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The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Pulse-chase experiments suggest that G is transported to the cell surface in two successive waves of clathrin-coated vesicles . The oligosaccharides of G protein carried in the early wave are of the "high-mannose" (G 1 ) form, whereas the oligosaccharides in the second, later wave are of the mature "complex" (G 2) form . The early wave is therefore proposed to correspond to transport of G in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, and the succeeding wave to transport from the Golgi apparatus to the plasma membrane . The G 1 -and G2-containing coated vesicles appear to be structurally distinct, as judged by their differential precipitation by anticoated vesicle serum .

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The thymidylate synthetase gene of B. subtifis bacteriophage Phi-3-T, when cloned in plasmids pSC101 or pMB9, is expressed in E. colt. Phi-3-T is a Bacillus subtilis temperate bacteriophage capable of lysogenic conversion of thy-B. subtilis clones to prototrophy (1): we describe here the cloning of the synthetase gene carried by Phi-3-T on the Escherichia coli plasmids pSC101 (2) and pMB9. The gene complements thymine-deficiency mutation in E. coli which indicates its correct transcription and translation in this new host. The promoter of the gene, cloned in pSC101, is likely to be contained within the inserted segment. Hybrid plasmid DNA transforms B. subtilis, although about 10fold less efficiently than the intact phage DNA. B. subtilis clones transformed with the hybrid plasmid, DNA do not contain detectable pSC101 sequences, but do show sequences homologous with part of the Phi-3-T genome.MATERIALS AND METHODS Bacterial Strains. E. coli strains used were W5443 thr-1 leu-6 thi-1 supE44 lacYl r-m-thy-str (tonB tryp-delta) (SB2 of D. Finnegan), W5469 thy-hs-argA metB leu-xyl-lacY strA polAts214 from D. Helinski, C600(pSCIOI) from S. Cohen, HB129 (pMB9) endo" rB+ mB+ gal-lac-strepr leu-prothi-from H. Boyer, and C600 (ColElAmp) (3) from V. Hershfield. B. subtilis strains included SB168 trypC2, SB591 thy-, SB1158 thyA (thyB ilvD6 delta) from this laboratory and SB168(Phi-3-T) from D. Dean.DNAs and Enzymes. Phage, purified by differential centrifugation followed by two CsCl density bandings, was used to prepare Phi-3-T DNA by phenol extraction. Plasmid DNAs were prepared by the clear lysis procedure (4).EcoRI nuclease was prepared according to an unpublished procedure (T. Landers, personal communication). T4 ligase was prepared and used as described (5). EcoRI cleavage was done as described (6). RNA Isolation of Tcr thy+ E. coli transformants Cleavage of the Phi-3-T DNA with EcoRI enzyme decreases the transforming activity of the thy gene about 1000 times (not shown). We have, therefore, attempted the cloning of that gene from an incompletely digested DNA sample, in which the transforming activity has been decreased only 10-fold. The sample was ligated to EcoRI cleaved pSC101 DNA and used to transform thymine-requiring E. coli cells by selecting either for thymine independence or tetracycline resistance. About 400 Tce clones were inspected for the presence of hybrid plasmids by colony hybridization (11). Nearly 8% of the clones contained sequences complementary to Phi-3-T cRNA. Two of the Tcr clones showed a thy + phenotype. Two more thy + clones have been obtained by selecting that phenotype directly: these were also Tcr. The frequency of phenotype occurrence for the di-4145 Abbreviation: pFT, a cloned plasmid chosen on the basis of its ability to hybridize with Phi-3-T cRNA.

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [OiOl:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A.