National Institutes of Health, a Component of the US Dept. of Health & Human Services

The United States of America As Represented by the Department of Health

Baylor College of Medicine

Donald L Court

Nina Costantino

Simanti Datta

James R Lupski

E-Chiang Lee

Jian-Dong Huang

Robert Allen Britton

Bradford S Powell

Xin-tian Li

Depei Liu

Daiguan Yu

Hilary M Ellis

Nancy A Jenkins

Neal G Copeland

Costantino Nina

Stanley Brown

Shyam K Sharan

Srividya Swaminathan

Lambda phages that can be used to introduce recombineering functions into host cells are disclosed. Also disclosed are plasmids that can be used to confer recombineering functions to a variety of strains ofand to other bacteria, including. These plasmids and phages can be isolated in vitro and can be used to transform bacterial cells, such as gram negative bacteria.

Plasmids and phages for homologous recombination and methods of use

Methods are disclosed herein for inducing homologous recombination in a host cell comprising a target nucleic acid, using a single-stranded nucleic acid molecule. The single-stranded nucleic acid molecule has a sufficient number of nucleotides homologous to the target nucleic acid to enable homologous recombination with the target nucleic acid. The host cell includes a de-repressible promoter operably linked to a nucleic acid encoding a single-stranded binding protein and is deficient for mismatch repair. Isolated host cells of use in this method are also disclosed.

Host cells deficient for mismatch repair and their use in methods for inducing homologous recombination using single-stranded nucleic acids

Disclosed herein are methods for generating recombinant DNA molecules in cells using homologous recombination mediated by recombinases and similar proteins. The methods promote high efficiency homologous recombination in bacterial cells, and in eukaryotic cells such as mammalian cells. The methods are useful for cloning, the generation of transgenic and knockout animals, and gene replacement. The methods are also useful for subcloning large DNA fragments without the need for restriction enzymes. The methods are also useful for repairing single or multiple base mutations to wild type or creating specific mutations in the genome. Also disclosed are bacterial strains and vectors which are useful for high-efficiency homologous recombination.

Enhanced homologous recombination mediated by lambda recombination proteins

The present invention provides for methods of screening for agents which delay the cell cycle and methods of delaying the cell cycle. Analogues of Era having arginine, histidine, or lysine at amino acid codon 17 are embodied by the present invention. Human and other homologs of bacterial Era amino acid and nucleic acid sequences are provided in the present invention. Vectors, host cells, protein preparations, cell cultures, and compositions comprising said analogue are also set forth in the present invention.

Methods of screening for agents that delay a cell cycle and compositions

This invention provides for improved means to produce binding specific proteins using concatamers of semi-randomly generated oligonucleotides inserted into genes encoding the external domains of bacterial outer membrane proteins. The genes are induced to express and the bacteria are then screened for the ability to bind to predetermined compositions. Those clones carrying the desired binding protein are isolated, cultured and the protein purified. Increased avidity of the binding specific proteins are achieved by reisolating the oligonucleotides which conferred binding affinity and mixing them with new semi-randomly generated oligonucleotides to generate a population enriched for oligonucleotides that had previously conferred to bacteria the desired binding affinity. The enriched population of oligonucleotides is then religated to the gene encoding the external domain of the bacterial protein, the gene is inserted into a bacterial cell and the cells analyzed for increased avidity for the predetermined composition.

Method for indentifying and expressing proteins that recognize and

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Frederick, MD, United States of America

Donald L Court