Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands,C-bands,and in situ hybridization

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase chromosomes with three restriction endonucleases (EcoRI, MboI and PstI). Banding patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.     

Expression of a thermostable restriction endonuclease in recombinant Escherichia coli cells and optimization of fermentation conditions

Taq I restriction endonuclease gene of the thermophilic eubacterium Thermus aquaticus YT-1 (ATCC 25104) was successfully cloned and expressed in recombinant Escherichia coli cells under the control of the lac promoter/operator system. Higher Taq I endonuclease specific activities and biomass yields were obtained from E. coli ER2508(pUCTaq) cells when they were induced at the late-exponential phase of their growth. Taq I endonuclease expression was found to be host strain-dependent such that, among the three different strains examined, E. coli XL1(pUCTaq) produced the highest specific Taq I endonuclease activities for longer induction periods. Decreasing the inducer concentration from 1 to 0.1 mM not only improved the specific enzyme activity yields but also is more economical, considering the high cost of isopropyl--D-thiogalactopyranoside (IPTG). The optimum culture temperature was found to be 37 °C. Taq I endonuclease specific activity recovered from E. coli XL1(pUCTaq) cells was 935 U/mg under optimum conditions.     

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

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

Characterisation of the novel restriction endonuclease <Emphasis Type="Italic">Sui</Emphasis>I from <Emphasis Type="Italic">Sulfolobus islandicus</Emphasis>

A restriction endonuclease activity from Sulfolobus islandicus REN2H1 was purified by phosphocellulose and cation exchange chromatography. The enzyme cuts DNA at the recognition site GCwGC as could be shown by restriction analysis of plasmids and short synthetic duplex DNA. The cleavage occurs after the first guanosine base and is inhibited by 5-methyl-cytosine methylation. The restriction activity is salt-sensitive and has an optimal activity around 70°C.     

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.     

Isolation and some properties of the site-specific endonuclease and methylase Bme2161 from Bacillus megaterium 216

The site-specific endonuclease Bme2161 was isolated as a homogeneous preparation by chromatography on phosphocellulose, hydroxyapatite and heparin-agarose. The molecular mass of the enzyme, determined by gel filtration and by electrophoresis under denaturing conditions, was found to be 60 kDa and 30 kDa respectively. These data indicate that the native enzyme consists of two identical subunits. The enzyme recognized the decreases pentanucleotide sequence 5'-GGACC-3' X 3'-CCTGG-5' and cleaves the sequence as indicated by arrows. The increases optimal concentration for endonuclease reaction is 6-7 mM Mg2+. The endonuclease relaxes its specificity in the presence of glycerol or dimethyl sulfoxide at low Mg2+ concentration (1-3 mM). Methylase Bme2161, which protects DNA against endonuclease Bme2161 action by DNA methylation, was isolated from the same bacterial strain.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of λ DNA have been compared. A common ^5′GAGACC_3′CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5 ′ G G T C T C N ↓ 3 ′ C C A G A G N N N N N ↑     

The mitochondrial genome of an exsymbiotic Chlorella-like green alga

Mitochondrial (mt) DNA from the unicellular, exsymbiotic Chlorella-like green alga, strain Nla was isolated and cloned. The mtDNA has a buoyant density of 1.692 g/ml in CsCl and an apparent G/C base composition of 32.5%. The genome contains approximately 76 kbp of DNA based on restriction fragment summation and electron microscopic measurements. A map of restriction endonuclease sites using Sst I, Bam I, Sal I and Xho I was generated. The genome maps as a circular molecule and appears as such under the electron microscope. Eight genes were assigned to the map by hybridization to specific restriction fragments using heterologous mt-encoded specific probes. These include the genes for subunits 6, 9, and alpha of the F₀-F₁ ATPase complex, the large and small subunit rRNAs, cytochrome oxidase subunits I and II, and apocytochrome b.     

Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice

Summary Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.Florida Agricultural Experiment Station Journal Series No. 8807. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable     

Comparison of Genomes in Streptococcus thermophilus Strains of Different Origin

According to DNA hybridization data, thermophilic streptococci used in Russia as starters in the dairy industry are divided into six different genomovars, with a degree of DNA homology not exceeding 20–50%. The analysis of genomes from these genomovars using SmaI restriction endonuclease and pulsed-field gel electrophoresis revealed wide variability of the genome size. In some strains, the genome size considerably exceeded 2000 kbp. Most of the strains studied contained plasmids about 120 kbp in size.     

Restriction fragment patterns of chloroplast and mitochondrial DNA of Dasypyvum villosum (L.) candargy and wheats

To elucidate the phylogenetic relationships and cytoplasmic types, restriction endonuclease fragment patterns of chloroplast (cp) and mitochondrial (mt) DNAs isolated from two different accessions of Dasypyrum villosum (L.) candargy were compared with those of tetraploid wheat (Triticum durum Desf., PI265007), hexaploid wheat (Triticum aestivum L., cv Chinese Spring), Aegilops longissimum (S. and M., in Muschli) Bowden and Hordeum vulgare L. T. aestivum and T. durum had identical restriction patterns for their cp and mtDNAs in digestions with four different enzymes. Likewise, no differences were found between the restriction fragment patterns of two accessions of D. villosum. But, there were distinct differences in chloroplast and mitochondrial DNA restriction fragment patterns between D. villosum and tetraploid and hexaploid wheats. A. longissimum (G609) showed a similar pattern to those wheats for PstI digestion of cpDNA. Organellar DNA from Hordeum vulgare (cv Himalaya) showed a distinctly different restriction pattern from those of wheat and D. villosum. These results suggest that D. villosum is unlikely to be the donor of cytoplasm to wheats, and its cytoplasmic organelles were also different from those of A. longissimum.Contribution No. 92-522-J from the Kansas Agricultural Experiment Station; Kansas State University, Manhattan, Kansas, USA     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV).

The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.     

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR–RFLP and nested-PCR of ribosomal DNA ITS1 region

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1–PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1–PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.     

Restriction endonuclease Sst 12I from a strain of Streptomyces sp. recognizing the nucleotide sequence 5'-CTGCAG-3'

A new restriction endonuclease Sst12I belonging to the II type and recognizing the sequence 5'-CTGCAG-3' was isolated from the bacterial strain Streptomyces sp. St-12. The enzyme hydrolyzes DNA between adenine and guanine residues; thus, it is a true isoschizomer of restrictase PstI. In contrast to PstI, the restriction endonuclease Sst12I hydrolyses DNA both at 37 degrees and 55 degrees C and remains active after long-term storage.     

Nucleotide polymorphism in chloroplast dna of Nicotiana debneyi

Summary EcoR1 restriction endonuclease analysis of chloroplast DNA isolated from several distinct populations of Nicotiana debneyi has revealed a naturally occurring polymorphism. The chloroplast DNA of seven of the nine populations analysed possessed an additional EcoRl site. The origin of the additional restriction endonuclease fragments was confirmed by hybridisation of [³² P]-cRNA to fractionated EcoRl restricted chloroplast-DNA fragments adsorbed to nitrocellulose filters. Reciprocal f₁ hybrids between plants carrying the variant chloroplast-DNA's confirmed maternal inheritance of chloroplast-DNA.Communicated by G. Melchers and D. von Wettstein     

Cloning and sequencing the HinfI restriction and modification genes

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).     

Enhanced expression of a distinct plastid DNA region in mustard seedlings by continuous far-red light

Fragments of chloroplast DNA from mustard (Sinapis alba L.) seedlings, generated by the restriction endonuclease Eco RI, were used to assess the frequency of complementary sequences in mustard RNA by DNA/RNA hybridization. A pronounced increase in hybridization to a single DNA fragment was found with RNA from seedlings irradiated with continuous far-red light, compared to RNA from dark-grown seedlings.     

Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome

A physical map of the unicellular cyanobacterium Synechococcus sp. strain PCC6301 genome has been constructed with restriction endonucleases PmeI, SwaI, and an intron-encoded endonuclease I-CeuI. The estimated size of the genome is 2.7 Mb. On the genome 49 genes or operons have been mapped. Two rRNA operons are separated by 600 kb and transcribed oppositely.     

Restriction Endonuclease Sst12I from a Strain of Streptomyces Recognizing the Nucleotide Sequence 5"-CTGCAG-3"

A new restriction endonuclease Sst12I belonging to type II and recognizing the sequence 5"-CTGCAG-3" was isolated from the bacterial strain Streptomycessp. St-12. The enzyme hydrolyzes DNA between adenine and guanine residues; thus, it is a true isoschizomer of restrictase PstI. In contrast to PstI, the restriction endonuclease Sst12I hydrolyses DNA both at 37 and 55°C and remains active after long-term storage.