The coding sequence of this protein was found in Drosophila melanogaster samples of different developmental stages as transcribed into mature messenger RNA by reverse transcription of the total mRNA pool into cDNA followed polymerase chain reaction with adequate primer oligonucleotides. The cDNA coding sequence corresponding to SEQ ID NO:6 was cloned into expression vectors as follows:
It was subcloned into vector pET22b (Novagen, EMD Biosciences, Darmstadt, Germany), using EcoRI-Xhol restriction sites originating in plasmid PETUDE. The coding sequence for a maltose binding protein was excised from plasmid pMal-c2E (New England BioLabs, Schwalbach, Germany) by Ndel-BamHI digestion, and was subcloned into plasmid PETUDE using Ndel-BamHI restriction sites originating in plasmid pETMalUDE. In plasmid pETMalUDE, the coding sequence of uracil-DNA endonuclease is at the 3′-end to the coding sequence of the maltose-binding protein. The nucleic acid segment encoding the uracil-DNA endonuclease coding sequence was also subcloned into vector PET19b (Stratagene), using Ndel-Xhol restriction enzyme sites originating in plasmid pETHisUDE. In pETHisUDE, the coding sequence of uracil-DNA endonuclease is at the 3′-end to the coding sequence of the ten-histidine affinity tag.
The expression vectors pETMalUDE and pETHisUDE were used to transform E. coli cells. Expression of the uracil-DNA nuclease protein with the affinity tag maltose binding protein or polyhistidine was performed by iso-propyl-thio-galactoside induction following general methods known to one skilled in the art. It will be evident to one skilled in the art that similar procedures without the exercise of inventive skill may easily result in expression vectors also containing the coding sequence of uracil-DNA nuclease. either with an affinity tag or without such tag. Such vectors may also be used for expression of the protein uracil-DNA nuclease using methods known in the art. Such equivalents are intended to be encompassed by the following claims.
Due to the known degeneracy of the genetic code, it will be also apparent to those skilled in the art that different, but equivalent nucleotide sequences which code for the uracil-DNA nuclease enzyme of the invention, as shown in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, may be isolated, synthesized or otherwise prepared without the exercise of the inventive skill. Such degenerate and equivalent coding sequences are included within the scope of the present invention.
Uracil-DNA nuclease activity of the recombinant uracil-DNA nuclease protein. The protein was expressed as described above. It was isolated from E. coli cell lysate and purified using the affinity tag with usual methods known in the art. Its activity was tested on uracil-DNA and normal DNA. Uracil-DNA was produced in the form of a plasmid isolated from dut-ung-double mutant E. coli cells. Normal DNA was produced in the form of plasmid from wild type E. coli cells. Plasmid preparations were according to usual methods known in the art. The recombinant protein, purified to approximataly 98% homogeneity, and containing the amino acid sequence constituted in SEQ ID NO:1 was incubated with normal DNA and uracil-DNA at 37.degree.C. for 10, 30, and 60 minutes in a solution containing 10 micrograms/ml uracil-DNA nuclease, 20 micrograms/ml DNA, 25 mmole/liter Hepes buffer, 150 mmole/liter sodium-chloride. Uracil-DNA was fragmented into small oligonucleotides while normal DNA showed practically no fragmentation at all. It was concluded that the protein enzyme uracil-DNA nuclease is strictly specific for uracil-DNA and is capable of fragmenting it into smaller oligonucleotides.
DNA binding affinity of recombinant uracil-DNA nuclease. The DNA binding affinity was tested on gel shift assay using usual methods known in the art. The recombinant uracil-DNA nuclease was shown to bind to uracil-DNA and normal DNA with comparable affinities. It was concluded that uracil-DNA nuclease has a general DNA binding ability which is not strictly specific for uracil-DNA.
Homologues of uracil-DNA nuclease. Homologue sequences of uracil-DNA nuclease from Drosophila melanogaster as contained in the amino acid sequence constituted in SEQ ID NO:1 or as contained in the nucleotide sequence constituted in SEQ ID No:6 were used to search for homologues in the usual databases known in the art. Four such homologues were identified, as contained in the amino acid sequence constituted in SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5, or as contained in the nucleotide sequence constituted in SEQ ID No:7, or SEQ ID No:8, or SEQ ID No:9, or SEQ ID No:10. All these homologues are present in genomes of metamorphing insects. It is concluded that the sequences contained in amino acid sequences constituted in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5. correspond to proteins specific to metamorphing insects and other organisms may only encode such distant relatives that are not evident at the present.
Novelty of the uracil-DNA nuclease enzyme protein. The protein as contained in the amino acid sequence constituted in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5. does not show significant homology to any of the known nucleases. Its active site may therefore constitute novel characteristics, exploitable in molecular biology application. Such an example may be that the enzymatic activity of uracil-DNA nuclease protein which is the subject of the present invention does not require the presence of divalent metal ions. This characteristics makes this enzyme a useful and rather unique tool for any molecular biology, or other applications where divalent metal ion-independent nuclease activity is used. Such applications are intended to be encompassed in the present invention.
Methods of use of uracil-DNA nuclease. The uracil-DNA nuclease of the present invention may be used in any circumstances where specific degradation of uracil-DNA is required. Such applications that require the protein enzyme uracil-DNA nuclease as contained in the amino acid sequence constituted in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5. are intended to be encompassed in the present invention. Due to the equivalent base-pairing capabilities of uracil and thymine, uracil-DNA and normal DNA may encode equivalent genetic information, if uracil is present only at thymine-replacing sites. One example for a useful application of uracil-DNA nuclease concerns biosafety.
Biosafety application of uracil-DNA nuclease. For specific degradation of recombinant DNA encoding potentially not desired, or even harmful, genetic information produced in vitro under laboratory circumstances, the use of uracil-DNA nuclease provides a rather simple and straightforward solution. Recombinant DNA is frequently produced in the laboratory and its escape from the laboratory is not always strictly ensured. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, it will be possible, using general methods known in the art, to produce all recombinant DNA with a significant uracil content. For in vitro production, inclusion of dUTP into the polymerase reactions will ensure the production of uracil-DNA. For cellular experiments in E. coli, the use of dut-ung-mutant E. coli strain will ensure the production of uracil-DNA. These uracil-DNA species will be equivalent in genetic coding to normal DNA of the same sequence where the deoxyuridine residue is replaced by thymine. Recombinant DNA with uracil content will be degradable by uracil-DNA nuclease with high efficiency, while normal DNA will not be degraded. Recombinant DNA will be therefore prevented from escaping the laboratory by the use of uracil-DNA nuclease.
Molecular diagnostics application of uracil-DNA nuclease. To ascertain the presence of a specific mutation in e.g. human or other genome, uracil-DNA nuclease may be applied. Some defined mutations in the human genome are well known to be involved in several pathogenic conditions. Molecular diagnostics techniques are known in the art to recognize such mutations by sequencing. With the use of uracil-DNA nuclease, a simple method with ease of use may be designed that does not require sequencing. In such a method, DNA oligomers hybridizing to the mutated site may be synthesized containing deoxyuridine residues. The oligomers may be labelled at the 5′ and 3′ end, with flurescent dyes capable of flurescence energy transfer. The labelling may be designed in such a way that within the intact oligomer, fluorescence is quenched due to the short distance between the two fluorescent labels as defined by the length of the oligonucleotide. However, fluorescence may be significantly increased if the distance between the two labels increase due to cleavage of the oligonucleotide. Such a deoxyuridine residue-containing double-labelled oligonucletide with its controlled sequence that is 100% complementary to the sequence containing the mutation to be investigated may hybridize to a site if it contains the mutation to be investigated. If the mutation is not present, hybridization will not occur. After the hydridization experiment, the hybrid double stranded DNA may be separated from single stranded DNA by usual methods known in the art. The double stranded DNA may then be treated with uracil-DNA nuclease that will result in cleavage of the uracil-containing oligonucleotide strand and therefore fluorescence intensity will increase. Increment in flurescent intensity, as detected by methods known in the art, will then reflect the existence of the mutation.
Persons skilled in the art will recognize, or will be able to design using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be included in the present invention.
Number | Date | Country | |
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60521645 | Jun 2004 | US |