Rational engineering of sequence specificity in R.MwoI restriction endonuclease

R.MwoI is a Type II restriction endonucleases enzyme (REase), which specifically recognizes a palindromic interrupted DNA sequence 5'-GCNNNNNNNGC-3' (where N indicates any nucleotide), and hydrolyzes the phosphodiester bond in the DNA between the 7th and 8th base in both strands. R.MwoI exhibits remote sequence similarity to R.BglI, a REase with known structure, which recognizes an interrupted palindromic target 5'-GCCNNNNNGGC-3'. A homology model of R.MwoI in complex with DNA was constructed and used to predict functionally important amino acid residues that were subsequently targeted by mutagenesis. The model, together with the supporting experimental data, revealed regions important for recognition of the common bases in DNA sequences recognized by R.BglI and R.MwoI. Based on the bioinformatics analysis, we designed substitutions of the S310 residue in R.MwoI to arginine or glutamic acid, which led to enzyme variants with altered sequence selectivity compared with the wild-type enzyme. The S310R variant of R.MwoI preferred the 5'-GCCNNNNNGGC-3' sequence as a target, similarly to R.BglI, whereas the S310E variant preferentially cleaved a subset of the MwoI sites, depending on the identity of the 3rd and 9th nucleotide residues. Our results represent a case study of a REase sequence specificity alteration by a single amino acid substitution, based on a theoretical model in the absence of a crystal structure.     

Cloning and expression of the MspI restriction and modification genes

The genes for the MspI restriction (R) and modification enzymes (recognition sequence CCGG) have been cloned into Escherichia coli using the vector pBR322. Clones carrying both genes have been isolated from libraries prepared with EcoRI, HindIII and BamHI. The smallest fragment that encodes both activities is a 3.6-kb HindIII fragment. Plasmids purified from the clones are fully resistant to digestion by MspI, indicating that the modification gene is functional in E. coli. The clones remain sensitive to phage infection, however, indicating that the endonuclease is dysfunctional. When the R gene is brought under the control of the inducible leftward promoter from phage lambda, the level of endonuclease increases and the level of methylase decreases, suggesting that the genes are transcribed in opposite directions.     

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

The gene coding for the sequence-specific modification methylase methM . BspI of Bacillus sphaericus R has been cloned in Escherichia coli by means of plasmid pBR322. The selection was based on the expression of the cloned gene which rendered the recombinant plasmid resistant to BspI restriction endonuclease cleavage. The gene is carried by a 9 kb BamHI fragment and by a smaller 2.5 kb EcoRI fragment derived from the BamHI fragment. The Bsp-specific methylase level was found to be higher in the recombinant clones than in the parental strain. The methylase gene is probably located on the Bacillus sphaericus chromosome, and not on a plasmid known to be carried by this strain. The recombinant clones do not exhibit an BspI restriction endonuclease activity.     

Restriction and modification in Bacillus subtilis: inducibility of a DNA methylating activity in nonmodifying cells.

The nonrestricting/nonmodifying strain Bacillus subtilis 222 (r-m-) can be induced to synthesize a DNA-modifying activity upon treatment with either mitomycin C (MC) or UV light. This is shown by the following facts. (i) Infection of MC-pretreated 222 cells with unmodified SPP1 phage yields about 3% modified phage that are resistant to restriction in B. subtilis R (r+m+). The induced modifying activity causes the production of a small fraction of fully modified phage in a minority class of MC-treated host cells. (ii) The MC-pretreated host cells contain a DNA cytosine methylating activity: both bacterial and phage DNAs have elevated levels of 5-methylcytosine. (iii) The MC-induced methylation of SPP1 DNA takes place at the recognition nucleotide sequences of restriction endonuclease R from B. subtilis R. (iv) Crude extracts of MC-pretreated 222 cells have enhanced DNA methyltransferase activities, with a substrate specificity similar to that found in modification enzymes present in (constitutively) modifying strains.     

A genetic dissection of the LlaJI restriction cassette reveals insights on a novel bacteriophage resistance system

Background  

Restriction/modification systems provide the dual function of protecting host DNA against restriction by methylation of appropriate bases within their recognition sequences, and restriction of foreign invading un-methylated DNA, such as promiscuous plasmids or infecting bacteriphage. The plasmid-encoded LlaJI restriction/modification system from Lactococcus lactis recognizes an asymmetric, complementary DNA sequence, consisting of 5'GACGC'3 in one strand and 5'GCGTC'3 in the other and provides a prodigious barrier to bacteriophage infection. LlaJI is comprised of four similarly oriented genes, encoding two 5mC-MTases (M1.LlaJI and M2.LlaJI) and two subunits responsible for restriction activity (R1.LlaJI and R2.LlaJI). Here we employ a detailed genetic analysis of the LlaJI restriction determinants in an attempt to characterize mechanistic features of this unusual hetero-oligomeric endonuclease.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

Molecular genetics of rotifers: preliminary restriction napping of the mitochondrial genome of Brachionus plicatilis

Methods are presented to extract and purify mitochondrial DNA from the rotifer Brachionus plicatilis. The mtDNA obtained is of sufficient purity for digestion with restriction endonucleases. EcoR I restriction patterns are presented for 4 geographically separated clones. A restriction map based on digestion with 5 different restriction enzymes is included for one of these clones. Finally, use of mtDNA analysis for studies on the population structure and biogeography of rotifers is discussed.     

Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

In this study, we used monoclonal antibodies to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cell free IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [3H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL 2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To our knowledge, this is the first demonstration of release or secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen.     

小麦6B染色体微切割及其不同片段的DNA文库构建

用Nd:YAG激光微束将处于丝分裂中期的小麦(Triticum aestivum L.)6B染色体微切割为四段,并用微细玻璃针将每个片段分别回收.将分离的染色体片段DNA用Sau3A接头介导的多聚酶链式反应(LA-PCR)分别扩增.Southem杂交证明4个特定区域的DNA确实来自于小麦基因组.用一系列(42对引物)位于6B染色体上的微卫星序列对微切割的染色体片段的PCR产物进行了验证.结果表明,获得的染色体片段的PCR产物来自于小麦6B染色体.将6B染色体4个片段的第二轮PCR产物克隆到pGET-vector中,建立了4个染色体特定区域的基因组文库,命名为R1、R2、R3和R4,分别包含2.1×105、2.74×105和2.93×105个重组子克隆.每个文库均随机挑选150个克隆进行质粒的小量制备和酶切验证.结果显示;插入片段大小在300～1800之间,平均大小为820～870bp,其中43%～48%的克隆为低/单拷贝序列,42%～47%为中/高拷贝序列.本研究为详细分析植物单染色体的不同片段的分子遗传学研究提供了基础.     

A novel class of DNA analogs bearing 5'-C-phosphonothymidine units: synthesis and physicochemical and biochemical properties

S(C) and R(C) diastereomers of 5'-C-(O,O-diethyl)-phosphonylthymidine ((R)T and (S)T) were used for the synthesis of the dimers T(R)T and T(S)T, respectively. These dimers were incorporated at selected sites in oligonucleotide constructs. Melting temperature (Tm) experiments demonstrated that relative to the unmodified oligodeoxyribonucleotide, the presence of the (R)T moiety reduced the thermal stability of the duplexes by approximately 5.0 degrees C per modification, whereas their (S)T counterparts only slightly destabilized the duplex structure (deltaTm < or = 1 degree C/modification). The stability of the triple-helical complexes containing one, two, or three modified thymidines is slightly higher than that of the parent complex. Nuclease resistance studies performed with snake venom phosphodiesterase, calf spleen phosphodiesterase, and 3'-exonuclease from human plasma showed that cleavage of the oligonucleotides at the site of the modification was completely suppressed regardless of the stereochemistry of the 5'-C-chiral center. The influence of the (R)T and (S)T modification in the recognition sequence of HindIII, EcoRI, and HpaI restriction endonucleases was also investigated. Although the catalytic activity of HindIII was not affected by the presence of the 5'-C-ethoxyphosphonyl modification, the activities of the two remaining restriction enzymes were partially suppressed depending on the site of modification or the stereochemistry of the modification or both ((R)T vs. (S)T).     

The pigeon cytochrome c-specific T cell response of low responder mice. I. Identification of antigenic determinants on fragment 1 to 65

An examination of the proliferative response to pigeon cytochrome c fragments 1 to 65 and 1 to 80 by T cells from mice that are low responders to the native molecule revealed that some of the strains could respond to antigenic determinants on these fragments. T cell clones derived from B10.A(3R) and B10.A(4R) mice were used to characterize the antigenic determinants on fragment 1 to 65. All of the clones recognized syngeneic A beta:A alpha Ia molecules as their restriction element. Three B10.A(3R) clones and six B10.A(4R) clones recognized fragment 39 to 65. Another four B10.A(4R) clones responded to fragment 1 to 38. By stimulating with a series of cytochrome c fragments from different species, as well as a synthetic peptide, it was possible to localize the antigenic determinant(s) recognized by the B10.A(3R) clones to residues 45 to 58. Each clone showed a unique pattern of responsiveness to the various fragments, suggesting a diversity of T cell receptors specific for the same peptide. One B10.A(3R) clone could be stimulated by many of the 1 to 65 fragments in association with allogeneic B10.SM presenting cells and by tuna fragment 1 to 65 in association with B10.M presenting cells, although the rank order of potency for several of the fragments was different than that observed with syngeneic antigen-presenting cells. In addition, the clone was poorly reactive to a synthetic peptide containing a conservative substitution, serine for threonine, at position 49. The implications of these results for subsite dissection (agretope and epitope) of the antigenic determinant recognized by this clone are discussed.     

Characterization of BseMII, a new type IV restriction-modification system, which recognizes the pentanucleotide sequence 5'-CTCAG(N)(10/8)/

We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5'-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3'-protruding end. Magnesium ions and S:-adenosyl-L-methionine (AdoMet) are required for cleavage. S:-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5'-CTCAG-3'/5'-CTGAG-3'. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5'-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-L-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.     

Molecular cloning of unintegrated visna viral DNA and characterization of frequent deletions in the 3' terminus

Visna viral DNA, like other retroviral DNA, exists in two circular forms in infected cells. The larger probably contains two copies of the LTR, the smaller, one copy. Recombinant DNA techniques were used to clone unintegrated circular visna viral DNA in the lambda WES . lambda B vector. Circular visna viral DNA was digested with the restriction enzyme SstI, which yields a 9.2-kb viral DNA fragment containing 90% of the viral genome colinear with the restriction map of linear viral DNA. This fragment extends from a site about 900 bp from the left (5') end of the viral DNA molecule, through the 3' region, including U3 and R sequences at its right (3') end. The recombinant clones isolated contain visna viral DNA inserts which range in size from 3.1 kb to 9.2 kb. All the clones contain the 5' region intact, but most had sustained deletions of varying lengths in the 3' terminal region of the cloned fragment.     

Caloric restriction augments radiation efficacy in breast cancer

Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.     

人R_O蛋白(60kD)编码序列的PCR产物在E.coli中的克隆与表达

通过聚合酶链反应 ( PCR)自人脾 c DNA扩增 RO 蛋白 ( 60 k D)编码 DNA片段 .将该片段定向插入麦芽糖结合蛋白 ( MBP)融合系统的 p MALTM- c载体中并转化 E.coli ( DH5α) ,通过酶联免疫印迹 ( IBT)筛选出具有 RO 抗原性的阳性克隆 .绝大部分阳性克隆表达完整的 RO 融合蛋白 ( 1 0 0k D) .但在传代过程中表达水平很快降低 ;少数阳性克隆虽然表达非完整 RO 融合蛋白 (分子量 <80k D) ,但表达水平却高而且稳定 .同时保留了很强的 RO 抗原性 .产生非完整融合蛋白的一个原因是 ,PCR碱基错配引起 RO 编码 DNA的序列缺失 ;另一原因是融合蛋白在表达过程中被降解     

Nucleotide sequence of a protamine component CII gene of Salmo gairdnerii.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".     

Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones.

The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.     

Connexin46 mutations in autosomal dominant congenital cataract

Loci for autosomal dominant "zonular pulverulent" cataract have been mapped to chromosomes 1q (CZP1) and 13q (CZP3). Here we report genetic refinement of the CZP3 locus and identify underlying mutations in the gene for gap-junction protein alpha-3 (GJA3), or connexin46 (Cx46). Linkage analysis gave a significantly positive two-point LOD score (Z) at marker D13S175 (maximum Z [Zmax]=>7.0; maximum recombination frequency [thetamax] =0). Haplotyping indicated that CZP3 probably lies in the genetic interval D13S1236-D13S175-D13S1316-cen-13pter, close to GJA3. Sequencing of a genomic clone isolated from the CZP3 candidate region identified an open reading frame coding for a protein of 435 amino acids (47,435 D) that shared approximately 88% homology with rat Cx46. Mutation analysis of GJA3 in two families with CZP3 detected distinct sequence changes that were not present in a panel of 105 normal, unrelated individuals. In family B, an A-->G transition resulted in an asparagine-to-serine substitution at codon 63 (N63S) and introduced a novel MwoI restriction site. In family E, insertion of a C at nucleotide 1137 (1137insC) introduced a novel BstXI site, causing a frameshift at codon 380. Restriction analysis confirmed that the novel MwoI and BstXI sites cosegregated with the disease in families B and E, respectively. This study identifies GJA3 as the sixth member of the connexin gene family to be implicated in human disease, and it highlights the physiological importance of gap-junction communication in the development of a transparent eye lens.