Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species, with Pleurotus spp. as an Example

A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.     

Restriction endonuclease banding of rainbow trout chromosomes

Rainbow trout chromosomes were treated with nine restriction endonucleases, stained with Giemsa, and examined for banding patterns. The enzymes AluI, MboI, HaeIII, HinfI (recognizing four base sequences), and PvuII (recognizing a six base sequence) revealed banding patterns similar to the C-bands produced by treatment with barium hydroxide. The PvuII recognition sequence contains an internal sequence of 4 bp identical to the recognition sequence of AluI. Both enzymes produced centromeric and telomeric banding patterns but the interstitial regions stained less intensely after AluI treatment. After digestion with AluI, silver grains were distributed on chromosomes labeled with [³H]thymidine in a pattern like that seen after AluI-digested chromosomes are stained with Giemsa. Similarly, acridine orange (a dye specific for DNA) stained chromosomes digested with AluI or PvuII in patterns resembling those produced with Giemsa stain. These results support the theory that restriction endonucleases produce bands by cutting the DNA at specific base pairs and the subsequent removal of the fragments results in diminished staining by Giemsa. This technique is simple, reproducible, and in rainbow trout produces a more distinct pattern than that obtained with conventional C-banding methods.     

Restriction fragment fingerprint and genome sizes of Staphylococcus species using pulsed-field gel electrophoresis and infrequent cleaving enzymes.

Large restriction fragments of genomic DNA from Staphylococcus species were separated by pulsed-field gel electrophoresis (PFGE). Five different strains of S. aureus (ISP8, SAU3A, PS96, ATCC 6538, ATCC 15564) and three representative strains of S. haemolyticus SM102, S. warneri MCS4, S. cohnii LK478 from human hosts, and one strain of S. aureus (ATCC 8432) from an avian host were used in this study. Since Staphylococcus is A + T rich (approximately 67%), restriction fragments were obtained by digesting chromosomal DNA with endonucleases that recognize GC-rich sequences. Five enzymes Csp I, Sma I, Ecl XI, Ksp I, or Sac II were used for generation of few (7 to 16) distinctly separated fragments, with average sizes in the range of 200-300 kb. The size distribution of restriction fragments for each enzyme for each strain produced a strain-identifying fingerprint, and the genome size of each strain was determined from such restriction fragments separated by PFGE.     

The majority of minicircle DNA in Crithidia fasciculata strain CF-C1 is of a single class with nearly homogeneous DNA sequence

DNA minicircles found within the kinetoplast of the trypanosomatid Crithidia fasciculata, like those of most other kinetoplastid species, are heterogeneous in sequence. The pattern of minicircle DNA fragments generated by cleavage of kinetoplast DNA with various restriction enzymes has been used to demonstrate this heterogeneity. Here we describe a strain of Crithidia fasciculata in which more than 90% of the DNA minicircles exhibit a common pattern of restriction enzyme cleavage sites. A map of cleavage sites within this major minicircle DNA class is presented for seven restriction enzymes with hexanucleotide recognition sequences. Sequence homogeneity at an even finer level is reflected in minicircle DNA digestion patterns generated by restriction enzymes with tetranucleotide recognition sites. Partial DNA sequence analysis of multiple clones from the major minicircle class shows nearly complete homogeneity at the nucleotide level. The existence of a near homogeneous complement of DNA minicircles in Crithidia should facilitate the study of their replication in this organism.     

Restriction endonucleases and their applications

Restriction endonucleases are deoxyribonucleases which cleave double-stranded DNA into fragments. With only one exception, all restriction endonucleases recognize short, non-methylated DNA sequences. Restriction endonucleases can be divided into two groups based on the position of the cleavage site relative to the recognition sequence. Class I restriction endonucleases cleave double-stranded DNA at positions outside the recognition sequence and generate fragments of random size. The cleavage sites of Class II restriction endonucleases are located, in most cases, within the recognition sequence. Most of the Class II restriction endonucleases recognize 4, 5, or 6 base pair palindromes and generate fragments with either flush ends or staggered ends. DNA fragments with staggered ends contain 3, 4, or 5 nucleotide single-stranded tails called ‘sticky ends’. DNA fragments produced by Class II restriction endonuclease cleavage can be separated on gels according to their molecular weight. The fragments can be isolated from the gel and used for sequence analysis to elucidate genetic information stored in DNA. Further, an isolated fragment can be inserted into a small extrachromosomal DNA, e.g. plasmid, phage or viral DNA, and its replication and expression can be studied in clones of prokaryotic or eukaryotic cells. Restriction endonucleases and cloning technology are powerful modern tools for attacking genetic problems in medicine, agriculture and industrial microbiology.     

Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales

A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation.     

Restriction of bacteriophage lambda by Escherichia coli K

Derivatives of phage lambda, for which the numbers and positions of the recognition sites for endonuclease R. Eco_k are known, were used as substrates for the Escherichia coli K restriction system in vivo and in vitro. A single unmodified recognition site was sufficient for a DNA molecule to be bound and broken by the K restriction enzyme. Although discrete fragments of DNA were not produced, the breaks were made preferentially in the proximity of the recognition site. Breakage of a DNA molecule with only one recognition site required a 10 to 40-fold higher concentration of restriction enzyme than breakage of a DNA molecule with two or more recognition sites, but these substrates were all equally effective in a binding assay for the enzyme.The polynucleotide kinase reaction provided no evidence for new 5′-terminal sequences generated by restriction in vitro; the 5′ termini were either refractory to the polynucleotide kinase reaction or had no sequence specificity.     

Determination of genome size of Haemophilus influenzae Sb: analysis of DNA restriction fragments.

Genomic DNA size was measured in clinical isolates of Haemophilus influenzae by Pulsed-Field Gel Electrophoresis of DNA restriction fragments. Because of the high (64%) A+T content of H. influenzae DNA, restriction enzymes that recognize sequences with at least four GC base pairs were expected to be rare cutters. Five enzymes that produced fragments greater than 200 kb in size were used to digest intact chromosomes and fragments resolved by TAFE and/or FIGE: ApaI (GGGCCC), EagI (CGGCCG), NotI (GCGGCCGC), RsrI (CGGA/TCCG), and SmaI (CCCGGG). All five had recognition sequences with at least six GC base pairs. The genomic DNA size of H. influenzae serotype b, estimated with ApaI, EagI, NotI, RsrII, and SmaI, is 1,950 kb.     

应用CRISPR/Cas13b编辑技术治疗TNNT2R141W转基因小鼠的扩张型心肌病

本研究旨在应用CRISPR/Cas13b系统对TNNT2R141W转基因扩张型心肌病（dilated cardiomyopathy,DCM）小鼠（DCM小鼠）进行探索性治疗,尝试发现治疗扩张型心肌病的一种新方式,为CRISPR/Cas13b系统在体内应用提供实验基础。随机设计11种Cas13b-TNNT2 gRNA并成功构建表达质粒,把它和人源TNNT2过表达质粒共同转染到293T细胞中,通过实时定量PCR（quantitative real-time PCR,Q-PCR）检测人源TNNT2 mRNA的表达水平。结果显示,gRNA 2引导Cas13b敲低目标基因的效率最高,达到80%（P<0.0001）。把gRNA2表达质粒包装到慢病毒载体中转导出生后1天的DCM小鼠原代心肌细胞,Q-PCR检测结果表明CRISPR/Cas13b系统对人源TNNT2 mRNA的敲低效率达到55%（P<0.01）。把PspCas13b和gRNA2的表达载体分别包装到AAV9病毒载体中,然后将200 μL 约1×1012 AAV9病毒颗粒通过尾静脉注射到4月龄DCM小鼠体内,待注射小鼠发育至5月龄时,Q-PCR检测结果显示,AAV9+DCM组TNNT2R141W表达水平较未注射组对照明显下降至40%（P<0.01）。对5月龄野生型（WT）、DCM（未注射病毒组）和AAV9+DCM（基因组编辑工具注射组）三组小鼠的心脏形态、心功能、心肌纤维化和心力衰竭等表型的观察结合显示：DCM小鼠的心脏形态异常,而AAV9+DCM小鼠心脏形态趋于正常;对三组小鼠的心脏进行超声心动图并对心功能指标进行统计发现,DCM组较WT组小鼠的左心室射血分数（left ventricular percent ejection fraction,LV EF%）、左心室短轴缩短率（left ventricular percent fractional shortening,LV FS%）分别下降了50.4%（P<0.0001）,55.1%（P<0.0001）,而AAV9+DCM组较DCM组小鼠的LV EF%、LV FS%分别上升了66.5%（P<0.01）,77.0%（P<0.01）;通过Q-PCR和天狼星红染色检测三组小鼠的心脏纤维化程度,结果显示DCM组较WT组小鼠的Col3a1和Postn两种纤维化基因,分别高表达5.2倍（P<0.001）、4.5倍（P<0.01）,而AAV9+DCM组较DCM组小鼠两种基因表达分别下降了2.0倍（P<0.05）、1.4倍（NS）,天狼星红染色结果显示纤维化区域明显下降;通过Q-PCR和蛋白质免疫印迹分别检测三组小鼠的心脏心力衰竭基因Nppb mRNA和Nppa蛋白质的表达水平,结果表明DCM组较WT组小鼠Nppb mRNA表达上升14.2倍（P<0.01）,而AAV9+DCM组较DCM组小鼠Nppb mRNA表达明显下降下降2.8倍（P<0.05）,Nppa蛋白质表达趋势与Nppb相同。把gRNA 5和含有R141W突变（gRNA 5T）和正常的TNNT2 mRNA（gRNA 5V）序列分别组合转染到293T细胞中,通过Q-PCR检测两种序列mRNA的表达水平。结果显示,gRNA 5T序列表达效率为30%（P<0.0001）,而并未检测到gRNA 5V mRNA的敲低。本研究通过设计靶向TNNT2R141W mRNA的gRNA,特异性敲低TNNT2R141W转基因小鼠体内突变的mRNA,有效改善了转基因小鼠的心功能,为临床进一步探索扩张型心肌病的治疗奠定了实验室基础。     

Additional Restriction Endonuclease Cleavage Sites on the Bacteriophage P22 Genome

We present complete restriction endonuclease cleavage site maps of the bacteriophage P22 chromosome for 16 enzymes with six base recognition sequences, thereby positioning 116 new sites on the chromosome. Twenty-four such restriction maps for P22 DNA, containing 162 sites, have now been completed, and three enzymes were found that did not cut P22 DNA. Our results are consistent with the ideas that ClaI does not cleave the methylated recognition sequence ATCGA(me)T or A(me)TCGAT and StuI does not cleave the methylated recognition sequence AGGCC(me)T.     

RSITE: a computer program to predict the recognition sequence of a restriction enzyme.

A computer program (RSITE) was developed which predicts the recognition sequence of a restriction endonuclease. The sizes of fragments experimentally determined on cleavage of a DNA of known sequence were input. Possible recognition sequences producing fragments of sizes matching those determined empirically were printed out. The program faithfully predicted the specificity of restriction enzymes of known recognition sequence and also determined the recognition sequence of a new restriction enzyme from Haemophilus influenzae GU (HinGU II).     

Restriction enzyme recognition sequence search program

A critical and difficult part of characterizing restriction enzymes and methylases is the identification of recognition sequences. To simplify this process, we have developed a plasmid transformation method along with a computer program named RM search that determines the exact recognition sequences for given restriction and modification systems.     

Recognition sequences of type II restriction systems are constrained by the G + C content of host genomes

I show that the recognition sequences of Type II restriction systems are correlated with the G + C content of the host bacterial DNA. Almost all restriction systems with G + C rich tetranucleotide recognition sequences are found in species with A + T rich genomes, whereas G + C rich hexanucleotide and octanucleotide recognition sequences are found almost exclusively in species with G + C rich genomes. Most hexanucleotide recognition sequences found in species with A + T rich genomes are A + T rich. This distribution eliminates a substantial proportion of the potential variance in the frequency of restriction recognition sequences in the host genomes. As a consequence, almost all restriction recognition sequences, including those eight base pairs in length (Not I and Sfi I), are predicted to occur with a frequency ranging from once every 300 to once every 5,000 base pairs in the host genome. Since the G + C content of bacteriophage DNA and of the host genome are also correlated, the data presented is evidence that most Type II "restriction systems" are indeed involved in phage restriction.     

计算机在确定牛病毒性腹泻病毒基因组常用限制性内切酶位点中的应用

应用计算机测定71种限制性内切酶在牛病毒性腹泻病毒NADL株(BVDV NADL)基因组的cDNA核苷酸序列中酶切位点,结果表明其中S8种酶的酶切位点所在的核苷酸序列数,也表明了其中13种限制性内切酶在核苷酸序列中无酶切位点。这些结果有助于进一步研究BVDV InL株的cDNA基因库。     

Restriction analysis of spacers in ribosomal DNA of Drosophila melanogaster.

The sequence arrangement of ribosomal DNA (rDNA) spacers in Drosophila melanogaster was analyzed with restriction endonucleases. Spacers, derived from cloned rDNA repeats and from uncloned purified rDNA, are internally repetitive, as demonstrated by the regular 250 base pairs interval between sites recognized by the enzyme Alu I. Length heterogeneity of spacers is due at least in part to varying numbers of repeated sequence elements. All spacers and analyzed, whether derived from X or from Y chromosomal rDNA, have a very similar sequence organization. The distance separating the repeated nontranscribed spacer sequences from the 5' end of the transcribed region is conserved in all ten cloned fragments examined, and is probably less than 150 base pairs, as measured by electron microscopy.     

Restriction endonucleases that bridge and excise two recognition sites from DNA

Most restriction endonucleases bridge two target sites before cleaving DNA: examples include all of the translocating Type I and Type III systems, and many Type II nucleases acting at their sites. A subset of Type II enzymes, the IIB systems, recognise bipartite sequences, like Type I sites, but cut specified phosphodiester bonds near their sites, like Type IIS enzymes. However, they make two double-strand breaks, one either side of the site, to release the recognition sequence on a short DNA fragment; 34 bp long in the case of the archetype, BcgI. It has been suggested that BcgI needs to interact with two recognition sites to cleave DNA but whether this is a general requirement for Type IIB enzymes had yet to be established. Ten Type IIB nucleases were tested against DNA substrates with one or two copies of the requisite sequences. With one exception, they all bridged two sites before cutting the DNA, usually in concerted reactions at both sites. The sites were ideally positioned in cis rather than in trans and were bridged through 3-D space, like Type II enzymes, rather than along the 1-D contour of the DNA, as seen with Type I enzymes. The standard mode of action for the restriction enzymes that excise their recognition sites from DNA thus involves concurrent action at two DNA sites.     

Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity

The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature.     

Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei

We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array.Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA.Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.     

Mitochondrial and Nuclear DNA Restriction Enzyme Analysis of the Closely Related Phytophthora Species P. infestans, P. mirabilis, and P. phaseoli

To determine relatedness of the phytopathogenic fungi Phytophthora infestans , P. mirabilis , and P. phaseoli restriction fragment patterns of mitochondrial DNAs of several isolates and hybridization patterns of nuclear DNAs after Southern hybridization with a specific homologous probe were analyzed.
All but two isolates of P. infestans and P. mirabilis show very similar restriction fragment patterns differing only in the length of one fragment due to small insertion/deletion(s). Two isolates of P. mirabilis have one additional site for Scr FI. On the contrary at least six sites differ in P. phaseoli when compared to the other two species. The mitochondrial genome of P. phaseoli is considerably smaller (approx. 6 kbp) than those of P. infestans and P. mirabilis .
A cloned 430 bp multicopy DNA sequence, derived from P. infestans , hybridized specifically with P. infestans, P. mirabilis , and P. phaseoli out of 61 species of Peronosporales ( Phytophthora, Halophytophthora, Pythium, Albugo, Bremia, Peronospora, Plasmopara ) tested and therefore has potential as a diagnostic probe. Restriction patterns revealed by this probe are invariant intraspecifi-cally but differ between the three species.
We consider P. mirabilis a forma specialis of P. infestans because of the very high similarly in its mitochondrial DNA restriction patterns.     

Ethidium binding sites on plasmid DNA determined by photoaffinity labeling

Photoaffinity labeling of pBR322 with ethidium monoazide (8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride) was used to provide evidence for the sequence specificity of ethidium binding to native DNA. DNA-drug interactions were examined at concentrations of eight covalently bound ethidium drugs per molecule of pBR322 (4363 base pairs). Restriction enzyme cutting was blocked by the covalent binding of a drug molecule at (or near) the enzyme recognition sequence. This phenomenon was observed with all restriction enzymes tested and was not limited to specific regions of the pBR322 molecule. Double-digestion experiments indicated that a drug molecule may bind 2 to 3 base pairs outside the recognition sequence and still block restriction enzyme digestion. Intact plasmid was treated with [3H]ethidium monoazide and digested with restriction enzymes. The amount of covalently-linked ethidium analog was quantitated for different restriction fragments and the G-C content of each fragment was determined from the DNA sequence. In approximately half of the fragments the drug appeared to preferentially bind at a G-C base pair. However, no preference for specific sequences such as 5'-C-G-3' was detected, as had been suggested by previous modeling studies with ethidium bromide. The other fragments were located in specific map regions of the plasmid and did not bind drug with a strict dependence on GC content suggesting that binding specificity may depend on more than one structural feature of the DNA.