Transformation of E. coli cells treated with CaCJ2 to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before antibiotic challenge. MATERIALS AND METHODSBacterial Strains and R Factors. The I-like R factor, R64-11 (16), which specifies resistance to tetracycline (Tc) and streptomycin (Sm), was obtained from R. Curtiss. R6 (17), an F-like R factor that carries resistance to kanamycin (Km), neomycin (Nm), chloramphenicol (Cm), sulphonamide (Su), streptomycin, and tetracycline, was obtained from T. Watanabe. R6-5, a spontaneous variant of R6 that lacks tetracycline resistance, was isolated in our laboratory (18). The bacterial strains used in these experiments have been described (7,22). DNA Preparations. In certain instances, covalently-closed R-factor DNA was isolated and purified from E. coli as described (6, 7). Alternatively, a Brij-lysis procedure (19) was used for initial R-factor DNA isolation, and preparations obtained by this method were subsequently purified by centrifugation in cesium chloride-ethidium bromide gradients. The catenated, closed circular, and noncircular forms of R-factor DNA used in experiments comparing the relative transforming ability of the various R-factor DNA species were isolated from E. coli minicells. R-factor DNA was denatured by heating it at 980 for 5 min in 15 mM NaCl-1.5 mM Na citrate followed by rapid cooling at 0°. Sonication of R-factor DNA to about 9S fragments was done for 15 see at 00 by a Branson model W185 D sonicator, and the size of the R-factor DNA fragments was confirmed by sucrose gradient centrifugation (7).Transformation Reaction Mixture. Transformation was done by a variation of the procedure of Mandel and Higa (15), as modified by Lobban, Masuda, and Kaiser (personal communication). E. coli strain C600 was grown at 370 in H1 medium (20) to an optical density of 0.85 at 590 nm. At this point, the cells 2110 Abbreviation: R factor, antibiotic resistance factor. * The previous paper in this series is ref. 18.

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColEl plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColEl, they were nevertheless compatible with ColEl. P15A-derived plasmids were not selftransmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColEl plasmid within the same cell.

The construction of new plasmid DNA species by in vitro joining of restriction endonucleasegenerated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins.Controlled shearing of antibiotic resistance (R) factor DNA leads to formation of plasmid DNA segments that can be taken up by appropriately treated Escherichia coli cells and that recircularize to form new, autonomously replicating plasmids (1). One such plasmid that is formed after transformation of E. coli by a fragment of sheared R6-5 DNA, pSC101 (previously referred to as Tc6-5), has a molecular weight of 5.8 X 106, which represents about 10% of the genome of the parent R factor. This plasmid carries genetic information necessary for its own replication and for expression of resistance to tetracycline, but lacks the other drug resistance determinants and the fertility functions carried by R6-5 (1).Two recently described restriction endonucleases, EcoRI and EcoRII, cleave double-stranded DNA so as to produce short overlapping single-stranded ends. The nucleotide sequences cleaved are unique and self-complementary (2-6) so that DNA fragments produced by one of these enzymes can associate by hydrogen-bonding with other fragments produced by the same enzyme. After hydrogen-bonding, the 3'-hydroxyl and 5'-phosphate ends can be joined by DNA ligase (6). Thus, these restriction endonucleases appeared to have great potential value for the construction of new plasmid species by joining DNA molecules from different sources. The EcoRI endonuclease seemed especially useful for this purpose, because on a random basis the sequence cleaved is expected to occur only about once for every 4,000 to 16,000 nucleotide pairs (2); thus, most EcoRI-generated DNA fragments should contain one or more intact genes. We describe here the construction of new plasmid DNA species by in vitro association of the EcoRI-derived DNA fragments from separate plasmids. In one instance a new plasmid has been constructed from two DNA species of entirely different origin, while in another, a plasmid which has itself been derived from EcoRI-generated DNA fragments of a larger parent plasmid genome has been joined to another replicon derived independently from the same parent plasmid. Plasmids that have been constructed by the in vitro joining of 3240EcoRI-generated fragments have been inserted into appropriately-treated E. coli by transformation (7) and have been shown to form biologically functional replicons that possess genetic properties and nucleotide base sequences of both parent DNA species. MATERIALS AND METHODSE. coli strain W1485 containing the RSF1010 plasmid, which carrie...

The nucleotide sequence of a 1,091-base pair cloned cDNA insert encoding bovine corticotropin-beta-lipotropin precursor mRNA is reported. The corresponding amino acid sequence indicates that the precursor protein consists of repetitive units and includes a third melanotropin sequence in its cryptic portion. Pairs of lysine and arginine residues separate the component peptides of the precursor.

An assay was developed to investigate the fate of specific segments of 13-lactamase (bla) and ompA gene transcripts in Escherichia coli. DNA probes cloned in bacteriophage M13 were treated with an. endonuclease capable of cleaving singlestranded DNA, the fragments produced were annealed with total cellular RNA, and the resulting RNADNA hybrids were subjected to SI nuclease treatment and gel fractionation. By using this assay, direct evidence was obtained for 3'-to-5' directionality in the decay of the long-lived mRNA encoded by the ompA gene, and no preferential stability was observed for translated versus untranslated mRNA segments. In the case of bla mRNA, initial cleavage of the full-length transcript was rate limiting, and no decay intermediates were detected. No difference in degradation rate was seen for bla transcripts having variant 3' or 5' termini.formation. with complementary RNA sequences are then fractionated by gel electrophoresis. This procedure has made it possible to determine the relative decay rates of different regions of the transcript and thus to investigate possible directionality; and segmental specificity of the decay process.We describe here experiments designed to elucidate the pattern and kinetics of decay of two different mRNA species in E. coli: (i) the RNA for the (&lactamase (bla) enzyme encoded by a Tn3 transposon-derived gene and (ii) the RNA for the abundant E. coli outer membrane protein II* (ompA). These RNA transcripts are both monocistronic, have roughly the same size, and encode proteins secreted through the bacterial cell inner membrane.The level of expression of a gene is, determined primarily by three elements: the, rate of transcription, the stability of the RNA transcript, and the efficiency oftranslation. In Escherichia coli, the factors influencing the efficiencies oftranscription and translation have been the subject ofconsiderable study, and the enzymes and nucleic acid sequences involved in these processes have been well characterized. It has been shown that the strengths of promoters span at least two orders of magnitude (1) and that translation rates can differ by as much as 10-fold (2).By contrast, the factors that determine mRNA stability are not well understood. Almost nothing is known about the initiation sites of mRNA degradation; although a number of RNases have been identified in E. coli (3), it is still unclear which are primarily responsible for determining the rate of mRNA decay in vivo.A major barrier to the detailed study of mRNA degradation has been the lack of an adequate assay system. Typically, one of three methods has been used to. monitor the rate ofdecay of individual mRNA species. One assay, which yields a so-called "functional half-life" ofa transcript, involves the pulse-labeling of proteins synthesized after the inhibition of transcription (4). Other methods are based on RNA blotting techniques or hybridization of pulse-labeled RNA. to DNA fragments (5).In order to investigate directly the factors that influence the decay of mRNA...

Growth arrest-specific (gas) genes are expressed preferentially in cells that enter a quiescent state. gas7, which we identified in serum-starved murine fibroblasts, is reported here to be expressed in vivo selectively in neuronal cells of the mature cerebral cortex, hippocampus, and cerebellum. gas7 transcripts encode a 48-kDa protein containing a structural domain that resembles sequences of OCT2, a POU transcription factor implicated in neuronal development, and synapsins, which have a role in modulating neurotransmitter release. Using in situ hybridization and immunocytochemical analysis, we show that GAS7 expression occurs prominently in cerebellar Purkinje cells and that inhibition of production in terminally differentiating cultures of embryonic murine cerebellum impedes neurite outgrowth from maturing Purkinje cells. Conversely, GAS7 overexpression in undifferentiated neuroblastoma cell cultures dramatically promotes neuritelike outgrowth. Collectively, our results provide evidence for an association between expression of this gas gene and neuronal development.

The construction and analysis of bacterial plasmids that contain and phenotypically express a mammalian genetic sequence are described. Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the antimetabolic drug trimethoprim.

Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSCl01; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA.Recombinant DNA molecules constructed in vitro from separate plasmids (1, 2) by the joining of DNA fragments having cohesive termini (3, 4) generated by the EcoRI restriction endonuclease (5, 6) can form biologically functional replicons when introduced into Escherichia coli by transformation (7). The E. coli tetracycline resistance plasmid, pSC101 (1, 8) (molecular weight 5.8 X 106), is useful for selection of recombinant plasmids in E. coli transformants, since insertion of a DNA segment at its single EcoRI cleavage site does not interfere with expression of its tetracycline resistance gene(s) or with the replication functions of the plasmid (1, 2).This report describes the in vitro linkage of pSC101 and eukaryotic DNA cleaved by EcoRI endonuclease, and subsequent recovery of recombinant DNA molecules from transformed E. coli in the absence of selection for genetic properties expressed by the eukaryotic DNA. The amplified rDNA (coding for 18S and 28S ribosomal RNA) of Xenopus laevis was used as a source of eukaryotic DNA, since it has been well characterized and can be isolated in quantity (9, 10). Recombinant plasmids containing both X. laevis and pSC101 DNA replicate stably in E. coli, where they are capable of synthesizing RNA complementary to X. laevi8 rDNA. MATERIALS AND METHODSDNA coding for ribosomal RNA of X. laevis, isolated by CsClgradient centrifugation, and 82P-labeled 18S and 28S X. laevis ribosomal RNA were the generous gifts of Dr. D. D.Brown. Bacterial strains and the tetracycline resistance plasmid pSC101 have been described (1, 2, 8). Covalently-closed circular plasmid DNA was isolated as described (8, 11), or Abbreviations: rRNA, ribosomal RNA; rDNA, amplified DNA containing the genes for 18S and 28S rRNA; EcoRI, the RI restriction and modification host specificity of E. coli controlled by the fi + plasmid, pHB1. (20) and E. coli ligase (the generous gift of Drs. P. Modrich and I. R. Lehman) (21) have been described. E. coli-X. laevis recombinant plasmids were constructed in vitro as follows: the reaction mixture (60 .A) contained 100 mM Tris-HCO (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1.0 jgg of pSC101 plasmid DNA, 2.5,g of X. laevis rDNA, and excess EcoRI restriction endonuclease (1 ,l, 2 units). After a 15-min incubation at 370, the reaction mixture was placed at 630 for 5 min to inactivate the EcoRI endonuclease. A 3-,ul sample was examined by electron microscopy to assess digestion. The remainder was refrigerated at 0.50 for 24 hr to allow association of the short cohesive termini; melting temperature (Tm) was ...