Very efficient template/primer-independent DNA synthesis by thermophilic DNA polymerase in the presence of a thermophilic restriction endonuclease

We have found that, in the presence of a thermophilic restriction endonuclease, thermophilic DNA polymerase efficiently synthesizes and amplifies DNA in the absence of any added template and primer nucleic acid under isothermal conditions. More than 10 microg of DNA can be synthesized by 1 unit of DNA polymerase in 1 h, and the reaction proceeds until available dNTPs are consumed. We used mostly the Tsp509I restriction endonuclease (recognition sequence: decreasing AATT), the TspRI restriction endonuclease (recognition sequence: NNCA(G/C)TGNN decreasing), and Vent (exo(-)) and Vent DNA polymerase. The synthesized double-stranded DNA has a highly repetitive palindromic sequence, e.g. (AAAAATTTTT)(n) and (ATACACTGTATATACAGTGTAT)(n). In every repeating unit, there are one or two recognition sites for the restriction enzyme. Our data show that the high efficiency of the restriction-endonuclease-DNA-polymerase (RE-pol) DNA synthesis results from an efficient exponential amplification involving digestion-elongation cycles: a longer DNA with numerous recognition sites for the restriction enzyme is digested to short fragments, and the short fragments are used as seeds for elongation to synthesize longer DNA. A possible role of RE-pol DNA synthesis in the evolutionary development of genetic materials is briefly discussed.     

Los Alamos sequence analysis package for nucleic acids and proteins.

An interactive system for computer analysis of nucleic acid and protein sequences has been developed for the Los Alamos DNA Sequence Database. It provides a convenient way to search or verify various sequence features, e.g., restriction enzyme sites, protein coding frames, and properties of coded proteins. Further, the comprehensive analysis package on a large-scale database can be used for comparative studies on sequence and structural homologies in order to find unnoted information stored in nucleic acid sequences.     

基因重组与基因合成实验设计软件系统的建立

本文报导了用于基因重组与基因合成实验设计的软件系统的建立.此系统由30个功能模块组成,为研究者提供了包括在DNA分子上寻找限制性内切酶位点、核酸分子片段之间同源性比较,基因化学合成的实验设计、特定顺序分析引物及核酸杂交探针的设计、阅读框的查找等功能.此外,本系统可以对外来数据库的资料进行援引和进一步分析,为分子生物学的研究提供有价值的信息.     

Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site

Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets.     

Identification of novel restriction endonuclease-like fold families among hypothetical proteins

Restriction endonucleases and other nucleic acid cleaving enzymes form a large and extremely diverse superfamily that display little sequence similarity despite retaining a common core fold responsible for cleavage. The lack of significant sequence similarity between protein families makes homology inference a challenging task and hinders new family identification with traditional sequence-based approaches. Using the consensus fold recognition method Meta-BASIC that combines sequence profiles with predicted protein secondary structure, we identify nine new restriction endonuclease-like fold families among previously uncharacterized proteins and predict these proteins to cleave nucleic acid substrates. Application of transitive searches combined with gene neighborhood analysis allow us to confidently link these unknown families to a number of known restriction endonuclease-like structures and thus assign folds to the uncharacterized proteins. Finally, our method identifies a novel restriction endonuclease-like domain in the C-terminus of RecC that is not detected with structure-based searches of the existing PDB database.     

DIGICALC: a restriction fragment analysis program.

DIGICALC is a program designed to aid in the acquisition, storage, and analysis of nucleic acid restriction fragment data. The chief considerations during program design were (i) ease of use for people with varying degrees of computer experience, (ii) minimal hardware requirements (e.g. an IBM PC), (iii) portability and ease of modification, and (iv) improved functionality in sizing and comparing restriction fragments over manual methods. The program accepts manual or digitizer input of nucleic acid fragment mobility, calculates the fragments' sizes, and provides the means to search the fragment database and to produce charts of fragment sizes.     

Computer-aided gene design.

A computer program, which runs on MS-DOS personal computers, is described that assists in the design of synthetic genes coding for proteins. The goal of the program is the design of a gene which (i) contains as many unique restriction sites as possible and (ii) uses a specific codon usage. The gene designed according to the criteria above is (i) suitable for 'modular mutagenesis' experiments and (ii) optimized for expression. The program 'reverse-translates' protein sequences into degenerated DNA sequences, generates a map of potential restriction sites and locates sequence positions where unique restriction sites can be accommodated. The nucleic acid sequence is then 'refined' according to a specific codon usage to remove any degeneration. Unique restriction sites, if potentially present, can be 'forced' into the degenerated nucleic acid sequence by using 'priority codes' assigned to different restriction sequences.     

Human apolipoprotein A-II: complete nucleic acid sequence of preproapoA-II

The complete cDNA nucleic acid sequence of preproapolipoprotein (apo) A-II, a major protein constituent of high density lipoproteins, has been determined on clones from a human liver ds-cDNA library. Clones containing ds-cDNA for apoA-II were identified in the human liver ds-cDNA library using synthetic oligonucleotides as probes. Of 3200 clones screened, 4 reacted with the oligonucleotide probes. The DNA sequence coding for amino acids ?17 to +17 of apoA-II were determined by Maxam-Gilbert sequence analysis of restriction fragments isolated from one of these clones, pMDB2049. The remainder of the cDNA sequence was established by sequence analysis of a primer extension product synthesized utilizing a restriction fragment near the 5'-end of clone pMDB2049 as primer with total liver mRNA. The apoA-II mRNA encodes for a 100 amino acid protein, preproapoA-II that has an 18 amino acid prepeptide and a 5 amino acid propeptide terminating with a basic dipeptide (Arg-Arg) at the cleavage site to mature apoA-II.     

SILMUT: a computer program for the identification of regions suitable for silent mutagenesis to introduce restriction enzyme recognition sequences.

We describe a set of IBM-compatible computer programs designed to selectively identify the potential sites for silent mutagenesis within a target DNA sequence. This program is based on a novel strategy of identifying amino acid motifs compatible with each restriction site (BioTechniques 12:382-384, 1991). The programs can be used to identify the suitability for the introduction of any 6-base nucleic acid sequences, such as restriction enzyme sites in cassette mutagenesis strategies. The Table program generates a table of multiple amino acid motifs for each restriction enzyme, obtained by translating each unique recognition sequence in all three reading frames. The Silmut program, which utilizes the features of Table, will further identify the presence of a match between any amino acid motif of each restriction enzyme and the input target sequence. Minor manipulations of the data base files will enable the individual researcher to identify the potential for introduction of any 6-base sequences by silent mutagenesis.     

序列同源性分析软件Blast的WEB界面构建及其应用

基于局域网(Intranet)内的PC/Linux服务器, 构建了序列同源性分析软件Blast的WEB界面. 局域网内的所有计算机均可通过WEB方式访问该服务器进行公共数据库和自建数据库的查询,具有保密、高效、免费的优点,能够满足实验室和研究院所的大规模、快速数据分析任务.     

Short-read reading-frame predictors are not created equal: sequence error causes loss of signal

ABSTRACT: BACKGROUND: Gene prediction algorithms (or gene callers) are an essential tool for analyzing shotgun nucleic acid sequence data. Gene prediction is a ubiquitous step in sequence analysis pipelines; it reduces the volume of data by identifying the most likely reading frame for a fragment, permitting the out-of-frame translations to be ignored. In this study we evaluate five widely used ab initio gene-calling algorithms--FragGeneScan, MetaGeneAnnotator, MetaGeneMark, Orphelia, and Prodigal--for accuracy on short (75-1000 bp) fragments containing sequence error from previously published artificial data and "real" metagenomic datasets. RESULTS: While gene prediction tools have similar accuracies predicting genes on error-free fragments, in the presence of sequencing errors considerable differences between tools become evident. For error-containing short reads, FragGeneScan finds more prokaryotic coding regions than does MetaGeneAnnotator, MetaGeneMark, Orphelia, or Prodigal. This improved detection of genes in error-containing fragments, however, comes at the cost of much lower (50%) specificity and overprediction of genes in noncoding regions. CONCLUSIONS: Ab initio gene callers offer a significant reduction in the computational burden of annotating individual nucleic acid reads and are used in many metagenomic annotation systems. For predicting reading frames on raw reads, we find the hidden Markov model approach in FragGeneScan is more sensitive than other gene prediction tools, while Prodigal, MGA, and MGM are better suited for higher-quality sequences such as assembled contigs.     

Location of spermine and other polyamines on DNA as revealed by photoaffinity cleavage with polyaminobenzenediazonium salts

Although polyamines interact strongly with nucleic acids, X-ray and NMR studies have not revealed much structural information about spermine-DNA complexes. Therefore, it was of interest to look at the binding of polyamines to 32P-labeled DNA restriction fragments by sequencing gel electrophoresis of the photoaffinity cleavage products induced by polyaminobenzendiazonium salts. The shift of cleavage patterns observed on opposite strands as well as competition experiments with distamycin shows polyamines to be located in the minor groove of B-DNA and to depend on the nucleic acid polymorphism, jumping to the major groove in the A-form. The sequence selectivities of various polycations (spermine, putrescine, and cobalt (III) hexaammine) are similar and slightly favor A,T-rich regions. Taken together, these results show that polycations which are not point charges are guided by the electronegative potential along the nucleic acid and suggest fast crawling of the polyamine within the minor groove, due to individual NH2+ jumping between multiple equidistant and isoenergetic bidentate hydrogen-bonding sites. Such a picture could be the clue to the unexpected NMR and to the frequently silent X-ray behavior of polyamines when bound to DNA.     

Most of the murine leukemia virus sequences in the DNA of NIH/swiss mice consist of two closely related proviruses, each repeated several times

The structure of the endogenous murine leukemia virus (MuLV) sequences of NIH/Swiss mice was analyzed by restriction endonuclease digestion, gel electrophoresis, and hybridization to an MuLV nucleic acid probe. Digestion of mouse DNA with certain restriction endonucleases revealed two classes of fragments. A large number of fragments (about 30) were present at a relatively low concentration, indicating that each derived from a sequence present once in the mouse genome. A smaller number of fragments (one to five) were present at a much higher concentration and must have resulted from sequences present multiple times in the mouse genome. These results indicated that the endogenous MuLV sequences represent a family of dispersed repetitive sequences. Hybridization of these same digested mouse DNAs to nucleic acid probes representing different portions of the MuLV genome allowed construction of a map of the sites where restriction endonucleases cleave the endogenous MuLV sequences. Several independent recombinant DNA clones of endogenous MuLV sequences have been isolated from C3H mice (Roblin et al., J. Virol. 43:113-126, 1982). Analysis of these sequences shows that they have the structure of MuLV proviruses. The sites at which restriction endonucleases cleave within these proviruses appeared to be similar or identical to the sites at which these nucleases cleaved within the MuLV sequences of NIH/Swiss mice. This identity was confirmed by parallel electrophoresis. We conclude that the apparently complex pattern of endogenous MuLV sequences of NIH/Swiss mice consists largely of only two kinds of provirus, each repeated multiple times at dispersed sites in the mouse genome.     

CHARACTERIZATION AND MOLECULAR PHYLOGENYOF A PROTEIN KINASE cDNA FROM THE DINOFLAGELLATE GONYAULAX (DINOPHYCEAE)1

Degenerate primers corresponding to conserved protein kinase motifs were used to amplify potential kinase DNA fragments from a Gonyaulax polyedra Stein cDNA library using PCR. One PCR fragment, potentially encoding a CAMP-dependent protein kinase, was used as a probe to isolate a near full-length cDNA from the library. The nucleic acid sequence of the entire cDNA clone had a high homology to the catalytic subunit of cAMP-dependent protein kinase (cAPK subfamily and affiliated members. Northern blot analysis showed that the corresponding mRNA had a size (about 1.4 kb) and a relative high abundance consistent with a cAPK homologue. Southern blot analysis showed that while there are roughly 30 copies of the kinase gene per genome, the pattern of restriction fragments is inconsistent with the hypothesis of a large gene family. Phylogenetic analyses comparing the deduced amino acid sequence from the Gonyaulax cDNA with other cAPK sequences place Gonyaulax close to the slime mold Dictyostelium discoideum. This is the first phylogenetic analysis of dinoflagellates based on protein sequence, and the results are in agreement with similar analyses based on rRNA sequences.     

Fast computer search for similar DNA sequences.

An extremely fast method of searching a nucleic acid sequence database against a probe sequence is described. The method is based on the detection of deviation from expected number and deviation from random spatial distribution of sub-sequences which are unique within a sequence, and shared between that sequence and the probe. On an IBM 3081 computer, total search of an encoded form of the EMBL nucleic acid sequence database with a 1 kbase probe sequence is completed in a few seconds. Previous best methods for a similar task required a few minutes.     

Homology between actin coding and its adjacent sequences in widely divergent species

Eco RI restriction endonuclease DNA fragments from several representatives of the kingdoms Protista and Animalia were electrophoretically separated and transferred to the nitrocellulose filters. These DNA's were hybridized with [32p]-labelled actin coding sequence from Drosophila melanogaster (Dm). The results indicate that the nucleic acid sequences of the genes coding for actin(s) has been highly conserved throughout evolution. Similar experiments were performed using the sequence derived from the 5' end of Drosophila actin gene as a probe. Cross-hybridization was observed between Drosophila and Acanthamoeba castellanii. This may indicate a functionally important region at the 5' end which has been conserved.     

A support vector machine approach to the identification of phosphorylation sites

We describe a bioinformatics tool that can be used to predict the position of phosphorylation sites in proteins based only on sequence information. The method uses the support vector machine (SVM) statistical learning theory. The statistical models for phosphorylation by various types of kinases are built using a dataset of short (9-amino acid long) sequence fragments. The sequence segments are dissected around post-translationally modified sites of proteins that are on the current release of the Swiss-Prot database, and that were experimentally confirmed to be phosphorylated by any kinase. We represent them as vectors in a multidimensional abstract space of short sequence fragments. The prediction method is as follows. First, a given query protein sequence is dissected into overlapping short segments. All the fragments are then projected into the multidimensional space of sequence fragments via a collection of different representations. Those points are classified with pre-built statistical models (the SVM method with linear, polynomial and radial kernel functions) either as phosphorylated or inactive ones. The resulting list of plausible sites for phosphorylation by various types of kinases in the query protein is returned to the user. The efficiency of the method for each type of phosphorylation is estimated using leave-one-out tests and presented here. The sensitivities of the models can reach over 70%, depending on the type of kinase. The additional information from profile representations of short sequence fragments helps in gaining a higher degree of accuracy in some phosphorylation types. The further development of an automatic phosphorylation site annotation predictor based on our algorithm should yield a significant improvement when using statistical algorithms in order to quantify the results.     

Support-vector-machine classification of linear functional motifs in proteins

Our algorithm predicts short linear functional motifs in proteins using only sequence information. Statistical models for short linear functional motifs in proteins are built using the database of short sequence fragments taken from proteins in the current release of the Swiss-Prot database. Those segments are confirmed by experiments to have single-residue post-translational modification. The sensitivities of the classification for various types of short linear motifs are in the range of 70%. The query protein sequence is dissected into short overlapping fragments. All segments are represented as vectors. Each vector is then classified by a machine learning algorithm (Support Vector Machine) as potentially modifiable or not. The resulting list of plausible post-translational sites in the query protein is returned to the user. We also present a study of the human protein kinase C family as a biological application of our method.