ABSTRACr Fragments of phage T7 DNA have been cloned in Escherichia coli by using the plasmid pMB9. Such cloned fragments are able to recombine with infecting phages, thus providing a means to integrate the physical and genetic maps of 17 DNA. Approximately. 65% of the 17 DNA molecule has been found in clones so far, and analysis of these clones has mapp genes 12-17 with an accuracy of about 1% the total length of T7 DNA. At least some cloned segments can supply 17 functions to infecting phages. Molecular cloning of DNA is a powerful tool for analyzing the structure and function of both prokaryotic and eukaryotic DNAs, and for amplifying specific gene products (1). We are applying molecular cloning techniques to the analysis of phage T7 DNA in order to refine the structural and genetic map of T7 DNA and to explore the usefulness of these techniques for studying the biochemical relationships between T7 and its host. T7 DNA is well suited for such analysis because most T7 genes have been identified and mapped genetically, and a wellcharacterized collection of mutants is available (2, 3). MATERIALS AND METHODSPhage and Bacterial Strains. Wild-type T7, T7 mutants, 9Escherichia coli B, suppressing strain E. coli 011' (Su+), and E. colh 011U1 have been described previously (2, 4). The Surecipient for transformations, E. coli HMS174 (rK12 mKl2+ recAl rifR Su-), was derived from the E. coil K-12 strains W31 10 (thy-) and KLL6-99 (5). The Su+ recipient was HB101 (rB-mB-pro-galh strR recAl Su+) (6).Cloning Procedures. Plasmid pmB9 (7) was used as the cloning vehicle. Fragments of T7 DNA were inserted at the single EcoRI cleavage site of pBM9 using poly(dA-dT) connectors (8-11). Fragments of T7 DNA were generated either by cleavage with Hpa I (12) Preparation and Analysis of DNA. T7 DNA was prepared by phenol extraction of purified phage particles. Plasmid DNA was prepared, after amplification in the presence of chloramphenicol, by gentle lysis of the cells with lysozyme and detergent, followed by centrifugation to remove chromosomal DNA (14). Further treatments included phenol extraction, ethanol precipitation, or banding in CsCl gradient containing ethidium bromide at 200 ,g/ml with subsequent removal of the ethidium bromide. Heteroduplexes between plasmid and T7 DNA were formed and analyzed by the method of Davis et al. (15) using deletion mutants to mark the left end of T7 DNA. Gel electrophoresis was carried out on agarose slab gels (12).Containment. All procedures were carried out under P1 physical containment with EK1 host-vector as specified by the National Institutes of Health Guidelines for Recombinant DNA Research. RESULTSCloning Random Fragments of 17 DNA. T7 is a strictly virulent phage, and certain portions of T7. DNA carry information lethal to the host cell. We sheared wild-type T7 DNA to fragments of 4% its full length, hoping to separate lethal from nonlethal regions and thereby to be able to clone a large portion of the genome. The fragments were inserted in pBM9 using poly(dA dT) connectors, and a set o...

Linear DNA with covalently closed ends is the predominant form of DNA in the spirochaete Borrelia burgdorferi. AH bacteria examined to date have small DNA-binding proteins related to the Escherichia coli IHF and HU proteins that appear to play roles in DNA compaction and replication, but such proteins had not been isolated from bacteria with linear genomes. We found a single gene in B. burgdorferi (named hbb) whose product (named Hbb) complements the defects for 1 DNA packaging found in Em coli strains mutant in the genes for IHF and HU. The sequence of the predicted B. burgdorferi protein is similar to those of HU and IHF-like proteins in other bacteria. The gene appears to be in an operon with the order rpsl-hbb+rf#, where the rpsr gene is a homologue of the Em coli gene encoding ribosomal protein S 2 0 and the orfH gene encodes a protein of unknown function. This operon is located upstream of the previously identified B. burgdorferi rho homologue.

The heat-shock proteins DnaJ, DnaK, and GrpE are involved in the replication of various species of DNA in Escherichia coli, in addition to their roles in other processes, including protein disaggregation and export. We have cloned the Borrelia burgdorferi homologues of these genes. DNA sequence analysis revealed an open reading frame encoding a protein that is 62% identical to the E. coli DnaK protein. Genes homologous to the E. coli grpE and dnaJ genes, encoding products 28% and 39% identical to their homologues, are located up- and downstream, respectively, of the B. burgdorferi dnaK gene. No obvious promoters were detected in the sequenced DNA, although a potential transcription terminator was found downstream of the dnaJ gene, so these three genes may form an operon, perhaps with a fourth gene located upstream of the grpE gene. The grpE homologue complemented an E. coli grpE mutant and the dnaJ homologue complemented an E. coli dnaJ mutant, whereas the B. burgdorferi dnaK gene did not complement dnaK mutants.

The Escherichia coli rho gene product causes termination ot transcription at specific sites, is essential for arowth and is an RNA-dependent adenosine triphosphatase (1). While searching for another B. butrgdo;frri gene. we isolated a clone that encoded an open reading frame (ORF) 58% identical to the Ecoli Rho protein (see Figure) (2). Many of the amino acids thought to be involved in ATP binding and hydrolysis are conserved between the two proteins (underlined in Figure) (3). These include lysines 180 and 183 and aspartate 264. which were showrn to be essential for ATPase activity in the E.coli protein (4). The degree of identity between these two proteins suggests that the two bacteriaL use similar transcription termination mechanismis.