Abstract: A crystal structure of a Non-LTR-retroelement reverse transcriptase and methods of using the same to identify enzymes with improved activity are provided. Mutant reverse transcriptase enzymes and methods of using the same are also provided.

Abstract: A crystal structure of a Non-LTR-retroelement reverse transcriptase and methods of using the same to identify enzymes with improved activity are provided. Mutant reverse transcriptase enzymes and methods of using the same are also provided.

Abstract: Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Abstract: A crystal structure of a Non-LTR-retroelement reverse transcriptase and methods of using the same to identify enzymes with improved activity are provided. Mutant reverse transcriptase enzymes and methods of using the same are also provided.

Abstract: Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse temperature. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Abstract: Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Abstract: Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Abstract: Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Abstract: The present disclosure provides methods and compositions for the integration of a target RNA or DNA into a DNA substrate. Also provided are methods of forming RNA-DNA bonds and enzymes for performing the same.

Abstract: Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse temperature. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Abstract: A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non-retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3? end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

Abstract: A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non-retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3? end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

Abstract: Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Abstract: The present invention provides a system and methods for analyzing the function of nucleotide integrases and modified group II introns. The system comprises a donor plasmid comprising a wild-type or modified group II intron, a recipient plasmid comprising a DNA recognition site and a promoterless reporter gene downstream of the DNA target site, and a host cell. The method comprises the steps of transforming a host cell with the donor and recipient plasmids, assaying for expression of the reporter gene, isolating plasmid DNA from the cotransformed cells, and analyzing the plasmid DNA to confirm that the group II intron has been inserted into the target sequence. The present invention also provides a method for simultaneously analyzing the activity of two or more modified nucleotide integrases. The present invention also relates to methods of preparing a library of donor plasmids containing a plurality of diverse modified group II intron DNA sequences.

Abstract: The present invention provides a system and methods for analyzing the function of nucleotide integrases and modified group II introns. The system comprises a donor plasmid comprising a wild-type or modified group II intron, a recipient plasmid comprising a DNA recognition site and a promoterless reporter gene downstream of the DNA target site, and a host cell. The method comprises the steps of transforming a host cell with the donor and recipient plasmids, assaying for expression of the reporter gene, isolating plasmid DNA from the cotransformed cells, and analyzing the plasmid DNA to confirm that the group II intron has been inserted into the target sequence. The present invention also provides a method for simultaneously analyzing the activity of two or more modified nucleotide integrases. The present invention also relates to methods of preparing a library of donor plasmids containing a plurality of diverse modified group II intron DNA sequences.

Abstract: Methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double-stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate are provided. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing an isolated nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on the top strand of the DNA substrate, and a group I-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate to permit the nucleotide integrase to cleave the top strand of the DNA substrate and to insert the group II intron RNA into the cleavage site.

Abstract: Methods for preparing nucleotide integrases are provided. The nucleotide integrases are prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as "exogenous RNA", with a group II intron-encoded protein. The exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises a group II intron sequence. In one embodiment, the group II intron-encoded protein is made by introducing into a host cell a DNA molecule that comprises at least the open reading frame sequence of a group II intron and then expressing the open reading frame sequence in the host cell. The DNA molecule may comprise the open reading frame sequence operably linked to a promoter, preferably an inducible promoter. Thereafter, the cell is fractionated and the protein is recovered and combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity.

Abstract: The present invention provides new methods, employing a nucleotide integrase, for cleaving double-stranded and single stranded DNA substrates at specific sites and for attaching nucleic acid molecules to the cleaved DNA substrates. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA and to concomitantly attach a nucleic acid molecule to the cleaved strand. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach a nucleic acid molecule to one strand of the DNA substrate. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach an RNA molecule to one strand of the substrate and for attaching a cDNA to the other strand of the substrate. Another method cleaves single stranded DNA with the concomitant insertion of a nucleic acid molecule at the cleavage point.

Abstract: The present invention provides new methods, employing a nucleotide integrase, for cleaving double-stranded and single stranded DNA substrates at specific sites and for attaching nucleic acid molecules to the cleaved DNA substrates. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA and to concomitantly attach a nucleic acid molecule to the cleaved strand. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach a nucleic acid molecule to one strand of the DNA substrate. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach an RNA molecule to one strand of the substrate and for attaching a cDNA to the other strand of the substrate. Another method cleaves single stranded DNA with the concomitant insertion of a nucleic acid molecule at the cleavage point.

Abstract: A functional extrachromosomal element capable of replication in filamentous fungi is provided. The extrachromosomal element employs (1) a mitochondrial replicating element or (2) a lower organism replication sequence recognized by the fungus, in combination with foreign DNA to provide replication, transcription, and translation of foreign regulatory elements and genes. The extrachromosomal element is exemplified by a mitochondrial replicating system from Neurospora.The cell strain E. coli HB101 containing the plasmid pALS-1-1 has been deposited at the A.T.C.C. on July 13, 1982, for patent purposes and given the designation ATCC 39157.The cell strain E. coli HB101 containing the plasmid pALS-2 has been deposited at the A.T.C.C. on July 13, 1982, for patent purposes and given the designation ATCC 39158.