Effect of restriction endonucleases on assessment of biodiversity of cultivable polar marine planktonic bacteria by amplified ribosomal DNA restriction analysis

To choose a suitable restriction endonuclease for quick assessment of bacterial diversity in polar environments by ARDRA, we investigated the effect of restriction enzymes on ARDRA patterns of cultivable marine planktonic bacteria isolated from polar region. Thirty-three isolates were analyzed by ARDRA using five enzymes (HinfI, HaeIII, AluI, and the mix AfaI/MspI), respectively, resulting in different groups, each group corresponding to a particular genotype. A comparison of the ARDRA patterns was carried out, and phylogenetic position of all thirty-three bacteria was obtained by 16S rDNA sequencing. Consistent with phylogenetic analysis, ARDRA pattern comparison revealed that AluI, being sensitive and reliable enough to generate species-specific patterns, was a suitable restriction enzyme used for evaluating bacterial diversity, suggesting a combination of ARDRA with AluI and 16S rDNA sequencing can provide a simple, fast and reliable means for bacterial identification and diversity assessment in polar environments.     

Phylogenetic relationships of subtribe Ecliptinae (Asteraceae: Heliantheae) based on chloroplast DNA restriction site data

Phylogenetic analysis of chloroplast DNA restriction site data for 76 of the 302 genera of Heliantheae sensu lato using 16 restriction endonucleases reveals that subtribe Ecliptinae is polyphyletic and that its genera are distributed in four different lineages. The ecliptinous genera Squamopappus, Podachaenium, Verbesina, and Tetrachyron (of the Neurolaeninae), along with other members of subtribe Neurolaeninae are the basalmost clades of the paleaceous Heliantheae. The mostly temperate species of subtribe Ecliptinae (exemplified by Balsamorhiza, Borrichia, Chrysogonum, Engelmannia, Silphium, Vigethia, and Wyethia) are strongly nested in a clade with the Mesoamerican monotypic genus Rojasianthe as basal. The genera characterized by marcescent ray corollas traditionally classified in subtribe Zinniinae constitute a strongly supported group sister to Acmella, Spilanthes, and Salmea. The largest clade of ecliptinous genera is the most recently derived group within Heliantheae sampled. This large group of mostly Neotropical lowland genera (variously characterized by their winged cypselae, foliaceous phyllaries, and opposite phyllotaxy and exemplified by Perymenium, Wedelia, and Zexmenia) has been and continues to be the most challenging group from a taxonomic standpoint. The study provides new insights as to their relationships that will have a positive impact in future monographic studies of the group. The genera of the Espeletiinae form a monophyletic clade and are sister to members of the Milleriinae and Melampodiinae. This result is consistent with their traditional taxonomic placement with genera such as Smallanthus with which they share a tendency for functionally staminate disc flowers. The phylogenetically enigmatic genus Montanoa is sister to Melampodium. Members of subtribe Galinsoginae are clustered in two main lineages that correspond to the traditional division of the subtribe based on pappus characteristics. There is no support for the monophyly of subtribe Galinsoginae, and the same results indicate some of its genera are paraphyletic.     

Phylogenetic analysis of the hard pines (Pinus subgenus Pinus,Pinaceae) from chloroplast DNA restriction site analysis

Phylogenetic analysis of plastid DNA restriction site and rearrangement mutations suggests a number of major revisions to taxonomy and phylogenetic concepts in the hard pines. Total genomic DNA from 18 species that sampled all nine subsections was digested with 19 restriction enzymes, blotted, and probed with 70% of the Douglas-fir (Pseudotsuga menziesii) chloroplast genome, or, with clones encompassing the entire chloroplast genome of Pinus contorta. A total of 204 site mutations and five rearrangement mutations were generated, of which 126 were phylogenetically informative. Wagner parsimony analyses revealed 11 clades that were strongly supported by bootstrap and decay index analyses. All North American species except P. resinosa formed a distinct monophyletic group that was strongly separated from the Eurasian species. Within the Eurasian clade subsect. Sylvestres was polyphyletic; its Mediterranean species were closely allied with members of sect. Pinea. Sect. Pinea appeared polyphyletic as well; both species of its subsect. Leiophyllae showed a close affinity to Mesoamerican pines of subsect. Oocarpae in sect. Pinus. Within the North American pines subsects. Ponderosae and Oocarpae were polyphyletic. Despite its shallow fossil record, subsect. Contortae emerged as a sister group to all of the North American pines apart from P. resinosa, which was allied with Eurasian species of subsect. Sylvestres. The remaining North American subsections formed two groups: a poorly resolved clade with subsects. Ponderosae and Sabinianae, and sequentially nested clades represented by: P. radiata; P. taeda; representatives of subsects. Oocarpae and Ponderosae from Mesoamerica, and subsect. Leiophyllae. We present estimates of divergence times for each of these major clades based on molecular clocks calibrated using two hard pine fossil observations.     

Chloroplast DNA variability in the genus Helianthus: restriction analysis and S1 nuclease mapping of DNA-DNA heteroduplexes

Chloroplast DNA (cpDNA) from 36 wild species of the genus Helianthus has been analysed with three restriction endonucleases (Bam HI, Hind III and Sst I). Out of the 71 restriction sites described on the reference cpDNA (sunflower cpDNA), three insertions/deletions and seven site modifications were detected during the survey of the other cpDNAs.Since restriction mapping showed only a very limited fraction of the DNA variability, we chose to adapt the S1 nuclease mapping technique to detect fine variations between chloroplast genomes. For this purpose, DNA-DNA heteroduplexes obtained between sunflower and wild-species DNAs were digested by S1 nuclease and the resulting mismatches were detected by classical endonuclease restriction and hybridization methods. The S1 nuclease mapping results were confirmed by sequencing one S1 nuclease-sensitive region detected between cultivated sunflower and two perennial wild-type species.As a result of these analyses, it appeared that the combination of restriction mapping and S1 nuclease mapping might be helpful to differentiate taxonomically close cytoplasms.     

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.     

Polymorphism of mitochondrial DNA in pigs based on restriction endonuclease cleavage patterns

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of pigs and Japanese wild boars were analyzed using 17 enzymes which recognize six nucleotides. The map of cleavage sites was made by double-digestion methods. Polymophism of mtDNA was detected in the digestion by BglII, EcoRV, ScaI, and StuI. The restriction cleavage patterns were identical among the breeds of Landrace, Hampshire, Duroc I, and Large White I (A type). The patterns of Large White II were the same as those of Japanese wild boars (B type). A difference between the A type and the B type of mtDNA was found in the case of three restriction enzymes, BglII, ScaI, and StuI, and the nucleotide alterations between them were estimated as more than six. On the other hand, a difference between mtDNA from almost all pigs and mtDNA from Duroc II was detected using EcoRV. We suggest that the difference of mtDNA between the A type and the B type of mtDNA could result from the different origin of boars, that is, whether they were of European or Asian origin.     

A phylogenetic analysis of American Zamiaceae (Cycadales) using chloroplast DNA restriction fragment length polymorphisms

A phylogenetic analysis of American cycad genera, all belonging to the family Zamiaceae, was attempted using chloroplast DNA restriction fragment polymorphisms.Ceratozamia mexicana Brongn.,Chigua restrepoi D. Stevenson,Dioon edule Lindley,Microcycas calocoma (Miq.) A. DC.,Zamia fischeri Miq., andZamia skinneri Warsz. ex A. Dietrich were used as representatives of the genera.Cycas revoluta Thunb., belonging to the family Cycadaceae, was used as an outgroup, following previous morphological works. One hundred and forty-one shared restriction fragments were scored for presence/absence and both Wagner and Dollo parsimony analyses were performed. The single, fully resolved, most parsimonious trees obtained from the analyses were topologically identical and perfectly matched previous morphology-based phylogenetic hypotheses. Statistical evaluation of the data showed a good reliability for the obtained phylogeny.Dioon edule, belonging to a different subfamily and more primitive on morphological grounds, proved to be the most primitive among American cycads as inferred from the molecular data;Chigua restrepoi, never analyzed before on cladistic grounds, was found to be the sister group of the genusZamia.     

Heteroplasmy of chloroplast DNA in Medicago

Two chloroplast DNA (cpDNA) regions exhibiting a high frequency of intra- or inter-species variation were identified in 12 accessions of the genus Medicago. Restriction maps of both regions were prepared for alfalfa, and the probable nature of the events causing the DNA differences was identified. Specific DNA fragments were then cloned for use in identification of variants in each region. Two each of M. sativa ssp. varia and ssp. caerulea and one of six M. sativa ssp. sativa single plants examined possessed cpDNA heterogeneity as identified by screening extracts for fragments generated by the presence and absence of a specific Xba I restriction site. Three plants of M. sativa ssp. sativa, two of each of sspp. varia and caerulea, and three M. scutellata were also examined for single-plant cpDNA heterogeneity at a hypervariable region where differences resulted from small insertion-deletion events. A single M. scutellata plant with mixed cpDNAs was identified. Sorting out was seen when one spp. sativa plant with mixed plastid types identifiable by the Xba I restriction site difference was vegetatively propagated. This indicated that the initial stock plant was heteroplastidic. Controlled crosses will be required in order to test whether heteroplasmy results from chloroplast transmission in the pollen and to examine the dynamic of sorting out. However, heteroplasmy is apparently not a rare situation in Medicago.Contribution No 88-547-J from the Kansas Agricultural Experiment Station, Manhattan.     

Paternal inheritance of chloroplast DNA in Larix

Restriction enzyme analysis was used to determine the inheritance of chloroplast DNA in conifers. The plant material studied included five individual trees of European larch (Larix decidua Mill.) and Japanese larch (Larix leptolepis Sieb. & Zucc.) and six hybrids from controlled crosses between these species. The chloroplast DNA fragment patterns generated by Bam-HI and Bcl-I were species-specific. Paternal inheritance of chloroplast DNA patterns was found in most Larix crosses. One hybrid showed maternal chloroplast DNA patterns. In addition, two other hybrids had mixed Bam-HI patterns suggesting recombination between maternal and paternal chloroplast DNA. The mechanisms favoring paternal inheritance in conifers are not known. Paternal inheritance of chloroplast DNA is suggested it to be a general phenomenon in conifers.     

A phylogenetic analysis of chloroplast DNA restriction site variation inPoaceae subfam.Pooideae

A phylogenetic analysis was conducted on chloroplast DNA restriction site variation in 34 genera of grasses (familyPoaceae), including 28 genera from subfam.Pooideae (representing tribesAveneae, Brachypodieae, Bromeae, Meliceae, Poeae, Stipeae, andTriticeae) and representatives of three other subfamilies,Arundinoideae, Oryzoideae, andPanicoideae. Analyses of all 34 genera always distinguishedPooideae as monophyletic, regardless of which nonpooid genus functioned as outgroup; six separate analyses of all 28 pooid genera, each including one of the six nonpooid genera as outgroup, resolved five identically-constituted clades withinPooideae (in four cases), or (in the other two cases) yielded results that were less well resolved, but not in conflict with those of the other four analyses. The four best-resolved analyses distinguishedMeliceae as the earliest diverging lineage withinPooideae, andStipeae as the next. Above the point of divergence ofStipeae is a dichotomy between supertribeTriticodae (including tribesBrachypodieae, Bromeae, andTriticeae), and a clade comprisingPoeae andAveneae. The analysis supports some tribal realignments, specifically the assignment ofBriza, Chascolytrum, Microbriza, andTorreyochloa toAveneae, andArctagrostis, Catabrosa, andSesleria toPoeae. The analysis also suggests that the pooid spikelet (i.e., glumes shorter than lemmas and florets two or more) is plesiomorphic inPooideae, and that spikelets with one floret, and those with glumes longer than the first lemma, each have evolved more than once withinPooideae. Results also indicate that small chromosomes and chromosome numbers based on x=c. 10–12 are plesiomorphic withinPooideae. Alternative states of these characters (chromosomes large, chromosome numbers based on x=7) are interpreted as synapomorphies or parallelisms of clades that includeTriticodae, Aveneae, andPoeae. Lanceolate lodicule shape may be a synapomorphy of the clade that includesStipeae, Triticodae, Aveneae, andPoeae, and loss of lodicule vascularization a synapomorphy of the entirePooideae.     

Chloroplast DNA restriction site variation and phylogeny of the Berberidaceae

Comparative restriction site mapping of the chloroplast genome was performed to examine phylogenetic relationships among 27 species representing 16 genera of the Berberidaceae and two outgroups. Chloroplast genomes of the species included in this study showed no major structural rearrangements (i.e., they are collinear to tobacco cpDNA) except for the extension of the inverted repeat in species of Berberis and Mahonia. Excluding several regions that exhibited severe length variation, a total of 501 phylogenetically informative sites was mapped for ten restriction enzymes. The strict consensus tree of 14 equally parsimonious trees indicated that some berberidaceous genera (Berberis, Mahonia, Diphylleia) are not monophyletic. To explore phylogenetic utility of different parsimony methods phylogenetic trees were generated using Wagner, Dollo, and weighted parsimony for a reduced data set that included 18 species. One of the most significant results was the recognition of the four chromosomal groups, which were strongly supported regardless of the parsimony method used. The most notable difference among the trees produced by the three parsimony methods was the relationships among the four chromosomal groups. The cpDNA trees also strongly supported a close relationship of several generic pairs (e.g., Berberis-Mahonia, Epimedium-Vancouveria, etc.). Maximum likelihood values were computed for the four different tree topologies of the chromosomal groups, two Wagner, one Dollo, and one weighted topology. The results indicate that the weighted tree has the highest likelihood value. The lowest likelihood value was obtained for the Dollo tree, which had the highest bootstrap and decay values. Separate analyses using only the Inverted Repeat (IR) region resulted in a tree that is identical to the weighted tree. Poor resolution and/or support for the relationships among the four chromosomal lineages of the Berberidaceae indicate that they may have radiated from an ancestral stock in a relatively short evolutionary time.     

Mitochondrial DNA polymorphism in native Philippine cattle based on restriction endonuclease cleavage patterns

An analysis of patterns of cleavage of mtDNA by restriction endonucleases was performed for nine individuals from the Philippine population of native cattle. MtDNA polymorphisms were detected in the restriction patterns generated by the following six enzymes,BamHI,BglII,EcoRV,HindIII,PstI, andScaI. The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine population of native cattle, indicating the existence of two separate types of mtDNA. These two types of mtDNA are very different from each other, at the level of subspecies. Since the native Philippine cattle are considered to represent an admixture of European and Indian cattle, the two types of mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and the nucleotide substitutions at some of the sites have also been estimated.     

Major lineages within Apiaceae subfamily Apioideae: a comparison of chloroplast restriction site and DNA sequence data

Traditional sources of taxonomic characters in the large and taxonomically complex subfamily Apioideae (Apiaceae) have been confounding and no classification system of the subfamily has been widely accepted. A restriction site analysis of the chloroplast genome from 78 representatives of Apioideae and related groups provided a data matrix of 990 variable characters (750 of which were potentially parsimony-informative). A comparison of these data to that of three recent DNA sequencing studies of Apioideae (based on ITS, rpoCl intron, and matK sequences) shows that the restriction site analysis provides 2.6–3.6 times more variable characters for a comparable group of taxa. Moreover, levels of divergence appear to be well suited to studies at the subfamilial and tribal levels of Apiaceae. Cladistic and phenetic analyses of the restriction site data yielded trees that are visually congruent to those derived from the other recent molecular studies. On the basis of these comparisons, six lineages and one paraphyletic grade are provisionally recognized as informal groups. These groups can serve as the starting point for future, more intensive studies of the subfamily.     

Scanning force microscopy of DNA translocation by the Type III restriction enzyme EcoP15I

Type III restriction enzymes are multifunctional heterooligomeric enzymes that cleave DNA at a fixed position downstream of a non-symmetric recognition site. For effective DNA cleavage these restriction enzymes need the presence of two unmethylated, inversely oriented recognition sites in the DNA molecule. DNA cleavage was proposed to result from ATP-dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. We used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two EcoP15I recognition sites in inverse orientation. In the presence of the cofactors ATP and Mg(2+), EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures. One of the origins of the protein-clipped DNA loops was shown to be located at an EcoP15I recognition site, the other origin had an unspecific position in between the two EcoP15I recognition sites. The data demonstrate for the first time DNA translocation by the Type III restriction enzyme EcoP15I using scanning force microscopy. Moreover, our study revealed differences in the DNA-translocation processes mediated by Type I and Type III restriction enzymes.     

Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels

We have studied with a number of bacterial restriction enzymes the conditions for digestion of DNA in agarose and polyacrylamide gels. The restriction endonucleases HpaII, MspI, HaeIII, HindIII, TaqI, HhaI, AluI, BamHI, EcoRI and SalI are capable of digesting DNA in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI, are also capable of digesting DNA in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible.     

极端嗜热古菌DNA修复核酸内切酶的研究进展

极端嗜热古菌由于生活在高温环境,其基因组DNA面临着严重的挑战,因此,它们如何维持其基因组稳定是本研究领域最为关注的科学问题之一。极端嗜热古菌具有与常温微生物相似的自发突变频率,暗示着它们比常温微生物具有更加有效的DNA修复体系进行修复高温所造成的基因组DNA损伤。目前,极端嗜热古菌DNA修复的分子机制尚不清楚。核酸内切酶在DNA修复途径中发挥着重要的作用。基因组序列显示极端嗜热古菌编码多种DNA修复核酸内切酶,但是其研究尚处于初期阶段。本文综述了极端嗜热古菌DNA修复核酸内切酶Nuc S、Endo V、Endo Q、XPF和Hjc的研究进展,并对今后的研究提出了展望。     

Pig mitochondrial DNA: Polymorphism,restriction map orientation,and sequence data

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) in pigs were analyzed using 18 enzymes which recognize six nucleotides and 1 four-nucleotide-recognizing enzyme. Pigs including Taiwan native breeds and miniature strains maintained in Japan were examined in this study; four commercial breeds of pigs and Japanese wild boars have been investigated earlier [Watanabe, T., et al. (1985). Biochem. Genet. 23:105]. mtDNA polymorphisms were observed in the cleavage patterns of five restriction enzymes, Bg1II, EcoRV, ScaI, StuI, and TaqI. The results support the previous hypothesis that pigs must be derived from two different maternal origins, European and Asian wild boars, and that a breed, Large White, arises from both European and Asian pigs. Two HindIII cleavage fragments were cloned into the HindIII site of M13mp10 and were partially sequenced by the dideoxynucleotide-chain termination method. Furthermore, DraI and StuI cleavage sites were newly determined on the restriction endonuclease map. On the basis of these results, the restriction endonuclease cleavage map of pig mtDNA was rewritten. Comparing sequence data of pig mtDNA at 237 positions with those of cow, human, mouse, and rat mtDNA, the sequence difference, silent and replacement changes, and transitions and transversions among mammalian species were estimated. The relationships among them are discussed.     

Targeting DNA 5mCpG sites with chimeric endonucleases

Cytosine modification of the dinucleotide CpG in the DNA regulatory region is an important epigenetic marker during early embryo development, cellular differentiation, and cancer progression. In clinical settings, such as anti-cancer drug treatment, it is desirable to develop research tools to characterize DNA sequences affected by epigenetic perturbations. Here, we describe the construction and characterization of two fusion endonucleases consisting of the ⁵mCpG-binding domain of human MeCP2 (hMeCP2) and the cleavage domains of BmrI and FokI restriction endonucleases (REases). The chimeric (CH) endonucleases cleave M.HpaII (C⁵mCGG)—and M.SssI (⁵mCpG)-modified DNA. Unmodified DNA and M.MspI-modified DNA (⁵mCCGG) are poor substrates for the CH-endonucleases. Sequencing cleavage products of modified λ DNA indicates that cleavage takes place outside the ⁵mCpG recognition sequence, predominantly 4-17 bp upstream of the modified base (/N_4-17⁵mCpG, where / indicates the cleavage site). Such ⁵mCpG-specific endonucleases will be useful to study CpG island modification of the regulatory regions of tumor suppressor genes, and for the construction of cell-specific and tumor-specific modified CpG island databases.