Timothy J. Close

Construction and characterization of the chloramphenicol-resistance gene cartridge: A new approach to the transcriptional mapping of extrachromosomal elements

Abstract A system has been developed to transcriptionally map plasmid DNA molecules in vivo. This system is based upon the activity of the Tn 9 Cm r gene whose product, chloramphenicol acetyltransferase (CAT), can be easily assayed enzymatically and identified phenotypically in Escherichia coli . The Cm r gene was excised as a single DNA fragment from the plasmid cloning vector, pBR328, and its ends were converted to hexamer-specific restriction enzyme recognition sites. These modified Cm r gene fragments were designated “CAT cartridges”. The CAT cartridges were expressed at high levels only when inserted in the proper orientation, downstream of a plasmid promoter. Using DNA restriction endonuclease cleavage sites as insertion points, the CAT cartridges were inserted into plasmid pBR327, and the levels of CAT activity produced in cells containing the various plasmids were determined. The CAT activities at each position and orientation were used to construct a transcriptional map of plasmid pBR327. The positions and strengths of promoters demonstrated in vivo using the CAT cartridge coincided with those predicted by in vitro studies. Using the CAT cartridges it should be possible to study gene expression on any extrachromosomal element in the appropriate host.

Design and development of amplifiable broad-host-range cloning vectors: Analysis of the vir region of Agrobacterium tumefaciens plasmid pTiC58

The construction of a set of new plasmids that are suitable as general cloning vectors in Escherichia coli and Agrobacterium tumefaciens is described. Plasmid pUCD2 is amplifiable in E. coli, replicates in a wide range of gram-negative hosts and contains a number of useful restriction endonuclear cleavage sites and antibiotic resistance genes. This includes unique sites for KpnI, SacI, SacII, PstI, ClaI, SalI, EcoRV, and PvuII and the genes for resistance to kanamycin, tetracycline, ampicillin, and spectinomycin/streptomycin. Derivatives of pUCD2 include pUCD4, which has a unique XbaI site and the cosmid pUCD5, which also contains a unique EcoRI site. Two smaller plasmids pUCD9P and pUCD9X, contain many of the same unique sites as pUCD2 and pUCD4, but carry only the pBR322 replication origin and therefore do not display the extensive host-range of pSa. These plasmids were used to isolate and manipulate fragments of the A. tumefaciens pTiC58 plasmid in both E. coli and A. tumefaciens. Fragments from the virulence (vir) region of pTiC58 inserted immediately upstream of the spectinomycin resistance gene of pUCD2 resulted in spectinomycin resistance levels that varied greatly depending on the particular fragment and its orientation of insertion. Using this property we find that a major portion of the vir region of pTiC58 is transcribed in A. tumefaciens and E. coli from left to right toward the T region.

Genetic complementation of Agrobacterium tumefaciens Ti plasmid mutants in the virulence region

SummaryMutants with Tn5 insertions in the vir region of the Agrobacterium tumefaciens TiC58 plasmid are unable to form crown-gall tumors. Complementation tests of these vir region mutants were carried out by constructing merodiploids in a recombination-deficient strain. Each merodiploid possessed a mutant TiC58 plasmid and a recombinant plasmid containing either the homologous wild-type DNA region or the homologous region containing a second Tn5 insertion. The analysis identified six complementation groups. Mutations in one of these complementation groups were not complemented in trans and represent a cis-dominant locus. The mutation in one complementation group showed variation in host range.

Isolation of the origin of replication of the IncW-group plasmid pSa

The origin of replication of the IncW plasmid pSa has been cloned and the function of this origin in Escherichia coli examined. A 1.9-kb region of DNA is required for efficient autonomous replication, and a 0.47-kb fragment within this region can initiate replication only in the presence of an autonomously replicating derivative of pSa. An Mr 35,000 protein (repA) is encoded adjacent to the origin and is required for efficient initiation of replication. The derivatives examined provide information suggesting a direct role of partition factors in plasmid replication and incompatibility.

Working with bacterial bioluminescence

I n t r o d u c t i o n Bioluminescence exists in a wide variety of life forms including worms, mollusks, insects, fish, diatoms, jellyfish, fungi and bacteria, and comprehensive reviews have been published addressing the ecology and biochemistry of bioluminescence (Hastings 1968; DeLuca et al., 1978; Hastings et al., 1985). In general, all bioluminescence results from the action of a luciferase enzyme upon its substrate, the luciferin. In bacteria the luciferin is an aldehyde which is synthesized from a fatty acid precursor by the action of a fatty acid reductase (Wall et al., 1984; Boylan et al., 1985; Byers and Meighen, 1985). The first published cloning of any type of luciferase genes was by Belas et al. (1982), who cloned the luciferase (lux) genes of Vibrio harweyi. Almost simultaneously another group reported similar success (Cohn et al., 1983). Functional fatty acid reductase genes were not present on these clones, and therefore, luciferin for the bioluminescence reaction has to be added exogenously. V harveyi luciferase genes have been used successfully to generate light in Rhizobium meliloti even inside functional root nodules (Legocki, 1987), in blue green algae (Schmetterer, 1986) and, after providing eukaryotic transcription signals, in plant protoplasts (Koncz et al., 1987). Engebrecht et al. (1983) reported the cloning of the V. fischeri luciferase genes as well as functional fatty acid reductase genes in a single 16 kb fragment. Further analysis demonstrated that five genes organized in a operon

M13 bacteriophage and pUC plasmids containing DNA inserts but still capable of β-galactosidase α-complementation

Abstract A DNA fragment encoding the transposen Tn9 chloramphenicol acetyltransferase gene ( cat ) was inserted into M13 phage and pUC plasmid cloning vehicles. When the cat gene was inserted in the same orientation as the lacZ gene, two new polypeptides were produced. One polypeptide possessed chloramphenicol acetyltransferase activity, while the other expressed β-galactosidase α-donor activity. Both new polypeptides were translated from a hybrid messenger RNA initiating from the lac promoter. These observations may help explain why not all inserts produce white plaques.