex and very thin context; a monomitic hyphal system in context; a dimitic hyphal system in trama; and broadly ellipsoid, hyaline and thick-walled basidiospores. Phylogenetically I. tenuicontextus clustered within Inonotus s. s. clade; moreover, it formed a well supported monophyletic subclade with Inonotus baumii, I. linteus, I. lonicericola, I. vaninii and I. weirianus. In morphology I. tenuicontextus distinguishes from I. linteus, also a species with duplex context, by its smaller basidiospores, while its duplex context makes it different from the other four species with homogeneous context. We proposed this subclade as a medicinal group for most of its members with medicinal functions. The phylogeny of the six species in this medicinal group was briefly discussed based on our results. An identification key to them is also provided.     

Genome analysis ofPropionibacterium freudenreichii by pulsed-field gel electrophoresis

Preparations of intact genomic DNA from 23 strains ofPropionibacterium freudenreichii were compared by digestion with restriction endonucleases and subsequent transverse alternating field electrophoresis (TAFE). Seven restriction enzymes,AsnI,DraI,HpaI,SnaBI,SpeI,SspI, andXbaI, produced DNA fragments useful for strain comparisons. A characteristic restriction fragment pattern was identified for 18 of the 23 strains. Estimates for the genome size of theP. freudenreichii strains ranged from 1.6×10⁶ to 2.3×10⁶ base pairs based on the sum of fragment sizes obtained with restriction digests. Restriction endonuclease patterns resolved by TAFE are useful for strain identification.     

Phylogeny of Isatis (Brassicaceae) and allied genera based on ITS sequences of nuclear ribosomal DNA and morphological characters

Systematics of the genus Isatis (Brassicaceae) is difficult and controversial, and previous studies were based solely on morphological characters. Sequence variation of the internal transcribed spacer (ITS) regions and the 5.8S gene of nuclear ribosomal DNA (nrDNA) were analyzed using parsimony and Bayesian methods. Twenty-eight taxa of Isatis and related genera of the tribe Isatideae were sampled, including 20 Isatis species representing almost all major morphological lineages, all three species of Pachypterygium, two of nine species of Sameraria, and monospecific Boreava, Myagrum, and Tauscheria. Two well-supported clades were resolved in the ITS tree, and they demonstrate the artificiality of the present delimitation of the tribe. One clade includes I. emarginata, I. minima, I. trachycarpa, P. brevipes, P. multicaule, P. stocksii, and T. lasiocarpa. The second clade includes I. buschiana, the polymorphic I. cappadocica with five subspecies, I. gaubae, I. kotschyana, I. leuconeura, I. pachycarpa, I. takhtajanii, I. tinctoria, and S. armena. Pachypterygium is polyphyletic and, together with Boreava, Sameraria, and Tauscheria, all are nested within Isatis. This study is a continuation of our recent systematic survey based on seed-coat microsculpturing ( Moazzeni et al., 2007. Flora 202, 447–454) and reveals that fruit characters mapped onto the molecular tree show considerable convergence. The reliance on fruit characters alone in the delimitation of genera may well lead to erroneous phylogenetic results and thus to incorrect taxonomic conclusions.     

A putative tropical American plant,Ipomoea nil (Convolvulaceae), in pre-Columbian Japanese art

The Heike Nôkyô, Japanese scrolls of Buddhist sutras created in 1164 AD, includes illustrations of anIpomoea that has long been identified by Japanese scholars asI. nil. What makes this occurrence ofI. nil in pre-Columbian Japan remarkable is that all of its closest relatives are American plants. We give a synopsis of the history of this economically important species. Then, using cladistic analysis, we show the relationships ofI. nil toI.eriocalyx,I. hederacea,I. indica,I. laeta,I. lindheimeri,I. meyeri, andI. pubescens. Six of these eight species inIpomoea seriesHeterophyllae are endemic to the New World.Ipomoea indica is pantropical, and may be carried by ocean currents. We offer four hypotheses as to how this putatively tropical American species may have arrived in Asia: 1)Ipomoea nil was introduced through long-distance dispersal by animals; 2)Ipomoea nil was introduced by humans in a pre-Columbian context; 3) TheIpomoea in theHeike Nôkyô scrolls does not representI. nil, but a different native Asian species; and 4)Ipomoea nil was introduced during post-Columbian times by Europeans. There are problems with accepting any of these possible alternatives.     

Purification and Biochemical Characterization of Mutacin I from the Group I Strain of Streptococcus mutans, CH43, and Genetic Analysis of Mutacin I Biosynthesis Genes

Previously, we reported isolation and characterization of mutacin III and genetic analysis of mutacin III biosynthesis genes from the group III strain of Streptococcus mutans, UA787 (F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:3880–3887, 1999). During the same process of isolating the mutacin III structural gene, we also cloned the structural gene for mutacin I. In this report, we present purification and biochemical characterization of mutacin I from the group I strain CH43 and compare mutacin I and mutacin III biosynthesis genes. The mutacin I biosynthesis gene locus consists of 14 genes in the order mutR, -A, -A′, -B, -C, -D, -P, -T, -F, -E, -G, orfX, orfY, orfZ. mutA is the structural gene for mutacin I, while mutA′ is not required for mutacin I activity. DNA and protein sequence analysis revealed that mutacins I and III are homologous to each other, possibly arising from a common ancestor. The mature mutacin I is 24 amino acids in size and has a molecular mass of 2,364 Da. Ethanethiol modification and peptide sequencing of mutacin I revealed that it contains six dehydrated serines, four of which are probably involved with thioether bridge formation. Comparison of the primary sequence of mutacin I with that of mutacin III and epidermin suggests that mutacin I likely has the same bridging pattern as epidermin.     

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters.     

Expression of the I-E target antigen for T-cell killing requires two genes

TheH?2I ^k region encodes at least two different target antigens for unrestricted T-cell mediated killing. The first is controlled by theI?A region alone and the second depends on a pair of alleles, one located to the left ofI?B (presumably inI?A) and the other to the right ofI?J (presumably inI?E). Hence, effector cells nominally specific for a product of theI?E region do not kill target cells with the sameI?E region as the stimulator unless theI?A region is also shared. Some effectors specific forH?2I ^k , such as A.TH anti-A.TL and B10.A(4R) anti-B10.A(2R), cross-react with B10.A(3R) and B10.A(5R) target cells. A product of theH?2 ^b haplotype was shown to complement products of theH?2 ^d orH?2 ^k haplotypes in forming this cross-reactive determinant. The results are consistent with recent biochemical data on the component chains of Ia antigens.     

Genomic Analysis of Clostridium botulinum Group II by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (nonproteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean ± standard deviation = 3,890 ± 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.     

Combined effects of three novel SNPs within goat LHX3 gene on milk performance

In this study, single nucleotide polymorphisms (SNPs) of LHX3 gene were detected by DNA sequencing based on DNA pools and PCR-RFLP method in 792 goats belonging to three Chinese indigenous breeds (Guanzhong dairy, Xinong Sannen dairy, Inner Mongolia white cashmere). The results revealed three novel mutations (AY923832:g.7778A>T; g.8035T>C; g.10592C>T), which were named as P2-DraI, P3-HinfI and P4-MspI loci, respectively. The linkage disequilibrium analysis demonstrated P3-HinfI and P4-MspI loci were strongly linked (r ²>0.33) in all the analyzed populations. Each SNP produced no significant (p>0.05) effects on milk performance. However, the two-loci and three-loci combined genotypes had more profound impacts on milk yield than in separation. The individuals with diplotype AATT (P2-DraI/P3-HinfI) showed significantly (p<0.05) higher milk yield than those with diplotypes ATTT, TTTT, ATTC, and AACC. Diplotype TTCCTT (P2-DraI/P3-HinfI/P4-MspI) carriers had significantly (p< 0.05) higher milk yield than those with diplotypes ATTTCC and AACCTC. These combined effects of LHX3 gene polymorphisms indicated that this gene had significant effect on milk performance and its corresponding combined diplotypes could be regarded as potential genetic markers of milk performance.     

Detection and Genetic Characterization of Relapsing Fever Spirochete Borrelia miyamotoi in Estonian Ticks

During the years 2008–2010 I. ricinus and I. persulcatus ticks were collected from 64 sites in mainland Estonia and on the island Saaremaa. Presence of B. miyamotoi was found in 0.9% (23/2622) of ticks. The prevalence in I. persulcatus and I. ricinus ticks differed significantly, 2.7% (15/561) and 0.4% (8/2061), respectively. The highest prevalence rates were in found South-Eastern Estonia in an area of I. persulcatus and I. ricinus sympatry and varied from 1.4% (1/73) to 2.8% (5/178). Co-infections with B. burgdorferi s.l. group spirochetes and tick-borne encephalitis virus were also revealed. Genetic characterization of partial 16S rRNA, p66 and glpQ genes demonstrated that Estonian sequences belong to two types of B. miyamotoi and cluster with sequences from Europe and the European part of Russia, as well as with sequences from Siberia, Asia and Japan, here designated as European and Asian types, respectively. Estonian sequences of the European type were obtained from I. ricinus ticks only, whereas the Asian type of B. miyamotoi was shown for both tick species in the sympatric regions.