The Escherichia coli dnaK gene product, originally defined by mutations that blocked lambda phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ ge...

DNA cloning is often used to select and amplify one DNA species from a mixture. However, the cloning process is complex and labor-intensive. We have developed a new two-step method for DNA sequencing directly from a mixture. The first is the introduction of a known oligonucleotide (common part) into the terminus of unknown DNA by ligation. The second is selective DNA sequencing using primers wi...

Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-bas...

The gene TOP2 encoding yeast topoisomerase II has been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector, lambda gt11. The ends of the message encoded by the cloned DNA fragment were delimited by the Berk and Sharp procedure (S1 nuclease mapping) for the 5' end and mapping of the polyA tail portion of a cDNA fragment for the 3' end. T...

We describe in this paper the isolation and characterization of a class of mutations, designated puq, that allow phage lambda to grow better under conditions that limit the synthesis of the phage Q gene product. These mutations were located between phage genes P and Q, a region of the lambda chromosome containing two gene N-independent mutations, nin5 and byp, that we also show to be puq mutati...

Holliday junctions are a central intermediate in diverse pathways of DNA repair and recombination. The isomerization of a junction determines the directionality of the recombination event. Previous studies have shown that the identity of the central sequence of the junction may favor one of the two isomers, in turn controlling the direction of the pathway. Here we demonstrate that, in the absen...

The Watson-Crick model for DNA duplex duplication proposes that the two parental chains separate and that each directs the synthesis of a complementary chain with which it is found associated after the duplication act. Previous experiments have left unchallenged alternative models which propose that in any single act of duplication only one of the two parental chains provides information for th...

Recombinant bacteriophage lambda from a human genomic library were screened to indentify human DNA inserts having only unique sequences. Unique human inserts were found in about 1% of the phage screened. One recombinant phage, P3-2, was studied in detail. It contains a human insert of 14.7 kilobases with four internal EcoRI cleavage sites. A restriction map was constructed for EcoRI and BamHI s...

The construction in bacteriophage lambda of a set of long DNA palindromes with paired changes in the central sequence is described. Identical palindrome centers were previously used by others to test the S-type model for cruciform extrusion in vitro. Long DNA palindromes prevent the propagation of carrier phage lambda on a wild-type host, and the sbcC mutation is sufficient to almost fully alle...