The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ∼ 100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P araBAD promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome.

It was demonstrated in this laboratory that poly G and other guanine-rich polynucleotides show differential affinity for the two complementary strands of various DNA's, indicating asymmetric distribution of poly G-binding sites.1' 2 Furthermore, it was postulated that these sites, probably deoxycytidine(dC)-rich clusters, might act as the initiation points of the DNA-to-RNA transcription.2' 3 For DNA which contains dC-rich clusters on both strands, as for instance coliphage X DNA (Fig. 1),'3 4 this hypothesis predicts that transcribing regions would be found on both strands. As will be shown, this prediction is confirmed for coliphage X, which provides the first example of in vivo transcription from both DNA strands, as documented by DNA-RNA hybridization techniques.3'5 This result agrees with the conclusions based on genetic experiments with X phage.6' I In earlier studies employing other phages, only one DNA strand was found to hybridize with phagespecific mRNA.8 55% 42% 46% G+C "LIGHT" LEFT ARM mRNA RIGHTARM W 5G . A B C D E F G H M L K J b2 a coNc, xyCr, OP QR C 5'A "DENSE" mRNA mRNA FIG. 1.-Genetic map of phage X. including A to R sus markers,9 "clear" markers Ci, {II, and CIII,24 central b2 region,25' 26 markers x and y,7 and marker a,27 all superimposed over X DNA.6 I The base compositions (% G + C) of both arms of X DNA and of the central b2 region are indicated.6' 25 5'G and 5'A identify the 5' terminal nucleotides23 and the polarity of the C and W strands.4 Symbol C ("DENSE") indicates the DNA strand which is denser in the poly G-containing CsCl gradient (and "lighter" in the alkaline CsCl gradient4 6) than strand W.2-4 The arrows (mRNA) indicate the orientation, the region, and the strand of preference for the DNA-to-RNA transcription, as discussed in this paper. The distribution of cytosine-rich clusters is indicated by the symbols (-E-), and is based on the data of Hradecna and Szybalski.4 Materials and Methods.-Bacterial and phage strains: Escherichia coli K12 strains included C600, which is permissive for Xsus mutants, and W3110 and W3350, which are nonpermissive for sus mutants.9 These were lysogenized or infected with appropriate X mutants as listed in Table 1. Most of the XcI, Xdg, and Xsus mutants and the lysogenic strains were obtained from Drs. . Strains T75'0 and T l [ = W3350-(Xtll)]7 were contributed by Dr. C. R. Fuerst. The subscript A-J indicates that genes A to J (entire left arm; Fig. 1) were deleted in XdgA -J and replaced by a part of the galactose operon.1' Biotin genes were substituted for deleted genes a-N or a-O in Xdba -N (=Xt75) and Xdba-o, respectively, the latter contributed by Dr. G. Kayajanian.The cultivation of bacteria, infection or induction of phages, preparation of phage stocks, and purification of phages by high-low speed sedimentation and CsCl density gradient centrifugation followed the published procedures.4' 6, 7,[9][10][11][12][13] The lysogenic cultures were induced by addition of 2 ,ug mitomycin C/ml.12

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In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E. coli. Using the yeast FLP/FRT site-specific recombination system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E. coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ--phoB and the 110-kb dapX--dsdC region of the E. coli MG1655 genome.

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