A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Dna mutagenesis by random fragmentation and reassembly

Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.

Methods and compositions for cellular and metabolic engineering

Everything you have ever wanted to know about Yin Yang 1......

Methods are provided for the evolution of proteins of industrial and pharmaceutical interest, including methods for effecting recombination and selection. Compositions produced by these methods are also disclosed.

Methods and compositions for polypeptide engineering

We attempted to use the polymerase chain reaction (PCR) to monitor in vitro recombination in a plasmid containing directly repeated sequences. Some of the plasmid preparations which had not been exposed to recombination conditions were however found to behave in the PCR test as if they had undergone homologous recombination. We show here that such false positives are attributable to a small degree of nicking and/or breaking of the DNA template. Presumably, such damage allows the formation of hybrid parental duplexes containing at least one truncated strand, the 3' end of which maps within the homology; extension of this 3' end by the polymerase then results in a linkage of sequences identical to that arising from homologous recombination.

DNA nicking favors PCR recombination

Decapsidation of polyoma virus: Identification of subviral species☆

POLYOMA and SV40 DNAs contain enough genetic information to code for half a dozen proteins. Very little is known about these proteins, although their recognition is of great interest in view of the oncogenic potential of the genomes. Recently, however, polyoma DNA from productively infected cells was isolated in the form of a protein–DNA complex distinct from the virus1 (our work in preparation). In an attempt to explore the nature and function of its protein moiety, we have investigated the effect of various inhibitors of protein synthesis on the formation and properties of this complex. We report here the results we obtained with puromycin.

Is a specific protein responsible for the supercoiling of polyoma DNA

Unwinding of replicating polyoma virus DNA

The replication of the ring-shaped polyoma DNA has been investigated. The replicative intermediate has been identified and characterized; it consists of molecules containing both circular and nicked strands, not longer than the strands of the DNA found in viral particles. The findings exclude certain proposed methods of replication of ring-shaped DNA and are compatible with Cairns' model.

Pierre Bourgaux

Papers

DNA nicking favors PCR recombination

Decapsidation of polyoma virus: Identification of subviral species☆

Is a specific protein responsible for the supercoiling of polyoma DNA

Unwinding of replicating polyoma virus DNA

The replication of the ring-shaped dna of polyoma virus, i. identification of the replicative intermediate