A set of Macintosh computer programs for the design and analysis of synthetic genes

Computer programs that can be used for the design of syntheticgenes and that are run on an Apple Macintosh computer are described.These programs determine nucleic acid sequences encoding aminoacid sequences. They select DNA sequences based on codon usageas specified by the user, and determine the placement of basechanges that can be used to create restriction enzyme siteswithout altering the amino acid sequence. A new algorithm forfinding restriction sites by translating the restriction endonucleasetarget sequence in all three reading frames and then searchingthe given peptide or protein amino acid sequence with theseshort restriction enzyme peptide sequences is described. Examplesare given for the creation of synthetic DNA sequences for thebovine prethrombin-2 and ribonuclease A genes Received on October 18, 1988; accepted on December 9, 1988     

用DREAM技术进行全长质粒快速定点突变

利用“设计限制酶辅助突变”(Designed Restriction Enzyme Assisted Mutagenesis, DREAM)进行全长质粒快速定点突变。根据突变位点附近氨基酸靶序列, 以简并密码子进行逆向推导, 这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体(Silent mutants), 这些突变体中包含大量的限制性酶切位点, 选择合适的酶切位点设计引物, 用Phusion超保真DNA聚合酶扩增全长质粒的DNA序列, 得到的PCR产物用T4多聚核苷酸激酶添加5￠磷酸基团后进行平末端连接, 转化大肠杆菌受体菌后用设计的酶切位点进行快速筛选。本研究用该方法成功地纠正了长约8 kb的质粒pcDNA3.1-pIgR中的突变碱基, 从而获得了多聚免疫球蛋白受体(pIgR)的野生型氨基酸序列。以上结果表明: 利用DREAM技术将限制性酶切位点引入目的基因而不改变目的蛋白质的氨基酸序列, 使突变体的筛选简单化; 配合使用高保真和高效率的Phusion DNA聚合酶可以进行长达8 kb的全长质粒的快速突变; 该方法无需使用定点突变试剂盒和特殊的受体菌, 同时避免了核酸杂交以及同位素的使用。     

The design of synthetic genes

Computer programs are described that aid in the design of synthetic genes coding for proteins that are targets of a research program in site directed mutagenesis. These programs "reverse-translate" protein sequences into general nucleic acid sequences (those where codons have not yet been selected), map restriction sites into general DNA sequences, identify points in the synthetic gene where unique restriction sites can be introduced, and assist in the design of genes coding for hybrids and evolutionary intermediates between homologous proteins. Application of these programs therefore facilitates the use of modular mutagenesis to create variants of proteins, and the implementation of evolutionary guidance as a strategy for selecting mutants.     

A prediction of the amino acids and structures involved in DNA recognition by type I DNA restriction and modification enzymes.

The S subunits of type I DNA restriction/modification enzymes are responsible for recognising the DNA target sequence for the enzyme. They contain two domains of approximately 150 amino acids, each of which is responsible for recognising one half of the bipartite asymmetric target. In the absence of any known tertiary structure for type I enzymes or recognisable DNA recognition motifs in the highly variable amino acid sequences of the S subunits, it has previously not been possible to predict which amino acids are responsible for sequence recognition. Using a combination of sequence alignment and secondary structure prediction methods to analyse the sequences of S subunits, we predict that all of the 51 known target recognition domains (TRDs) have the same tertiary structure. Furthermore, this structure is similar to the structure of the TRD of the C5-cytosine methyltransferase, Hha I, which recognises its DNA target via interactions with two short polypeptide loops and a beta strand. Our results predict the location of these sequence recognition structures within the TRDs of all type I S subunits.     

A Macintosh computer program for designing DNA sequences that code for specific peptides and proteins

A computer program (PINCERS) is described for use in the design of synthetic genes and mixed-probe DNA sequences. A protein sequence is reverse translated with generation of synonymous codons at each position producing a degenerate sequence. In order to locate potential restriction enzyme sites, the degenerate sequence is searched with a library of restriction enzymes for sites that utilize any combination of synonymous codons. These sites are indicated in a map so that they may be incorporated into the synthetic gene sequence. The program allows the user to select the appropriate codon usage table for the organism of interest and then to set a threshold usage frequency below which codons are not generated. PINCERS may also be used to assist in planning the synthesis of mixed-probe DNA sequences for cross-hybridization experiments. It can identify regions of specified length with the protein sequence that have the least overall degeneracy, thereby minimizing the number of probes to be synthesized and, therefore, maximizing the concentration of a given probe sequence.     

Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites

A method is described for the efficient insertion of mutagenic oligodeoxynucleotide cassettes which allow saturation of a target amino acid codon with multiple mutations. Restriction sites are introduced by oligonucleotide-directed mutagenesis procedures to flank closely the target codon in the plasmid containing the gene. The restriction sites to be introduced are chosen based on their uniqueness to the plasmid, proximity to the target codon and conservation of the final amino acid coding sequence. The flanking restriction sites in the plasmid are digested with the cognate restriction enzymes, and short synthetic duplex DNA cassettes (10-25 bp) are inserted. The mutagenic cassette is designed to restore fully the wild-type coding sequence, except over the target codon, and to eliminate one or both restriction sites. Elimination of a restriction site facilitates selection of clones containing the mutagenic oligodeoxynucleotide cassette. To make the cassettes, single-stranded oligodeoxynucleotides and their complements are synthesized in separate pools containing different codons over the target. This method has been successfully applied to generate 19 amino acid substitutions at position 222 in the subtilisin protein sequence.     

用DREAM技术纠正人工合成基因中的突变

目的：介绍一种简便、有效的定点突变技术。方法：根据突变位点附近的DNA序列推导出氨基酸序列,再以此氨基酸序列进行逆翻译,这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体（silent mutants）,这些突变体中包含大量的限制性内切酶位点,选择合适的酶切位点设计引物用PCR技术扩增两侧DNA片段,然后以相应酶切融合这两个片段即可完成定点突变。结果：用该方法成功地在人工合成的含有缺失的可溶性组织因子基因的472位插入C,T两个碱基,校正了阅读框架,获得了预期的目的基因。结论：该方法简便、有效, 避免了多轮PCR和合成长引物导致突变的可能性,这种改进的PCR 定点诱变技术我们称之为“设计限制酶辅助突变”（Designed Restriction Enzyme Assisted Mutagenesis, DREAM）。此技术简单方便, 诱变的成功率高, 适于实验室常规应用。     

A general DNA analysis program for the Hewlett-Packard Model 86/87 microcomputer.

A program is described to perform general DNA sequence analysis on the Hewlett-Packard Model 86/87 microcomputer operating on 128 K of RAM. The following analytical procedures can be performed: 1. display of the sequence, in whole or part, or its complement; 2. search for specified sequences e.g. restriction sites, and in the case of the latter give fragment sizes; 3. perform a comprehensive search for all known restriction enzyme sites; 4. map sites graphically; 5. perform editing functions; 6. base frequency analysis; 7. search for repeated sequences; 8. search for open reading frames or translate into the amino acid sequence and analyse for basic and acidic amino acids, hydrophobicity, and codon usage. Two sequences, or parts thereof, can be merged in various orientations to mimic recombination strategies, or can be compared for homologies. The program is written in HP BASIC and is designed principally as a tool for the laboratory investigator manipulating a defined set of vectors and recombinant DNA constructs.     

EcoKI with an amino acid substitution in any one of seven DEAD-box motifs has impaired ATPase and endonuclease activities.

For type I restriction systems, recently determined nucleotide sequences predict conserved amino acids in the subunit that is essential for restriction but not modification (HsdR). The conserved sequences emphasize motifs characteristic of the DEAD-box family of proteins which comprises putative helicases, and they identify a new candidate for motif IV. We provide evidence based on an analysis of Eco KI which supports both the relevance of DEAD-box motifs to the mechanism of restriction and the new definition of motif IV. Amino acid substitutions within the newly identified motif IV and those in six other previously identified DEAD-box motifs, but not in the original motif IV, confer restriction-deficient phenotypes. We have examined the relevance of the DEAD-box motifs to the restriction pathway by determining the steps permitted in vitro by the defective enzymes resulting from amino acid substitutions in each of the seven motifs. Eco KI purified from the seven restriction-deficient mutants binds to an unmethylated target sequence and, in the presence of AdoMet, responds to ATP by undergoing the conformational change essential for the pathway of events leading to DNA cleavage. The seven enzymes have little or no ATPase activity and no endonuclease activity, but they retain the ability to nick unmodified DNA, though at reduced rates. Nicking of a DNA strand could therefore be an essential early step in the restriction pathway, facilitating the ATP-dependent translocation of DNA, particularly if this involves DNA helicase activity.     

Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.     

insilico.mutagenesis: a primer selection tool designed for sequence scanning applications used in directed evolution experiments

Several primer prediction programs have been developed for a variety of applications. However none of these tools allows the prediction of a large set of primers for whole gene site-directed mutagenesis experiments using the megaprimer method. We report a novel primer prediction tool (insilico.mutagenesis), accessible at www.insilico.uni-duesseldorf.de, developed for the application to high-throughput mutagenesis used in directed evolution or structure-function dependency projects, which involve the subsequent mutagenesis of a large number of amino acid positions (e.g., in whole gene saturation or gene scanning mutagenesis experiments). Furthermore, the program is suitable for all site-directed (saturation) mutagenesis approaches, such as saturation mutagenesis of promoter sequences and other types of untranslated intergenic regions. In anticipation of downstream cloning steps, the primer design tool also includes a restriction site control feature alerting the user if unwanted restriction sites have been introduced within the mutagenesis primer. The use of our tool promises to speed up the process of site-directed mutagenesis, as it instantly allows predicting a large set of primers.     

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.     

青藏高原5种害鼠D型肉毒素抗性相关基因VAMP1的序列分析

突触小泡相关膜蛋白1基因(VAMP1)的变异是导致鼠类对D型肉毒梭毒素灭鼠剂产生抗性的主要原因。本研究利用转录组测序的方法,分析青藏高原地区5种主要害鼠:高原鼠兔(Ochotona curzoniae)、高原鼢鼠(Eospalax baileyi)、长尾仓鼠(Cricetulus longicaudatus)、青海松田鼠(Neodon fuscus)和喜马拉雅旱獭(Marmota himalayana)的VAMP1序列信息。同时,分别采集来自5个地理种群的58只高原鼠兔和59只高原鼢鼠,对VAMP1基因第二外显子区序列进行分析。结果显示,从转录组组装文件中成功获得5种动物的VAMP1基因全序列,长度均为357 bp,共检测到46个核苷酸变异位点和4个氨基酸变异位点,但未发现与D型肉毒素抗性相关的氨基酸位点。对高原鼠兔群体和高原鼢鼠群体的VAMP1基因第二外显子序列的分析显示,高原鼠兔所有个体的序列高度保守,而在高原鼢鼠中则存在一个同义突变位点,但两物种在D型肉毒素抗性相关位点上都未监测出位点变异。该研究结果提示,D型肉毒杀鼠剂在青藏高原地区害鼠防治方面应该可以长期发挥重要作用。     

C.U.R.R.F. (Codon Usage regarding Restriction Finder): A Free Java?-Based Tool to Detect Potential Restriction Sites in Both Coding and Non-Coding DNA Sequences

The synthesis of complete genes is becoming a more and more popular approach in heterologous gene expression. Reasons for this are the decreasing prices and the numerous advantages in comparison to classic molecular cloning methods. Two of these advantages are the possibility to adapt the codon usage to the host organism and the option to introduce restriction enzyme target sites of choice. C.U.R.R.F. (Codon Usage regarding Restriction Finder) is a free Java(?)-based software program which is able to detect possible restriction sites in both coding and non-coding DNA sequences by introducing multiple silent or non-silent mutations, respectively. The deviation of an alternative sequence containing a desired restriction motive from the sequence with the optimal codon usage is considered during the search of potential restriction sites in coding DNA and mRNA sequences as well as protein sequences. C.U.R.R.F is available at http://www.zvm.tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/mikrobiologie/allgemeine_mikrobiologie/currf .     

Chemical synthesis of a biologically active gene for human immune interferon-gamma. Prospect for site-specific mutagenesis and structure-function studies

A 453-base pair DNA duplex consisting of a gene coding for human interferon-gamma and initiation and termination signals plus appropriate restriction enzyme sites for plasmid insertion has been totally synthesized. The synthesis involved preparation of 66 oligodeoxynucleotides by a modified, solid phase phosphite procedure and enzymatic ligation of the oligonucleotides. The gene, when inserted into a previously constructed expression vector, was expressed in Escherichia coli, demonstrating functional activity for the synthetic gene. Several strategically located restriction cleavage sites have been introduced into the sequence. This provides a convenient system for site-specific mutagenesis for structure-function studies.     

Creating new restriction sites by silent changes in coding sequences

We present methods for identifying a useful type of DNA site--one that can be mutated to create a new restriction site within a coding region without changing the amino acid sequence. These "latent sites" are abundant--silent mutations creating one of 44 different 6-bp or 8-bp recognition sites were found at relatively high density, roughly one latent site per 9 bp, in the eleven genes tested. Our analysis suggests that site-directed mutagenesis can be used to refashion coding sequences at will for flexible analysis.