text:
|Publication number
|Publication type
|Application number
|Publication date
|Feb 4, 2003
|Filing date
|Nov 28, 2000
|Priority date
|Feb 12, 1998
|Fee status
|Also published as
|Publication number
|Daniel Dupret, Jean-Michel Masson, Fabrice Lefevre
|Original Assignee
|Proteus S.A.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (10), Non-Patent Citations (15), Referenced by (1), Classifications (14), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention has for its object a process permitting separation of the functions potentially present in a biological sample containing nucleic acids and the characterization of said function, by protein expression in vitro after transcription and translation of DNA fragments. The characterization of a function according to the process of the invention can be done among other things by the biochemical analysis of this function and optionally by the cloning or the sequencing of the polynucleotide sequence corresponding to each of the functions possibly present in the studied sample.
In this way, the process according to the present invention more particularly permits the identification and the isolation of genes or of the corresponding proteins possessing a determined or determinable function.
The search for new genes and/or proteins encoded thereby constitutes an important objective of numerous molecular biology laboratories. In effect, the genetic and cellular therapy or the creation of transgenic animals or plants has caused new hopes for human or animal health, diagnoses and feeding, but requires, among other things, the identification of numerous genes and/or protein activities. Thus, numerous techniques exist for the isolation and the screening of genes by cloning and expression.
One of these techniques called "genomics" comprises the sequencing of a part or the totality of the organism's genome, then searching for homology with already identified sequences in data libraries of potentially coding sequences. Once identified, these sequences are to be subcloned and expressed in order to verify that they effectively code for the sought-after property. In addition to the time necessary for its implementation, this technique presents the disadvantage of being capable of identifying only functions homologous to those that are already known and referenced in the databases.
Proteomics is a second possible approach for looking for interesting properties. It consists of extracting the proteins expressed by a microorganism and then purifying them. Each purification fraction is then tested by detecting which one contains the sought-after property. The primary disadvantage of this technique resides in the fact that it does not permit having a direct link between the detected property and the gene that permits the expression of this property. The second disadvantage of this approach is that it does not permit the detection of the properties that have not been induced in the starting microorganism.
A third technique for detecting a function expressed by a microorganism consists of using expression cloning. This method has as a principle the extraction of the DNA of the starting microorganism, fragmenting it and inserting it in an expression vector in vivo that is transformed in a host. This host is selected for its ability to express the genes of the starting microorganism. In the same fashion the vector is always selected for its compatibility with the expression host. The expression cloning technique is used in context of the search for a function in a microorganism having genetic characteristics close to one of the existing host (same codon usage, same GC percentage). In the case of the search for a function starting with a large number of microorganisms of genetically varied origin, the expression cloning method becomes non-usable, the hosts no longer being capable of expressing the heterologous genes that are provided to them.
Additionally, expression cloning like other prior art processes for isolating and screening of genes presents a number of disadvantages:
the cellular toxicity of the transcription and translation products, which can induce genetic recombinations in order to mitigate these toxicity problems,
the poor representativity of the library used,
the implementation time,
the low compatibility between the sequence of the cloned gene and the punctuation of the expression of the vector used,
highly variable expression levels,
codon usage problems,
refolding and post-translational problems,
the problem posed by the influence of the physiological state of a cell on the expression level of the proteins when the protein activity is sought directly in the original cellular extracts,
a high difficulty of automation.
The present invention precisely aims to offset these disadvantages by offering a fast and effective method for the identification of a function associated with a polynucleotide sequence contained in a biological sample containing nucleic acids, and permitting the isolation of said sequence.
This goal is achieved thanks to a process for the separation and the characterization of the functions potentially present in a biological sample containing nucleic acids, characterized in that it includes the following steps:
a) the preparation of nucleic acid fragments starting from said sample,
b) the association of each of said fragments with a vector molecule,
c) the isolation of each fragment associated with a vector molecule or with a part of each construction composed of a fragment associated with a vector molecule of step (b),
d) the in vitro treatment of each fragment associated with a vector molecule or of a part of each construction composed of a fragment associated with a vector molecule from step (c) in order to obtain transcripts,
e) the test of a function of the transcripts obtained at step (d) or of proteins for which they code after translation of said transcripts.
The process of the invention offers the advantage of enabling the carrying out of:
a test of the properties of the transcripts of step (d), when they have advantageous properties of the tRNA, rybozyme type, or
a test of the properties of the proteins coded for by said transcripts.
In each case where the functions of the proteins encoded by the transcripts obtained at step (d) are tested, the process of the invention comprises at step (d) the treatment of said transcripts in vitro with a cellular extract permitting their translation to protein, then the test of a function of said proteins by any appropriate means.
By function in the sense of the present invention is understood more particularly the property that can be encoded by a polynucleotide sequence. This property can for example be an enzymatic activity or an affinity if said sequences codes for a protein, or an endonuclease activity for example if said sequences codes for a catalytic mRNA.
Thus, the process of the invention permits not only the detection of a function, but also the characterization of this function from a biochemical point of view. If the function is an enzymatic activity for example, it can relate to the analysis of the optimal conditions of functioning (pH, temperature, salts concentration), of the kinetic parameters (Vm, Km), of the inhibition parameters (Ki). If the function is an affinity, it can relate to the determination of the Kd, or of the molecule having the most affinity for this protein. It can also relate to the determination of the size of the translated protein or of the mRNA transcript, and optionally of the sequencing of the corresponding gene.
By biological sample is understood any sample liable to contain nucleic acids, such as a soil, plant, blood, human or animal, water, microbial, cellular or viral culture, biopsy, etc. sample, but these samples can equally correspond to amplification products (PCR, NASBA, etc . . . ), genomic DNA, synthetic DNA, mRNA, or any product of nucleic acids resulting from treatments currently used by a person skilled in the art.
By vector molecule is understood one or several polynucleotide sequences comprising at least a transcription promoter for step (d) and possibly a substance facilitating the isolation of the step (c) fragment. Optionally this substance can be one or several molecules of streptavidine or of biotin, of the polypyrol group, of antibodies, a single or double-stranded polynucleotide sequence, a DNA plasmid vector preferably not containing sequences permitting the in vivo expression of the associated fragment, or any other compound permitting the isolation of the fragment associated with step (c).
By isolation at step (c) of the process of the invention is understood the subdivision of the batch of fragments obtained at step (b) in subsets, a subset that can be composed of a single or of several nucleic acid fragments.
Step (a) for the preparation of the nucleic acid fragments of the process of the invention can comprise an extraction phase if the nucleic acids are not directly accessible in the biological sample, for example in the case of nucleic acids contained in cells, viruses, blood, organic elements, etc . . . This extraction phase is thus included in step (a) for preparation of nucleic acid fragments. Similarly in the case where the biological sample is composed of mRNA, a step of RT-PCR is necessary to prepare the nucleic acid fragments of step (a).
According to a particularly preferred embodiment of the invention, the vector molecule is composed of two polynucleotide sequences each comprising at least one transcription promoter. Each one of these sequences is associated with an end of one of the fragments obtained at step (a).
According to an advantageous embodiment of the process of the invention, the transcription promoter or promoters carried by the vector molecule is/are a promoter or promoters of the strong type, such as the RNA polymerase transcription promoter of the T7, SP6, Qβ or λ phage.
Thus, a particular application of the process of the invention permits identification of a polynucleotide sequence and/or the corresponding protein, possessing a function, starting from a sample containing nucleic acids. This goal is attained according to a process characterized in that it comprises the following steps:
a) the preparation of nucleic acid fragments starting from said sample,
b) the insertion of each one of said fragments in a vector so as to create recombinant vectors,
c) the isolation of each recombinant vector or of a part of this recombinant vector by any appropriate means,
d) the in vitro treatment of the vector or of a part of the recombinant vector isolated at step (c) to obtain transcripts,
e) the test of a function of the transcripts obtained at step (d) or of the proteins encoded thereby by any appropriate means after translation of said transcripts.
By recombinant vector in the application of the above process of the invention is understood a vector, for example, plasmidic, in which a fragment has been introduced, and by part of this vector, the part of the recombinant vector containing the fragment obtained at step (a) and the elements necessary for the implementation of the steps (d) and optionally (e).
The steps of the process of the invention can be carried out successively without interruption by the same operator, advantageously on an automated device integrating each of the steps, or can be carried out in a discontinuous fashion, optionally by different operators.
The transcription step (d) and the translation phase of step (e) of the fragments are also conjointly designated In Vitro Protein Expression reaction, also designated EPIV reaction. The EPIV reaction can be simultaneous, which means that the step (e) translation phase is carried out simultaneously with the step (d) transcription, or broken down into two distinct steps, transcription (d) and translation (e).
The uncoupling of the steps (d) and (e) permits optimization of the yields of each step, and thus production of greater quantities of proteins, which finds significant utility in the case of enzymes of weak specific activity.
This uncoupling also permits normalization of the formation of the step (e) products and enables later comparison of the different expressed functions.
The uncoupling out of the step (d) transcription and the step (e) translation equally permits avoiding the problems of degradation of the DNA template by nucleases if it was prepared by PCR. In effect, the components of the transcription reaction are less contaminated by the nucleases, in contrast to the translation extracts.
The uncoupling moreover permits the use of different translation extracts according to the origin of the DNA screened. In effect, the step (e) translation phase of the transcripts obtained during step (d) is advantageously carried out with a translation extract of the same origin or of a close origin to that of the biological sample on which the process of the invention is practiced. Thus, the correspondence between the origin of the transcript translation signals and the cellular extract is optimized for optimal translation effectiveness. By way of example, there can be cited the use of a translation extract of an extremophilic organism for the screening of a DNA library of the same organism or of another extremophilic organism (thermophiles, halophiles, acidophiles, etc . . . ) or also a translation extract of eukaryotic cells for the screening of a eukaryotic DNA library. These respective extracts are likely to improve the effectiveness of the process. These extracts are selected for their capacity to translate the transcripts at step (e).
The process of the invention is notable in that it implements a correspondence between the expression punctuation of the transcripts of step (d) and the translation extracts used. These extracts are also characterized in that either they do not contain the sought-after property, or they contain it but it is not detectable under the conditions of the test carried out for detecting the sought-after function. It concerns for example the use of a translation extract containing a mesophilic beta-galactosidase activity permitting translation of a thermophilic beta-galactosidase mRNA and the detection of the activity of the latter at high temperature, which eliminates the mesophilic beta-galactosidase activity.
Depending of the genetic origin of the fragments obtained at step (a), (i.e. DNA of Gram positive or negative, eukaryotic, viral, etc. microorganisms), and of the function tested, different translation extracts can be used.
A particular embodiment of the process of the invention consists of using at step (a) a translation extract that is in fact a mixture of several translation extracts. It relates for example to a translation extract of E. coli over-expressing a chaperon A protein mixed with a translation extract of E. coli over-expressing a chaperon B protein. Any type of mixture can be contemplated from the moment that it corresponds to the characteristics described above. In the same manner, it is possible to use a translation extract in which one or several specific tRNAs of one or several codons are added. The translation extracts obtained in this way thus permit translation of the mRNA containing specific codons, such as for example the translation of an mRNA containing an amber codon by adding in the translation extract one of tRNA suppressor(s).
The treatment of step (e) with a translation extract can also be carried out with a universal translation extract whatever be the origin of the sample such as for example an extract of E. coli and/or any other cellular extract or extracts supplemented or not by molecules of interest such as those, for example, indicated previously (tRNA, chaperon . . . ).
It is equally possible to add to the translation extract of step (e) one or several substances favoring a more effective refolding or maturation of the expressed proteins, such as for example chaperons, detergents, sulfobetaines, membrane extracts, etc . . .
The test of a function of the proteins synthesized at step (e) can be carried out by any appropriate means permitting for example the detection of the sought-after enzymatic activity or activities. This embodiment will more particularly be applied during the search for enzymes with original properties, such as for example thermostable enzymes active at high temperature, in an acidic medium, in a medium of high salt concentration, etc . . . , starting from a DNA sample resulting from an extremophilic organism or organisms. These extremophilic enzymes, often active under conditions close to the physiological conditions of the strains which produce them (temperatures, salinity, pH, etc . . . ) are particularly interesting tools for numerous industrial processes (agribusiness, animal nutrition, paper, detergents, textile industries etc . . . ), where they can be substituted for their mesophilic homologues.
The process of the invention offers the advantage of being capable of carrying out:
a test of the properties of the step (d) transcripts, when they have advantageous properties of the tRNA, rybozyme type, or
a test of the properties of the proteins encoded by said transcripts.
In the case of a ribozyme having an endonuclease activity for example, it is possible to detect this activity by using a nucleotide matrix having a fluorescent group at one end and a "quencher" group at the other. In case of cutting of the matrix by the ribozyme, the fluorescent group is separated from the "quencher" group, which frees the fluorescence from the first.
In the case of a tRNA, it is possible to use for example a fraction of the reaction potentially containing this tRNA and to put it in an in vitro translation reaction containing the mRNA of a reporter gene of which one of the codons can be read only by the sought-after tRNA. If the activity of the reporter is detected, this means the tRNA was present in the initial fraction and that it permitted the in vitro translation of the reporter mRNA.
In the context of demonstrating an enzymatic activity, any type of specific substrate can be contemplated by a person skilled in the art for demonstrating the presence or the absence of the sought-after function at step (e). All transformation(s) of the substrate(s) by the sought-after function(s) can be detected by any method known by a person skilled in the art (fluorimetry, colorimetry, absorbance, viscosity, etc . . . ).
In the case of the demonstration of an affinity, it is possible for example to use, according to the sought-after affinity: antibody-antigen, double stranded DNA binding protein, receptor-ligand, etc . . . , tests such as radio-labeled ligand fixation, an immunological detection comprising the immobilization of an antigen, its specific detection with the sought-after antibody, and the revelation of this sought-after antibody thanks to an anti-antibody antibody coupled to a reporter activity which can be revealed, or an antigen detection by using a goat antibody fixed on a support capable of recognizing the antigen, the antigen being able to be detected by a second rabbit antibody (sandwich formation) indirectly coupled or not to a reporter activity (alkaline phosphatase or peroxidase type).
One particular embodiment of the process consists at step (e) of testing, in parallel or in a simultaneous or non-simultaneous manner, different properties of the same or several functions.
Thus, the process of the invention is notable in that it not only permits the detection of the functions potentially contained in a biological sample, but also:
their quantification, when these properties permit it such as for example enzymatic activities or affinities.
their characterization, notably biochemical, such as for example the optimal conditions of functioning at temperature, pH, saline concentration, etc . . . , molecular weight, protein sequencing, and optionally sequencing of the corresponding polynucleotide sequence.
The process of the invention is also notable in that it is totally independent of the in vivo expression of the proteins. The cellular hosts, like microorganisms, if they are used, are used only for the isolation and amplification of the heterologous fragments. Consequently, the process of the invention permits avoiding all of the problems previously reported with expression cloning methods of the prior art. In particular, the process of the invention is of great interest in the identification of a gene expressing a cytotoxic protein. In this sense, the vector molecule associated with each fragment of step (b) can be a plasmidic vector preferably not permitting the expression of said fragment in vivo. By way of example of such a vector one can cite: pBR322 or pACYC184.
Advantageously, the fragments prepared at step (a) of the process of the invention are obtained by the action of one or several endonucleases on the nucleic acids of the biological sample or on the PCR products of said nucleic acids. These fragments can also be obtained by a mechanical action on theses nucleic acids, for example by passage in a syringe needle, disruption under pressure, sonication, etc.
In the particular embodiment of the process of the invention where the nucleic acids of the sample do not require fragmentation, such as for example genomic libraries, step (a) consists of preparing the fragments of this sample for step (b), for example by extraction, and/or purification and/or preparation of the ends for the association with vector molecules, for example the insertion in the plasmidic vector.
In a particular embodiment of the process of the invention, the fragments prepared at step (a) have a size of 1 to several dozen kb, preferably from 1 to 40 kb and advantageously from 1 to 10 kb when the sample comes from a prokaryote organism. Preferably, the fragments prepared at step (a) have a size on the order of 5 kb. In effect, the average size of a prokaryote gene is about 1000 base pairs. By using fragments of 5000 base pairs, it is possible to obtain clones carrying the complete gene with their proper ribosome-binding site. In the case where the DNA is of eukaryotic origin, the size of the fragments prepared at step (a) is much more important, advantageously on the order of several dozens to several hundreds of kilobases.
It can be noted that the fragments prepared at step (a) can carry a partial or entire operon.
The biological sample that the fragments of step (a) are prepared from can come from one or several identical or different prokaryotic organisms or eukaryotic cells or even viruses. It can for example be a sample of nucleic acids of a microorganism or of a mixture of microorganisms, or of eukaryotic tissue cells or of identical or different organisms. But the sample of nucleic acids can equally be composed of a sequence or of a library of a nucleic acid or of nucleic acids. The biological sample can be composed of known or unknown organisms and/or of known or unknown nucleic acids. The sample can also advantageously be composed of synthetic nucleic acids.
In the case of a eukaryotic DNA library, the transcription reaction can be completed by in vitro splicing and maturation reactions of the mRNA by using for example a nuclear extract (3).
The process of the invention assures a direct link between the function demonstrated at step (e) and the corresponding polynucleotide sequence. This process is therefore most particularly suited for detecting functions and identifying the corresponding genes starting from genomic DNA. By gene is understood a DNA fragment or sequence associated with a biological function.
As indicated previously, according to a particular embodiment of the process of the invention, the vector molecule associated with the nucleic acid fragments at step (b) is a plasmidic vector. In this case, at step (b) of the process of the invention, each fragment is inserted in a vector at the level of a cloning site or of a restriction cassette. This plasmidic vector is characterized in that it comprises an RNA polymerase promoter at one side of the cloning site and optionally an RNA polymerase terminator at the other side. It is also possible to design a vector comprising a cloning site surrounded by two different or identical RNA polymerase promoters and possibly flanked on both sides by a corresponding RNA polymerase terminator or terminators. These promoters and possibly terminators preferentially have the characteristic of not functioning in the microorganism that can be used for the separation of the recombinant vectors at step (c).
In the case where the vector does not possess an RNA polymerase promoter or promoters and/or optional terminator or terminators, or in the case where the RNA polymerase promoter or promoters and possible terminator or terminators are not adequate for carrying out step (d), this promoter or promoters and possible terminator or terminators can be inserted at step (c) of the process of the invention by any appropriate means. An advantageous embodiment of this insertion consists of carrying out a PCR with a set of primers carrying the sequences of the promoter(s) and terminator(s).
According to a particular embodiment of the process of the invention, the promoter(s) and possible terminator(s) are of the strong type such as for example those of the T7 RNA polymerase.
In the case where the vector molecule is a plasmidic vector, the isolation of the recombinant vector at step (c) can be carried out by transformation of host cells by the entirety of the recombinant vectors obtained at step (b) so as to create a library of clones, then by carrying out an extraction of the recombinant vector or of a part of the vector contained by each clone of the library by any appropriate means.
The extraction of the recombinant vector or of a part of the recombinant vector flanked by the RNA polymerase promoter(s) and possibly terminator(s) can be carried out by any method known by a person skilled in the art, such as by plasmid miniprep and possibly digestion or by PCR. An advantageous alternative consists of carrying out this PCR with oligonucleotides protected from 5′ nucleasic attacks, notably from the nucleases contained in the translation medium, by phosphorothioate groups.
As previously indicated, the isolation at step (c) can be carried out by any physical, mechanical or chemical means such as for example a simple extreme dilution of the entirety of the fragments associated with the vector molecule at step (b). However, the isolation can also advantageously be carried out by using the properties of a specific substance included in the vector molecule, such as an antibody molecule, and the isolation of the fragment is carried by using the antibody-antigen affinity, or a biotin, and the isolation is carried out by using the biotin-streptavidine affinity, etc. . .
According to a particular embodiment of the process of the invention, a sorting of the fragments or the part of them each associated with a vector molecule obtained at step (c) is effected. For this, an EPIV reaction is carried out for each of the fragments or the part of them associated with a vector molecule obtained at step (c) by incorporating in the reaction mixture of the translation phase of step (e) a label of protein synthesis (biotinylated tRNA, modified amino acid, etc . . . ). Each EPIV reaction product is then analyzed, for example by ELISA, for the presence of an expressed protein. The fragments or the part of them associated with a vector molecule for which the EPIV reaction is negative are determined. These fragments do not possess an ORF on their insert. Following this pre-screening procedure, these fragments are eliminated from the following activity identification screenings linked to a protein. Such a pre-screening procedure permits a savings in time and in reactants in the case of a screening that is repetitive and not simultaneous from the same library.
According to a particular embodiment, the process of the invention is entirely carried out on a solid chip type support or a membrane or a nanotitration plate. The chip type support can be a glass plate, a nitrocellulose membrane or any other support known to a person skilled in the art. The fragments associated with the vector molecule are isolated on this chip type or nanotitration plate support, and the reactants permitting the implementation of the process of the invention are deposited on this support. The test of the sought-after function or functions can be directly conducted on the support after a possible washing of the latter. In the case where the vector molecule is a plasmidic vector and/or the process of the invention is carried out on a support, the colonies transformed by the recombinant vectors are transferred separately from the others on a same support, then lysed in situ (3) such that each colony can liberate on the support the copies of the recombinant vector that it contains. Another embodiment consists to separately loading on a same support each recombinant vector or part it. It is thus possible to deposit reactants permitting the carrying out of an EPIV reaction on the support having the deposited DNA according to one of the techniques described above. The test of a function can be directly conducted on the support after an optional washing of the latter.
The invention equally has for an object:
a not yet known nucleic acid sequence identified and selected by the process of the invention,
a fragment containing this nucleic acid sequence associated with a vector molecule,
a plasmidic vector containing this sequence, a cellular host transformed by this nucleic acid sequence or by this vector, or
a protein encoded by this sequence.
The invention equally relates to any library composed of:
nucleic acid sequences isolated by the process of the invention,
vector molecules associated with fragments containing these sequences,
vectors containing said sequences,
cellular hosts transformed by one (or some) of these sequences or by one of these vectors, or
proteins encoded by said sequences.
The process of the invention offers the advantage as compared to the methods of the prior art of being capable of being automated. In the case where the vector molecule is a plasmidic vector, this automation can be conducted, for example, in the following manner:
Each recombinant vector of the library formed at step (b) can be put in culture on a support, in a microplate well by a Colony Picker type robot.
This culture can be used for a plasmidic extraction step carried out by a Biorobot 9600 (QIAGEN) type robot, or for a PCR amplification step implemented by a MultiProbe type machine (PACKARD) on a PTC 200 or PTC 225 (automated lid-MJ RESEARCH) type automatic thermocyler.
The optional purification of the PCR products can be conducted by the BioRobot 9600 automatic machine.
The EPIV reaction of steps (d) and (e) can be directed entirely by the MultiProbe robot. The tests of the functions of the transcripts obtained at step (d) can be effected on the robot pipetor and the reading of the results is obtained on a corresponding reader. If the transcription reaction is separated out from the translation reaction, the optional purification of the mRNA can be carried out by the BioRobot 9600.
The tests of the activity of the proteins synthesized at step (e) are carried out by the robot pipetor, and the reading of the results is obtained on the reader (spectrophotometry, colorimetry, fluorimetry, etc . . . , according to the test carried out) of micro plaques or by any other appropriate means.
Consequently, the invention also relates to an automated device for the implementation of the processes described previously comprising a layout of one or several supports, robots, automatic machines and readers optionally permitting the preparation of the sample at step (a) and optionally the carrying out of the step of association of the fragments with the vector molecule at step (b), optionally the isolation of said fragments associated with the vector molecule at step (c), and to carry out the implementation of the steps (d) and (e). By assuring an increased repetition flow of the process of the invention, or of a part of the process of the invention (steps (d) and (e)) this layout of automatic machines permits rapid searching of the functions starting from different nucleic acid samples.
Any plasmidic vector having the characteristics defined previously can be used in the process of the invention. By way of example can be cited a vector represented at FIG. 2 and constructed in the following manner:
A plasmidic replication origin.
A cloning site surrounded by two identical promoters, such as that of the T7 RNA polymerase, or of any other strong RNA polymerase promoter, such as Qβ, T3, SP6, etc., and optionally flanked on both sides by the same RNA polymerase terminator. These promoters and terminator if it is present, preferably do not function in the microorganism used to separate the recombinant vectors. Such a construction permits transcription of a DNA fragment inserted in the cloning site, regardless of its sense of insertion. The probability of finding a good clone is therefore multiplied by two. The average size of a prokaryotic gene is about 1000 bp. By using prokaryotic DNA fragments of about 5000 bp to generate the library, it is highly probable that clones carrying the complete genes will be obtained, with their proper ribosome binding site (or RBS for Ribosome Binding Site). With this double promoter system, the gene is located in the worst case 2000 bases from the beginning of the mRNA, which permits effective expression of the corresponding protein by the process of the invention (as reported in the experimental hereinafter on the β-lactamase activity).
Optionally, some specific sequences on both sides of the terminators can be used as hybridization sites for a PCR amplification of the nucleic acid fragment carried by the vector.
A selection gene composed of a tRNA gene (4). Optionally, in parallel, an antibiotic resistance gene (or another type of selection gene) is inserted in the cloning site. This antibiotic selection is used only for the preparative amplification of the cloning vector. In effect, during the insertion of each one of said fragments of step (b), a DNA fragment is substituted for this resistance gene. This system has the advantage of not depending on an antibiotic selection, which raises problems of contamination and degradation of the antibiotic, and permits obtaining a recombinant vector not possessing an ORF other than that possibly introduced by the heterologous fragment. On the other hand, it permits a very rapid evaluation of the level of negative clones, by practicing a parallel spreading of a fraction of the library on minimum medium and on a medium containing the selection antibiotic.
The process of the invention is notable in that it permits looking in crude samples for nucleic acid functions and to characterize them and identify the corresponding nucleic acid sequence. In effect, it avoids the isolation of each microorganism present in this sample. The isolation of the microorganisms contained in a sample is known to give limited results, for only a few percent of the diversity of the microorganisms present are recovered. Moreover, the process of the invention offers a considerable time savings. The method of the invention permits the realization of a better result, since the screened biodiversity is of the order of 100%, in a time period of 5 to 10 days. Whereas the prior art methods permit isolation of around of 5% of the biodiversity of a sample over several weeks or even several months, as much time is necessary to screen these strains in order to detect a protein activity. The process of the invention is equally advantageous for searching for the protein activities in a given germ or cell. A genomic or cDNA library can be easily prepared and screened according to the process of the invention. In the case of a cDNA library, the vector molecule comprises a translation initiation sequence corresponding to the translation extract used at step (e). In addition to its rapidity, the process of the invention permits a freeing from the cellular physiology. It is thus possible to detect the protein activities without having to resolve problems of culture and of physiological states. The process of the invention permits directly going back up to the gene starting from the detection of a function of the organism. It also permits looking for a function in an organism potentially containing said function. This technique permits, thanks to its speed and its effectiveness, the screening of one or several functions of a large number of organisms from a collection and therefore to bring out its microbiological biodiversity. This process will find much of its utility in the identification of protein activities of industrial interest starting from isolated extremophilic organisms, or also from crude samples of said organisms.
Finally, the process of the invention finds an interest in the area of genomics. From its conception, it permits the identification of new genes without going through sequencing in the first step, because it permits going directly from the detection of a function to the sequence of the corresponding nucleic acid. Applied to the totality of the genomic DNA of an organism, the process of the invention permits attainment of the characterization of the total phenotype of this organism, which introduces the notion of "phenomics."
The invention also relates to a kit for the implementation of the process of the invention described previously. This kit comprises:
the means necessary for the preparation of the nucleic acid fragments
at least one vector molecule
at least one polymerization agent
optionally at least one cellular translation extract
the means necessary for the test of one or several functions
the buffers necessary for the carrying out of the different steps
This kit can be packaged in one or different containers.
Other advantages and characteristics of the invention will appear from the examples of carrying out the invention that follow and that refer to the annexed drawings in which:
FIG. 1 is a schematic representation of an example of an embodiment of the process of the invention.
FIG. 2 represents an example of a cloning vector for the construction of a library according to the process of the invention.
FIG. 3 represents an example of the plasmids pADH, PTEM, pET26-Tfu2, pLIPet pGFP used in the scope of the examples of carrying out the process of the invention.
FIG. 4 represents the activity of the intein 2 of a DNA polymerase of Thermococcus fumicolans (Tfu) obtained in vivo by expression cloning. Track A: molecular weigh marker 1 Kb. Track B: reaction without enzyme. Track C: reaction with intein 2.
FIG. 5 represents the activity of the GFP produced by EPIV reaction (Emission of fluorescence followed by an exposure at about 400 nm) with mesophilic translation extracts. Tube A: EPIV reaction with water (control). Tube B: EPIV reaction with the vector pGFP.
FIG. 6 represents the activity of the intein 2 of the Tfu DNA polymerase produced by EPIV reaction with mesophilic-type translation extracts. Track 1: molecular weigh marker 1 Kb. Track 2: T− (reaction without enzyme). Track 3: Blank (reaction with EPIV extract without DNA). Track 4: T+ (reaction with intein 2 produced in vivo). Track 5: Test (reaction with intein 2 produced by EPIV (pET26-Tfu2)).
FIG. 7 represents the detection of the activity of a thermophilic enzyme by using a mesophilic translation extract containing said activity. Tube A: EPIV reaction with water (control) incubated at 37° C., Tube B: EPIV reaction incubated at 37° C., Tube C: EPIV reaction with water (control) incubated at 70° C. after centrifugation. Tube D: EPIV reaction incubated at 70° C. after centrifugation.
1) Strains and Plasmids
The vector pET26b+ is part of the family of pET vectors developed by Studier and Moffatt (8) and commercialized by the NOVAGEN Corporation. This vector permits expression of the genes under the control of the T7 phage promoter. The PinPoint™ (PROMEGA) vector carries the cat gene (coding for the chloramphenicol acetyltransferase) under the control of the T7 phage RNA polymerase. The vector pHS2-22-21 was constructed by introducing by reverse PCR the cutting site recognized by the intein 2 of the Tfu DNA polymerase positioned in the polycloning site of the plasmid pUC19. The vector pHS2-22-21 corresponds to pUC 19 containing the homing site (43 bp) of the intein or the site in which the intein gene is inserted.
The strain XL1-Blue [Tn10 proA+B+ laclq Δ (lacZ)M15/recA1 endA1 gyrA96 (Nalr) thi hsdR17 (rk −mk +)supE44 relA1 lac) was used for the amplification of the plasmidic DNA.
2) Reagents
The restriction and modification enzymes reported in Table I below were used according to the recommendations of the suppliers.
Sca I
10 U/μl
Appligene Oncor
Sal I
8 U/μl
New England Biolabs
Nde I
20 U/μl
New England Biolabs
Bam HI
20 U/μl
New England Biolabs
Eco RI
20 U/μl
New England Biolabs
50 U/μl
New England Biolabs
T4 DNA ligase
400 U/μl
New England Biolabs
The buffers used are described in Table II below.
Tris HCl 10 mM pH 8.0
T7 RNA polymerase
Tris HCl 400 mM pH 7.9, 60 mM MgCl2, 20
MM spermidine, 10 mM DTT
Tris-Oac 500 mM pH 8.0, 750 mM
Mg(OAc)2, 100 mM NH4OAc
Glycine 100 mM (NaOH pH 10), 20 mM
Butanol 1, 0.9 M NaCl, 4 mM NADP
KH2PO4 0.1 M, pH 6.8, dioxane 5%,
Thesit 5%
NaP 50 mM pH 7.0, 100 μg/ml
Nitrocephine, 0.25 mM DMSO
Ligation 10X
Tris HCl 500 mM pH 7.5, 100 mM MgCl2,
100 mM DTT, 10 mM ATP, 250 μg/ml BSA
1) Construction
The gene of the intein 2 of the Tfu DNA polymerase (itfu2) (Accession number in Gene library: Z69822) was inserted between the restriction sites Nde I and Sal 1 of the vector pET26b+ in order to create the plasmid pET26-Tfu2 represented in FIG. 3. In similar fashion, the alcohol dehydrogenase gene (adh) of Thermococcus hydrothermalis was inserted between the restriction sites Nde I and Bam HI of the vector pET26B+ in order to create the vector pADH, and the genes of the P-lactamase TEM-1 (bla) of Escherichia coli (9), and of the Green Fluorescent Protein (gfp) of Aequorea Victoria (6) and of the lipase B of Candida Antarctica (lipB) (Accession number Y14015 in Gene library) were inserted respectively between the restriction sites Nde I and Eco RI of the vector pET26b+, in order to create the plasmids pADH, pTEM, pGFP and pLIP represented in FIG. 3. For each one of these four genes, the restriction site Nde I is over located with the codon ATG of the translation initiation.
Each construction was verified by several restriction profile analyses. 200 μl of XL1-Blue chimiocompetent cells (1) were transformed with 10 ng of each plasmid by a thermal shock (2), and the cells thus transformed were spread out on a solid LB medium containing 60 μg/ml of kanamycin and 12.5 μg/ml of tetracycline. Starting from a clone of each one of these transformations, a plasmidic DNA maxipreparation was carried out with a TIP100 type column (QIAGEN). After precipitation in isopropanol, each plasmidic DNA sample was resuspended in 100 μl of buffer T. The concentration of these plasmidic DNAs was evaluated by a spectrophotometric measurement at 260 nm. The purity of each plasmidic DNA was verified by depositing 0.2 μl of each of the vectors on agarose gel TBE 1%.
2) In Vivo Expression Tests-activity Tests
-pTEM: 200 μl of BL21 DE3 (pLysS) chimiocompetent cells were transformed with 10 ng of the pTEM plasmid by a thermal shock, and the cells thus transformed were spread out on a solid LB medium containing 60 μg/ml of kanamycin, 20 μg/ml of chloramphenicol, 32 μg/ml of IPTG and 100 μg/ml of ampicillin. After incubation one night at 37° C., very numerous colonies could be observed on the Petri dish, thereby revealing that the TEM-1 gene of the plasmid pTEM is expressed and functional, and that it confers ampicillin resistance.
-pGFP: 200 μl of BL21 DE3 (pLysS) chimiocompetent cells were transformed with 10 ng of the plasmid pGFP by a thermal shock, and the cells thereby transformed were spread out on a solid LB medium containing 60 μ g/ml of kanamycin, 20 μg/ml of chloramphenicol and 32 μg/ml of IPTG. After incubation one night at 37° C., very numerous colonies could be observed on the Petri dish. All of these reacted to ultraviolet excitation (at about 400 nm) by emitting a green fluorescent light, which permitted verification that the gfp gene of the plasmid pGFP is expressed and functional.
-pET26-Tfu2: 200 μl of BI21 DE3 (pLysS) chimiocompetent cells were transformed with 10 ng of the plasmid pET26-Tfu2 by a thermal shock, and the cells transformed thereby were spread out on a solid LB medium containing 60 μg/ml of kanamycin and 20 μg/ml of chloramphenicol. A culture carried out starting from a clone of this transformation was induced at DO600 nm=0.5 with 0.5 mM of IPTG for two hours at 37° C. After centrifugation, the bacterial precipitate was resuspended in 20 mM sodium phosphate buffer pH 7.5, and a cellular lysate was obtained after to several cycles of freezing/thawing. The intein 2 was then purified on a Qfast-Flow column with a NaCl gradient. The activity of this enzyme was tested according to the following protocol: 1 μl of the elution fraction having the highest pure enzyme concentration was diluted one hundred times. One μl of this dilution was incubated 15 minutes at 70° C. with 3 μl of IT2 buffer and 220 ng of the pHS2-22-21 plasmid, linearized with the restriction enzyme Sca I, in a final volume of 30 μl. 15 μl of this digestion mixture were deposited on an agarose gel TBE 1%. After migration and staining with ethidium bromide, the gel was exposed to ultraviolet. As shown in FIG. 4, the analysis of this gel reveals the presences of two bands of respectively 934 bp and 1752 bp, corresponding to the cutting of the pHS2-22-21 vector (Sca I linearized) by the intein 2. The gene itfu2 of the plasmid pET26-Tfu2 is therefore expressed and its product, the intein 2, is active.
-pADH: 200 μl of BL21 DE3 (pLysS) chimiocompetent cells were transformed with 10 ng of the pADH plasmid by a thermal shock, and the cells thus transformed were spread out on a solid LB medium containing 60 μg/ml of kanamycin and 20 μg/ml of chloramphenicol. A culture carried out starting from a clone of this transformation was induced at DO600 nm=0.6 with 1 mM of IPTG for three hours at 37° C. After centrifugation, the bacterial precipitate was taken up in a sodium phosphate buffer 50 mM-MgCl2 10 mM pH 8.0, and the cellular lysate was obtained by incubating 30 minutes over ice in the presence of 1 mg/ml of lysozyme, 10 μg/ml of RNAse A and 100 μg/ml of DNAse I. The centrifugation supernatant of this extraction step was incubated 30 minutes at 50° C., and centrifuged again. The supernatant of this last step was used as an enzymatic extract for the measurements of activity. A negative control was made up in parallel by carrying out a similar extraction on a culture of BL21 DE3 (pLysS) cells excluding plasmid. The alcohol dehydrogenase activity was tested by following with a spectrophotometer the reduction kinetics of the NADP to NADPH at 340 nm. For this, 10 μl of the enzymatic extract, or of the control, were incubated at 50° C. for five minutes with 500 μl of TADH buffer and 490 μl of water. Under these conditions, an activity of 15.6 UDO/miniml of enzymatic extract was detected, against 0 UDO/min/ml for the control. The pADH plasmid gene adh is therefore expressed and its product, the alcohol dehydrogenase, is active.
4 μg of each vector were precipitated in the presence of one tenth volume of sodium acetate 3 M pH 6.0, and two volumes of absolute alcohol. The precipitates were rinsed with 70% ethanol in order to eliminate any trace of salts. Each precipitated DNA was resuspended in 4 μl of buffer T.
This DNA is incubated two hours at 37° C. in a protein expression mixture in vitro containing 0.1 mM of each one of the 20 amino acids, 20 μl of "S30 Premix" extract and 15 μl of "T7 S30 extract" (PROMEGA) in a final volume of 50 μl. The "S30 Premix" and "T7 S30 extract" extracts contain all the elements necessary for an in vitro transcription reaction coupled with a translation reaction, notably: T7 RNA polymerase, CTP, UTP, GTP, ATP, tRNAs, EDTA, folic acid and appropriate salts. The translation extract was produced according to the procedure described by Zubay (10) from an Escherichia coli B strain deficient in endoproteinase OmpT and in Ion protease, which better stabilizes the proteins expressed in vitro.
A negative control was prepared by incubating ultra pure sterile water in place of the DNA in the transcription-translation mixture. The positive control was formed by incubating 2 μg of PinPoint™ plasmid.
The EPIV reactions were preserved on ice until the enzymatic activity of each sample could be evaluated.
1) Mesophilic Enzymes
a) GFP Activity
FIG. 5 shows the tube containing the product of the EPIV reaction with the pGFP vector that was exposed to ultraviolet at about 400 nm, beside the tube containing the control reaction. Only the tube containing the EPIV reaction of the pGFP plasmid emitted a green fluorescent light. The EPIV reaction therefore permits the production of a GFP protein having a fluorescent activity.
b) β-lactamase Activity
The β-lactamase activity was evaluated by following by spectrophotometry the degradation kinetics of nitrocephine, a chromogenic cephalosporin, at 486 nm (5). For this, 5, 10, or 20 μl of the EPIV reaction with the pTEM vector are incubated 2 minutes at 37° C. in a BETA buffer, final volume 1 ml. The average activity of the EPIV reaction with the pTEM vector could be estimated at 8.9 UDO/min/ml of extract, against 0.6 UDO/min/ml of extract with the control EPIV reaction. The protein expression reaction in vitro therefore permitted the synthesis of an active β-lactamase, capable of degrading the nitrocephine in vitro.
The control PinPoint™ vector carries the bla gene under the control of its promoter. However, the β-lactamase activity of the EPIV reaction with this vector was tested and evaluated at 6 UDO/min/ml of extract. It is useful to note that the addition of rifampicine at 1 ng/μl, the RNA polymerase inhibitor of E. coli, does not significantly modify the in vitro expression of the β-lactamase with the PinPoint™ vector. The bla gene of the PinPoint™ vector, located 2123 bp downstream of the T7 promoter is therefore transcribed and translated effectively. This implies that a gene located 2000 bp downstream of a T7 promoter is effectively transcribed and translated during an EPIV reaction.
2) Thermophilic Enzymes
a) Intein 2 Activity
The EPIV reaction with the pET26-Tfu2 vector was tested in order to know if an active intein 2 could be produced. For this, 5 μl of this EPIV reaction were incubated 20 minutes at 70° C. with 220 ng of pHS2-22-21 vector, linearized with Sca 1, and 3 μl of IT2 buffer in a final volume of 30 μl. A negative control was formed by replacing the 5 μl of the EPIV reaction with water. The positive control contained 1 μl of the purified intein 2 fraction produced in vitro diluted to {fraction (1/100)}th. Finally, a specificity control was made by incubating 5 μl of the EPIV reaction not having received any DNA.
After incubation, the four trials underwent a phenol-chloroform extraction and an ethanol precipitation, followed by rinsing with 70% ethanol in order to eliminate salts. Each precipitate was taken up in 10 μl of buffer T, and 8 μl were deposited on an agarose TBE 1% gel. After migration and staining with ethidium bromide, the gel was exposed to ultraviolet in order to analyze the restriction profiles. The appearance of bands at 934 and 1752 bp on track 5 of FIG. 6, identical to the bands of track 4 (positive control) reveals that the intein 2 is indeed produced by the EPIV reaction, and that this enzyme is active. In addition, the EPIV reaction is specific since no other digestion band can be observed on the control of track 3.
b) Alcohol Dehydrogenase Activity
Using spectrophotometry, the alcohol dehydrogenase activity was tested by following the reduction kinetics from NADP to NADPH at 340 nm. For this, 5, 10, or 15 μl of the EPIV reaction with the vector pADH, or without DNA, were incubated at 50° C. for five minutes with 500 μl of TADH buffer in a final volume of 1 ml. Under these conditions, the average activity of the EPIV reaction with the pADH vector was estimated at 2.3 UDO/min/ml of extract, against 0.32 UDO/min/ml of extract for the control (EPIV reaction with water). The ADH of Thermococcus hydrothermalis (accession number Y14015 in Gene library) was therefore produced in an active form during the EPIV reaction.
3) Psychrophyle Enzyme
The lipase activity was tested by following, the spectrophotometric degradation kinetics of a chromogenic lipid (1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufin ester) at 572 nm. For this, 5 μl of the EPIV reaction with the PLIP vector, or without DNA were incubated at 70° C. for fifteen minutes in the reaction buffer in the presence of 100 μg of substrate. Under these conditions, the activity of the EPIV reaction with the pLIP vector was estimated at 0.50 UDO/min/ml of extract against 0.04 UDO/min/ml of extract for the control. The lipase B of Candida Antarctica (Eukaryotic organism) was therefore produced in active form during the EPIV reaction.
The beta-galactosidase gene of Thermotoga neapolitana was inserted in a vector containing the T7 RNA polymerase transcription promoter. The vector thus obtained was used for carrying out an EPIV reaction with a translation extract of an E. coli strain possessing a beta-galactosidase activity. In parallel, an EPIV reaction without DNA was carried out. By incubating a fraction of each one of the EPIV reactions at 37° C. in the presence of Xgal at 37° C. in a sodium phosphate buffer (50 mM, pH 7), the two tubes changed to a blue color in minutes (cf FIG. 7 tubes A and B). By incubating a fraction of each one of the EPIV reactions in the presence of Xgal under the same conditions but at 70° C., only the tube corresponding to the EPIV reaction with the plasmid coding for the thermophilic beta-galactosidase changed to the blue color pellet (cf FIG. 7 tubes C and D: these two tubes were centrifuged in order to color the proteins of the mesophilic translation extract which precipitated as a consequence of their thermic denaturation during the activity test at 70° C.). The mesophilic beta-galactosidase activity is no longer detectable at this temperature (thermal denaturation of the mesophilic beta-galactosidase). It is therefore possible to use a translation extract containing a property similar to the sought-after property if this property is not detectable under the detection conditions of the sought-after property.
Translation extracts of other organisms and in particular of extremophilic organisms can be prepared starting from cells according to one of the procedures described by Zubay (1973) (10) or by Pratt (1984) (7). The speeds of centrifugation, the conditions of cell breakage and the different reaction or preparation buffers will be adjusted for each type of translation extract by systematic trials. By thus practicing the range of translation extracts, it becomes possible to translate a gene whatever be its genetic origin: the process of the invention therefore permits having a correspondence between the translation system and the punctuation of expression of the gene coding for the sought-after property.
1) Hanahan D., (1985), Techniques for transformation of Excherichia coli, in DNA cloning: a practical approach. Glover D. M. (Ed.), IRL Press, Oxford vol. I, 109-135.
2) Maniatis T., Fristch E. F. and Sambrook J., (1982). Molecular cloning. A laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
3) Masson J. M., Meuric P., Grunstein M., Abelson J. and Miller H. J., (1987), Expression of a set of synthetic suppressor tRNAPhe genes in Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. UAS, 84, 6815-6819.
4) Normanly J., Masson J. M., Kleina L, G., Abelson J. and Miller J. H., 1986, Construction of two Escherichia coli amber suppressor genes: tRNAPheCUA and tRNACysCUA, Proc, Natl. Acad. Sci. USA, 83, 6548-6552.
5) O'Callaghan C. H., Morris A., Kirby S. M. and Shingler A. H., 1972, Novel methel for detection of beta-lactamase by using a chromogenic cephalosporin substrate, Antimicrob. Agents Chemother., 1. 283-288.
6) Prasher D. C.. Eckenrode V. K., Ward W. W., Prendergast F. G. and Cormier M. J., 1992 Primary structure of the Aequorea Victoria green-fluorescent protein, Gene, 111, 229-233.
7) Pratt J. M., 1984, Coupled transcription-translation in prokaryotic cell-free systems, Transcription and Translation: A practical approach, Hames B. D. and Higgins S. J. Eds, JRL Press.
8) Studier F. W. and Moffatt B. A., 1986, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol, 189, 113-130.
9) Sutcliffe J. G., 1978, Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322, Proc. Natl. Acad. Sci. USA, 75. 3737-3741.
10) Zubay G., 1973, In vitro synthesis of protein in microdial systems. Ann. Rev. Geneti., 7, 267-287.
|Cited Patent
|Filing date
|Publication date
|May 8, 1991
|Nov 5, 1996
|The University Of Virginia Patents Foundation
|Method for the cell-free synthesis of proteins
|May 11, 1994
|Jul 1, 1997
|Trustees Of Boston University
|Methods for the detection and isolation of proteins
|Sep 24, 1996
|Mar 14, 2000
|Selective Genetics, Inc.
|Compositions containing nucleic acids and ligands for therapeutic treatment
|Aug 25, 1999
|Oct 16, 2001
|Amber Gen. Inc.
|N-terminal and C-terminal markers in nascent proteins
|Jun 17, 1985
|Oct 9, 1986
|Marc Ballivet
|Method for obtaining dna, rna, peptides, polypeptides or proteins by means of a dna recombinant technique
|Apr 8, 1994
|Oct 27, 1994
|Research Development Foundation
|Cell free system for protein synthesis and use of chaperone proteins therein
|Apr 10, 1996
|Oct 17, 1996
|Trustees Of Boston University
|Solid phase sequencing of biopolymers
|Apr 29, 1998
|Nov 5, 1998
|Appligene-Oncor (S.A.)
|Archaebacterium intein of the thermococcus fumicolans species
|Dec 21, 1998
|Jul 1, 1999
|The Carnegie Institution Of Washington
|Genetic inhibition by double-stranded rna
|May 5, 1999
|Nov 11, 1999
|Gene Logic, Inc.
|A process to study changes in gene expression in t lymphocytes
|Bowers et al., "Isolation Sequencing and Expression of the Gene Encoding a Major Protein from the Backteriophage Associated with Bartonella Henselae", Gene, vol. 206, pp. 49-52; (1998).
|Craig et al., "Plasmid cDNA-Directed Protein Synthesis in a Coupled Eukaryotic In Vitro Transcription-Translation System", Nucleic Acids Research, vol. 20, No. 19; pp. 4987-4995; (1992).
|Fincham, J.R.S., "Genes as DNA Sequences", Genetics, pp. 385-398; (1983).
|Kruger et al., "Characterization of the mcrBC region of Escherichia coli K-12 ild-type and mutant strains", Gene, (1992), vol. 114, pp. 1-12.
|Mackow, E.R., et al., "DNA Amplification-Restricted Transcription-Translation: Rapid Analysis of Rhesus Rotavirus Neutralization Sites", Proc. Natl. Acad. Sci. USA; vol. 87; pp. 518-522; (1990).
|Negishi et al., "Identitfication and cDNA cloning of single-stranded DNA binding proteins that interact with the region upstream of the human c-myc gene," 1994, Oncogene, vol. 9, pp. 1133-1143.*
|Niehaus et al., "Cloning and Characterisation of a Thermostable alpha-DNA Polymerase From the Hyperthermophilic Archaeon Thermococus sp. TY", Gene, vol. 204, pp. 153-158; (1997).
|Niehaus et al., "Cloning and Characterisation of a Thermostable α-DNA Polymerase From the Hyperthermophilic Archaeon Thermococus sp. TY", Gene, vol. 204, pp. 153-158; (1997).
|Ohuchi et al., "In Vitro Method for the Generation of Protein Libraries Using PCR Amplification of a Single DNA Molecule and Coupled Transcription/Translation", Nucleic Acids Research, vol. 26, No. 19, pp. 4339-4346; (1998).
|Pratt, J.M., "Coupled Transcription-Translation in Prokaryotic Cell-Free Systems", IRL Press, Oxford XP002102865, pp. 179-209 (Chapter 7); (1992).
|Promega Corp.: "Promega: 1997 Catalog", Promega Corp., NSDOCID XP002102864, pp, 2, 117-136 (1997).
|Southworth, M.W et al., "Control of Protein Splicing by Intein Fragment Reassemby", The EMBO Journal, vol. 17, pp. 918-926;(1998).
|Southworth, M.W. et al., "Cloning of Thermostable DNA Polymerases from Hyperthermophilic Marine Archaea with Emphasis on Thermococcus sp.9°N-7 and Mutations Affecting 3'-5' Exonuclease Activity", Proc. Natl. Acad. Sci. USA, vol. 93, pp. 5281-5285; (1996).
|Southworth, M.W. et al., "Cloning of Thermostable DNA Polymerases from Hyperthermophilic Marine Archaea with Emphasis on Thermococcus sp.9°N-7 and Mutations Affecting 3′-5′ Exonuclease Activity", Proc. Natl. Acad. Sci. USA, vol. 93, pp. 5281-5285; (1996).
|Stratagene: "Stratagene: 1995 Catalog", Stratagene USA, NSDOCID XP002102863, pp. 284-285 (1995).
|Citing Patent
|Filing date
|Publication date
|Jun 23, 2011
|Abbott Laboratories
|Sorf constructs and multiple gene expression
|U.S. Classification
|International Classification
|Cooperative Classification
|European Classification
|May 14, 2001
Owner name: PROTEUS, FRANCE
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text: Home > Metro Manila Doctors, staff of QC hospitals hold mass walkout ABS-CBN News Posted at Oct 25 2012 03:12 PM | Updated as of Oct 25 2012 11:15 PM Share Facebook Twitter GPlus LinkedIn Pinterest MANILA, Philippines – The doctors and staff of three major public hospitals in Quezon City held a lunch time mass walkout to protest the proposed privatization of 26 public hospitals in the country. Doctors and staff of East Avenue Medical Center, Philippine Heart Center and Philippine Orthopedic Center are opposing the two bills in both chambers of Congress seeking to privatize 26 government-owned and -controlled hospitals. The doctors joined militant groups in a lunch break protest. They said privatizing the hospitals would deprive poor people of affordable medical services as it would raise fees. The protesters were referring to House Bill 6069 by Rep. Anthony Rolando T. Golez and Senate Bill 3130 by Sen. Franklin Drilon. – report from Johnson Manabat, dzMM Share Facebook Share on Twitter GPlus LinkedIn Pinterest Read More: Philippine Heart Center East Avenue Medical Center Philippine Orthopedic Center hospital privatization /video/news/08/29/16/duterte-itatabi-muna-ang-desisyon-ng-arbitral-tribunal/video/entertainment/08/29/16/nicholas-hoult-ng-x-men-nag-enjoy-sa-pilipinas/video/news/08/29/16/8-miyembro-ng-maute-group-nakatakas/video/news/08/29/16/andanar-hinamon-ang-international-media/entertainment/08/29/16/till-i-met-you-premiere-sets-twitter-on-fire

subset: commoncrawl


text:
- Black Catholics gather to prepare for upcoming national congress
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Not since the aftermath of 9/11 has America's national security and foreign policy seemed to resonate so heavily with the electorate during a U.S. presidential election, according to several political scientists.Read More


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Camp Latgawa Is The Cheese To My Macaroni! by Camp Latgawa - 12 months ago
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Camp HAWC by Camp HAWC - about 1 year ago
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2005 SDMO J125UC-Diesel-Generator Equipment in Sylvania, OH
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When I went to Winooski to vote this morning, I stood in line near my neighbor, Nari Penson, an Obama supporter.
Nari got to the polls around 6:45 a.m. She was #9 in line. She took the day off work to vote, and then to drive voters to the polls.
"I always try to make sure I vote early on election day," she told me.
Nari's family is from Georgia; her parents live in Athens. She explained that years ago, when her grandmother went to vote in Fayette County, Georgia, she was not allowed into the building because she was black. "She stood on a box to vote through a window," Nari explained. "It was really humiliating, but she did it."
Nari's father, who is now 90 years old, had a similarly difficult experience trying to vote in Macon County, Alabama. "He had to get a federal judge to register him because the Macon County officials wouldn't do it," Nari said.
Both her parents called her repeatedly in the last few weeks to urge her to vote early, but she said she likes to wait until election day. It's exciting, she said. Nari is an elementary school teacher at a private school, and she's been encouraging people at school to vote today. "They'll be able to tell their children and grandchildren stories about how they were there for this historic election," she said.
Nari, who is 54, said she always thinks about her family's experiences when she votes. She was proud to be voting for an African-American candidate for president. "Today," she said, "this feels really special."
Terence Campbell: on 10/31/2010. I called 911.The dispatcher sent the ambulance to the wrong TOWN! The responding EMS was so…
jhilt123: Where's the outrage toward the state's negligence regulating the agricultural runoff in VT?
Rita Ann: Homelessness can happen to anyone . Including "UglyTown". This Shelburne road is filled with car dealerships, restaurants, and…
Chrystal Ann: Come on, who dumps hundreds of thousands on friends with no promise of repayment. You were scammed. Maybe…


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Verified ID
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Reviewed7 Reviews
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About Me
- Languages
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- Genna's Airbnb Symbol
Hey, I'm Genna!
San Diego, California, United States · Member since July 2011
I grew up in a San Diego beach city and after going to university in Berkeley and San Francisco I have moved to the downtown of San Diego. I work as a speech-language pathologist in nursing homes. I love travel, good food, music, podcasts, design, yoga, and gardening. I am a social introvert and need my alone time to recharge but absolutely love meeting new people and spending time in groups.
I have used Airbnb often in the past for travel, and I started hosting Couchsurfers this past year - I love this armchair travel! Now time for my own...


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Monday Morning Lyrics by A Day At The Fair
Monday Morning by A Day At The Fair
Long before my stomping grounds got trampled on
I sat and felt the greatest song
That every painter - every poet couldn't create.
And words they opened doors
From what my parents had wished for
When the had a child and raised a kid
That I came to this.
And How good does life feel in times like this?
And How good is my shot before I close my eyes and miss?
These feelings exist.
Let it rain on monday morning
Right before the world is awake
I will ly there and just think about the weather.
Let my blood beat from my chest
And put my veins up to it's test
I will breathe in and know what it feels to feel alive.
I'm alive.
About the time our tree house built fell on the lawn
We sat and heard the first of songs
That every rocking chair and shoe box would create.
It's a world that's grown to be so careless with it's memories.
Only benevolence can capture what I mean.
But how good is this picture when the background's gone?
When I still feel great about standing tall when everything went wrong,
And I am all alone.
Let it rain on monday morning
Right before the world is awake
I will ly there and just think about the weather.
Let my blood beat from my chest
And put my veins up to it's test
I will breathe in and know what it feels to feel alive.
I'm alive.
Let it rain on my rooftop
So I can hear the sounds,
Of passing winds through blowing tree's
That say "I'll see you around."
The seasons can say things that I never can.
These words describe nothing,
When I come home again.
Well I guess I must have lost it,
In a line of my luck.
It said "this is you're life now, and you're done with growing up."
Well I missed my mark,
And I miss those tree's,
And I miss lying in bed tonight to picture these things.
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Los Angeles, CA
Comedy Central Certified
Cort McCown
Cort McCown is an American actor and comedian.
McCown debuted on the 1985 hit film Teen Wolf with a role as one of Michael J. Fox's basketball teammates. He followed that with a supporting role as Quint on the 1987 hit teen film Can't Buy Me Love. In 1989 he had roles in the films Beverly Hills Brats and One Man Force. And 1992 comedy/horror film Auntie Lee's Meat Pies, with Pat Morita.
McCown has also appeared on television shows like Hunter, JAG, Beverly Hills, 90210, and on the soap opera's Passions and The Young and Restless. In 2010, he returned to TV with a role on the FX sitcom The League. He recently completed a Feature Film with the Sundance Award
Winning Barnes Brothers.
In 2001 McCown began touring as a Stand Up Comedian, opening for acts like Andrew Dice Clay and Harland Williams. He is a regular at Los Angeles clubs The World Famous Comedy Store and Laugh Factory.
In 2003 McCown created The Playboy Comedy Tour that was at The Palms Hotel in Las Vegas for 9 years, winning "Best of Las Vegas" several times. He was also the Morning Show host for Playboy Radio when it debuted on Sirius Satellite Radio.
McCown was recently featured in Vince Vaughn's Wild West Comedy Festival in Nashville.
In 2014 McCown performed at the prestigious Edinburgh Fringe Festival in the show American…ish, and The Amsterdam International Comedy Festival, as well as a Tour in England.
Currently McCown has a Podcast with fellow Comedian Joe Bartnick titled "Insensitivity Training" on All Things Comedy Network. He can also be seen touring clubs across the country performing Standup Comedy.
After this Cort began performing Stand-up Comedy at clubs across the nation. Cort is a paid regular at "The World Famous Comedy Store" on The Sunset Strip. Cort is also the Co-Creator of "The Playboy Comedy Tour" at The Palms Casino in Las Vegas. The show was twice voted "Best Comedy Club" in Las Vegas.
Cort was recently on SIRIUS SATELLITE RADIO where he Co-Hosted "THE PLAYBOY MORNING SHOW" for PLAYBOY RADIO CHANNEL 198. He was also featured on "The Girls Next Door" when Hef came to Vegas to see "Playboy Comedy".
Cort McCown's Upcoming Events
Cort McCown's Videos
Upcoming Shows
- Thu 8.25.16 - 10:00 PM At The Lab: The Late Night Show with Stuart Thompson & Luke Schwartz featuring Brent Morin, Jerrod Carmichael, Tommy Johnagin, Jessica Michelle Singleton, Caleb Synan & The Chroma Keys Buy Tickets
- Thu 8.25.16 - 10:00 PM Running Lines with Lenoci featuring Bobby Lee, Jimmy Carr, Adam Ray, Amir K, & Tiffany Haddish Buy Tickets
- Fri 8.26.16 - 8:00 PM At The Lab: What Now? with Bret Ernst, Emily Heller, Chris Redd, Brad Silnutzer, Matt Donaher, Noah Findling & Conner McNutt Buy Tickets
- Fri 8.26.16 - 8:00 PM Owen Benjamin & Friends with Jimmy Carr, Kevin Hickerson, Skyler Stone & more! Buy Tickets
- Fri 8.26.16 - 10:00 PM At the Lab: Fun with (Andy) Dick Buy Tickets
- Fri 8.26.16 - 10:00 PM Tonight at the Improv for the Leukemia & Lymphoma Society with Adam Devine, Iliza Shlesinger, Greg Fitzsimmons, Wayne Federman, Tommy Johnagin, Kyle Dunnigan, Greg Behrendt, & Josh Fadem! Buy Tickets
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- {{matchup.date.fullday}}{{matchup.date.month + ' ' + matchup.date.day}}
Tampa Bay hammers Kansas City
The Tampa Bay Devil Rays scored early and often Sunday afternoon earning a 9-5 win over the Kansas City Royals at Tropicana Field.
The Devil Rays were -115 favorites at home in the game. The contests total runs were OVER the posted total of 10.
Ty Wigginton, Damon Hollins and Jonny Gomes all homered for the Devil Rays in the victory. Tampa Bay had 14 total hits in the game.
Shane Costa hit his third homer of the year for the Royals in a losing effort.


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|By Marketwired .
Article Rating:
|April 22, 2014 10:18 AM EDT
MELVILLE, NY -- (Marketwired) -- 04/22/14 -- Secure Infrastructure & Services (SIaS), a managed services provider to businesses using or considering moving to a cloud-based model, announced today that it has expanded its operations by selecting Xand, a premier facilities-based provider of data center managed services and technology solutions, as one of its new data center locations.
Xand's Westchester County, N.Y. data center is a 50,000 square-foot facility that was custom built with the latest in redundant power, environmental control and networking technology. SIaS recently expanded its computing infrastructure in the newest build within the facility enabling its clients to benefit from its highly scalable infrastructure, rock-solid technology and the confidence in the availability of their data and systems.
"It is essential that we provide our clients with the highest performing and most reliable computing infrastructure possible and partnering with Xand to leverage their state-of-the-art facility is a key component to serving our clients," said Thomas Kempster, VP of Services and Technologies at SIaS. "In addition to their advanced technologies, we also chose Xand because of their close proximity to our offices giving us the ability to periodically meet with them face-to-face, their responsiveness, and the lack of red tape often associated with other data center facilities."
"As a managed services provider hosting mission-critical systems and applications for companies of all sizes, Secure Infrastructure & Services requires top-level security, performance and availability to meet its clients' needs," said Denoid Tucker, vice president of Technology at Xand. "Our hybrid cloud and managed data center solutions help Secure Infrastructure & Services minimize risk and maximize operational efficiencies with tailored solutions for unique and continually evolving technology requirements."
About Xand
Xand is the Northeast's premier provider of highly resilient cloud, managed services, colocation, and disaster recovery. Since 1999, Xand has designed, built, and managed IT infrastructure services which allow enterprises to maximize their critical application performance and availability. With data centers in New York, Pennsylvania, Connecticut, and Massachusetts, Xand offers colocation, cloud, enterprise hosting, managed services, business continuity, disaster recovery and managed networking throughout the region via best-in-class facilities, engineering expertise, and commitment to customer service excellence. For more information, please visit http://www.xand.com.
Secure Infrastructure & Services
Secure Infrastructure & Services (SIaS) is a managed services provider to businesses using or considering moving to a cloud-based model. The company's core offerings include: SecureHOST Infrastructure-as-a-Service (IaaS) for production and test workloads; SecureHA fully managed replication and availability services; and SecureDR virtualized recovery services. As a result of its expert technical staff, secure and robust infrastructure of networks and data centers and its streamlined processes it is able to deliver its services at a lower cost for its clients. As an IBM MSP Business Partner, SIaS provides its services for IBM AIX and OS/400 and other industry-leading platforms, such as: Microsoft Windows. The company is headquartered in Melville, N.Y. and can be located on the web at www.siasmsp.com.
Media Contact:
Karen Higgins
A&E Communications, Inc.
Tel: 610-831-5723
Email Contact
SYS-CON Events announced today that 910Telecom will exhibit at the 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Housed in the classic Denver Gas & Electric Building, 910 15th St., 910Telecom is a carrier-neutral telecom hotel located in the heart of Denver. Adjacent to CenturyLink, AT&T, and Denver Main, 910Telecom offers connectivity to all major carriers, Internet service providers, Internet backbones and ...
Aug. 31, 2016 09:00 PM EDT Reads: 2,076
SYS-CON Events announced today that Numerex Corp, a leading provider of managed enterprise solutions enabling the Internet of Things (IoT), will exhibit at the 19th International Cloud Expo | @ThingsExpo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Numerex Corp. (NASDAQ:NMRX) is a leading provider of managed enterprise solutions enabling the Internet of Things (IoT). The Company's solutions produce new revenue streams or create operating...
Aug. 31, 2016 08:15 PM EDT Reads: 352
The Internet of Things can drive efficiency for airlines and airports. In their session at @ThingsExpo, Shyam Varan Nath, Principal Architect with GE, and Sudip Majumder, senior director of development at Oracle, will discuss the technical details of the connected airline baggage and related social media solutions. These IoT applications will enhance travelers' journey experience and drive efficiency for the airlines and the airports. The session will include a working demo and a technical d...
Aug. 31, 2016 07:00 PM EDT Reads: 303
Although it has gained significant traction in the consumer space, IoT is still in the early stages of adoption in enterprises environments. However, many companies are working on initiatives like Industry 4.0 that includes IoT as one of the key disruptive technologies expected to reshape businesses of tomorrow. The key challenges will be availability, robustness and reliability of networks that connect devices in a business environment. Software Defined Wide Area Network (SD-WAN) is expected to...
Aug. 31, 2016 06:15 PM EDT Reads: 337
Developing software for the Internet of Things (IoT) comes with its own set of challenges. Security, privacy, and unified standards are a few key issues. In addition, each IoT product is comprised of (at least) three separate application components: the software embedded in the device, the back-end service, and the mobile application for the end user's controls. Each component is developed by a different team, using different technologies and practices, and deployed to a different stack/target –...
Aug. 31, 2016 05:15 PM EDT Reads: 327
Internet of @ThingsExpo, taking place November 1-3, 2016, at the Santa Clara Convention Center in Santa Clara, CA, is co-located with 19th Cloud Expo and will feature technical sessions from a rock star conference faculty and the leading industry players in the world. The Internet of Things (IoT) is the most profound change in personal and enterprise IT since the creation of the Worldwide Web more than 20 years ago. All major researchers estimate there will be tens of billions devices - comp...
Aug. 31, 2016 04:45 PM EDT Reads: 3,861
As cloud adoption continues to transform business, today's global enterprises are challenged with managing a growing amount of information living outside of the data center. The rapid adoption of IoT and increasingly mobile workforce are exacerbating the problem. Ensuring secure data sharing and efficient backup poses capacity and bandwidth considerations as well as policy and regulatory compliance issues.
Aug. 31, 2016 03:15 PM EDT Reads: 389
Why do your mobile transformations need to happen today? Mobile is the strategy that enterprise transformation centers on to drive customer engagement. In his general session at @ThingsExpo, Roger Woods, Director, Mobile Product & Strategy – Adobe Marketing Cloud, covered key IoT and mobile trends that are forcing mobile transformation, key components of a solid mobile strategy and explored how brands are effectively driving mobile change throughout the enterprise.
Aug. 31, 2016 03:00 PM EDT Reads: 814
Data is the fuel that drives the machine learning algorithmic engines and ultimately provides the business value. In his session at Cloud Expo, Ed Featherston, a director and senior enterprise architect at Collaborative Consulting, will discuss the key considerations around quality, volume, timeliness, and pedigree that must be dealt with in order to properly fuel that engine.
Aug. 31, 2016 02:30 PM EDT Reads: 2,105
19th Cloud Expo, taking place November 1-3, 2016, at the Santa Clara Convention Center in Santa Clara, CA, will feature technical sessions from a rock star conference faculty and the leading industry players in the world. Cloud computing is now being embraced by a majority of enterprises of all sizes. Yesterday's debate about public vs. private has transformed into the reality of hybrid cloud: a recent survey shows that 74% of enterprises have a hybrid cloud strategy. Meanwhile, 94% of enterpri...
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Data is an unusual currency; it is not restricted by the same transactional limitations as money or people. In fact, the more that you leverage your data across multiple business use cases, the more valuable it becomes to the organization. And the same can be said about the organization's analytics. In his session at 19th Cloud Expo, Bill Schmarzo, CTO for the Big Data Practice at EMC, will introduce a methodology for capturing, enriching and sharing data (and analytics) across the organizati...
Aug. 31, 2016 11:00 AM EDT Reads: 518
SYS-CON Events announced today that Pulzze Systems will exhibit at the 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Pulzze Systems, Inc. provides infrastructure products for the Internet of Things to enable any connected device and system to carry out matched operations without programming. For more information, visit http://www.pulzzesystems.com.
Aug. 31, 2016 10:45 AM EDT Reads: 499
SYS-CON Events announced today Telecom Reseller has been named "Media Sponsor" of SYS-CON's 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Telecom Reseller reports on Unified Communications, UCaaS, BPaaS for enterprise and SMBs. They report extensively on both customer premises based solutions such as IP-PBX as well as cloud based and hosted platforms.
Aug. 31, 2016 09:30 AM EDT Reads: 1,148
Almost two-thirds of companies either have or soon will have IoT as the backbone of their business in 2016. However, IoT is far more complex than most firms expected. How can you not get trapped in the pitfalls? In his session at @ThingsExpo, Tony Shan, a renowned visionary and thought leader, will introduce a holistic method of IoTification, which is the process of IoTifying the existing technology and business models to adopt and leverage IoT. He will drill down to the components in this fra...
Aug. 31, 2016 09:00 AM EDT Reads: 610
Pulzze Systems was happy to participate in such a premier event and thankful to be receiving the winning investment and global network support from G-Startup Worldwide. It is an exciting time for Pulzze to showcase the effectiveness of innovative technologies and enable them to make the world smarter and better. The reputable contest is held to identify promising startups around the globe that are assured to change the world through their innovative products and disruptive technologies. There w...
Aug. 31, 2016 08:45 AM EDT Reads: 949
There is growing need for data-driven applications and the need for digital platforms to build these apps. In his session at 19th Cloud Expo, Muddu Sudhakar, VP and GM of Security & IoT at Splunk, will cover different PaaS solutions and Big Data platforms that are available to build applications. In addition, AI and machine learning are creating new requirements that developers need in the building of next-gen apps. The next-generation digital platforms have some of the past platform needs a...
Aug. 31, 2016 07:45 AM EDT Reads: 990
With so much going on in this space you could be forgiven for thinking you were always working with yesterday's technologies. So much change, so quickly. What do you do if you have to build a solution from the ground up that is expected to live in the field for at least 5-10 years? This is the challenge we faced when we looked to refresh our existing 10-year-old custom hardware stack to measure the fullness of trash cans and compactors.
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The emerging Internet of Everything creates tremendous new opportunities for customer engagement and business model innovation. However, enterprises must overcome a number of critical challenges to bring these new solutions to market. In his session at @ThingsExpo, Michael Martin, CTO/CIO at nfrastructure, outlined these key challenges and recommended approaches for overcoming them to achieve speed and agility in the design, development and implementation of Internet of Everything solutions wi...
Aug. 31, 2016 02:15 AM EDT Reads: 2,320
Today we can collect lots and lots of performance data. We build beautiful dashboards and even have fancy query languages to access and transform the data. Still performance data is a secret language only a couple of people understand. The more business becomes digital the more stakeholders are interested in this data including how it relates to business. Some of these people have never used a monitoring tool before. They have a question on their mind like "How is my application doing" but no id...
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Cloud computing is being adopted in one form or another by 94% of enterprises today. Tens of billions of new devices are being connected to The Internet of Things. And Big Data is driving this bus. An exponential increase is expected in the amount of information being processed, managed, analyzed, and acted upon by enterprise IT. This amazing is not part of some distant future - it is happening today. One report shows a 650% increase in enterprise data by 2020. Other estimates are even higher....
Aug. 31, 2016 01:45 AM EDT Reads: 3,146


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Out Of Love lyricsRunaway lyricsPtolemy lyricsPlease Don't Leave Me lyricsShe Knows lyricsCry For Love lyricsI Need An Angel lyricsShouldn't Have Let You Go lyricsWe All Fall Down lyricsSave My Love lyricsSex Child lyricsItchycoo Park lyricsLove Child lyricsJelly Roll lyricsRiot lyricsValley Of The Kings lyricsBlack-hearted Woman lyricsBilly lyricsBlue Murder lyricsCold Sweat lyricsDance lyricsDancin' In The Moonlight lyricsStill Of The Night lyricsI'm On Fire lyrics
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Related Information for Blue Murder There is more than one artist with this name:1) British hardrock band2) Dutch guitarpop band3) British folk group 1) Blue Murder was the hard rock brainchild of ex-Whitesnake and Thin Lizzy guitarist John Sykes. The original 1988 lineup of the band, also included ex-The Firm bass player Tony Franklin and drummer Cozy Powell (although he was almost immediately replaced by Carmine Appice). Ray Gillen was also briefly in the 1988 line-up before Sykes decided to take the vocal duties himself.. read more