Herbert W. Boyer

Construction and characterization of new cloning vehicles. II. A multipurpose cloning system

In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain. Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.

A complementation analysis of the restriction and modification of DNA in Escherichia coli.

Abstract A complementation analysis of host-controlled modification and restriction of DNA by Escherichia coli has been carried out by examining the restriction and modification phenotypes of partial, permanent diploids containing various arrangements of wild type and mutant restriction and modification alleles. Intercistronic complementation was observed between three classes of restriction and modification mutants of E. coli B, indicating that at least three cistrons (the ram cistrons) are involved in the genetic control of the [restriction and modification of DNA. Mutations in one cistron ( ramA ) result in a loss of restriction activity but not in modification activity (r − m + ). Mutations in a second cistron ( ramC ) result in a loss of restriction and modification activities (r − m − ). Mutations in a third cistron result in a loss of modification activity and appear to be lethal unless accompanied by a mutation in the ramA or ramC cistrons. A fourth class of mutations, which are linked to the other ram cistrons and are expressed phenotypically as r − m − mutants, are trans dominant to the wild-type ram alleles. It is not known if this latter class of mutants represents a fourth cistron of the ram locus. Complementation was observed between E. coli K12 and B ramA and ramC mutations and the host specificity of the restored restriction activity was dependent on an intact ramC cistron. However, complementation was not detected between the P1 and K12 or P1 and B ram alleles. A general model for the genetic control of the restriction and modification properties of E. coli strains and their episomes is presented.

Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9

In vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap resistance (Apr) gene resulted in the construction of a new cloning vehicle, the plasmid pBR313. This vector was derived from a ColE1-like plasmid and, while it does not produce colicon E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101 respectively. During the construction of pBR313, the TnA component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a molecular weight of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which (EcoRI, SmaI, HpaI, HindIII, BamHI and SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these six enzymes. The restriction sites for the latter three enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for cells carrying recombinant plasmid molecules. An enrichment method for AprTcS cells carrying recombinant plasmid molecules is described.

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Abstract Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with Eco RI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of ( Eco RI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules. Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i , and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j . The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i . When j = i , equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10 −2 μm) is smaller than the random coil segment value b (7·17 × 10 −2 μm) was observed, while circular structures of the dimer of the same molecule (12 × 10 −2 μm) were detected.

Protein expression in E. coli minicells by recombinant plasmids

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.

Restriction and modification of a self-complementary octanucleotide containing the EcoRI substrate.

In order to study the interactions of the EcoRI restriction endonuclease and modification methylase with DNA, we have synthesized the self-complementary octanucleotide, pT-G-A-A-T-T-C-A, which contains the nucleotide sequence of the EcoRI substrate. This octamer can act as a substrate for both the endonuclease and methylase; the enzymatic alteration of this molecule is the same as that of DNA, with cleavage and methylation both occurring at the same positions on either substrate. The optimum temperature for the reaction of the octanucleotide with the endonuclease is 15°C and that with the methylase is 12·5°C. The optimum temperature for the reactions of both of the enzymes with DNA is 37°C. The low temperatures for reactions with the octanucleotide reflect the conditions under which this molecule can serve as a substrate for the enzymes. The Km values and turnover numbers for the EcoRI endonuclease using both the synthetic octanucleotide and simian virus 40 (SV40) DNA as substrates were determined. These constants are, respectively, 7 × 10−6 m and 4 min−1 for the endonuclease-octamer reaction and 3 × 10−8 m and 3 min−1 with SV40 DNA. Although the Km does not measure affinity directly, the similarity of turnover numbers on both substrates allows us to conclude that the affinity of the enzyme for SV40 DNA is approximately 200 times greater than for the octanucleotide. The synthetic octanucleotide is self-complementary and can form a duplex structure. Computer analysis of initial reaction-rate data shows that in order to act as a substrate the octamer must be a dimer. This observation, together with the correspondence of the temperature optima for both enzymatic reactions to the tm value for the double-stranded form of the octanucleotide (17 to 19°C), lead us to conclude that the substrate for both EcoRI enzymes is a double-helical segment of DNA containing the central hexamer of the octanucleotide. It is unlikely that any cruciform rearrangement is required for enzyme activity.

CONSTRUCTION AND CHARACTERIZATION OF CLONING VEHICLES

ABSTRACT A series of in vitro recombination experiments with restriction endonuclease fragments of pSClOl and a col El-like plasmid resulted in the recovery of a plasmid containing the best cloning characteristics of each plasmid. The newly constructed plasmid, designated pMB9, is similar to col El in its replication properites, its immunity to colicin E1 but does not produce colicin E1, and does not appear to form a relaxation complex. It carries a segment of DNA derived from the pSClOl plasmid which contains a functional tetracycline resistant gene. The plasmid has one restriction site for the Eco RI, Hind III, Bam I and Sal I restriction endonucleases. The latter three sites are located in the gene for tetracycline resistance. The plasmid has a molecular weight of about 3.5 × 10 6 daltons. A derivative of pMB9 containing an ampicillin resistant gene was constructed by in vivo translocation of this gene from p2124 to pMB9. This new plasmid, designated pBR312, contains two BamI sites and was used as a parent for the construction of several plasmids which can serve as cloning vehicles for Hind III, Sal I and Bam I endonuclease generated fragments by providing ampicillin resistance as a selective marker. Transformants containing recombinant plasmids with DNA fragments inserted at any of these three sites can be identified or selected from transformant populations by their response to tetracycline.

Solubility and dialysis limits of DNA oligonucleotides.

Abstract Oligonucleotides, 3–17 nucleotides in length, were generated from mouse L cell DNA and their dialysis and solubility limits were determined under a variety of conditions. After dialysis for 24-h oligonucleotides more than 4 nucleotides in length were retained. Ethanol precipitates at least 10–20 % of all classes of oligonucleotides and 65 % of high molecular weight DNA under our conditions; oligonucleotides longer than 15–16 nucleotides are precipitated to about the same extent as high molecular weight DNA. HClO 4 and trichloroacetic acid are equally effective in precipitating oligonucleotides: 50 % precipitation occurs at 17 nucleotides for HClO 4 and 16 nucleotides for trichloroacetic acid. Acid precipitation of oligonucleotides adsorbed on glass or paper fiber filters is different from that observed with solutions. The efficiency of binding of oligonucleotides increases with increasing chain length: 50 % binding occurs at 5–6 bases with paper and 9–15 with glass. These numerical estimates are important with reference to some current techniques and results concerning DNA repair.

In vitro maturation of germinal vesicle oocytes in stimulated intracytoplasmic sperm injection cycles.

In vitro enzymatic alteration of plasmid phenotype and in vitro construction of recombinant plasmids containing genetic information derived from the plasmid pSC101 have been used to investigate the mechanism of function of tetracycline resistance determined by the plasmid pSC101. The resistance has been shown to be inducible and involves the increased synthesis of membrane-associated polypeptides of 34,000, 26,000 and 14,000 daltons that are encoded for by the plasmid. The 34,000 dalton polypeptide along with another plasmid-encoded polypeptide of 18,000 daltons function in an ATP-independent manner to prevent the accumulation of tetracycline by the cell. These polypeptides are sufficient for resistance. A second component of plasmid-determined resistance involves the 14,000 dalton polypeptide and reduces the initial adsorption of tetracycline by sensitive cells, but is not alone sufficient for the generation of resistance. The role of the 26,000 dalton polypeptide in tetracycline resistance has not been identified.

The structure of cauliflower mosaic virus: I. A restriction endonuclease map of cauliflower mosaic virus DNA

A physical map of the genome of cauliflower mosaic virus (CaMV) has been constructed on the basis of the cleavage of CaMV-DNA by restriction endonucleases. Digestion of circular native viral DNA (1) with Eco RI endonuclease produces three fragments, (2) with Hin dIII endonuclease produces nine fragments, and (3) with Bam HI endonuclease produces three fragments from the average virus DNA molecule in the population. The restriction fragments have been ordered by examining the overlapping fragments and the partial digestion products generated by these three restriction endonucleases. Restriction fragments of the virus DNA generated by Eco RI and Bam HI endonucleases have been molecularly cloned in bacterial plasmids in Escherichia coli in order to facilitate restriction mapping of the virus DNA. Evidence for nucleotide sequence heterogeneity in the native virus DNA population is apparent in the form of uncommon restriction endonuclease sites found in only a small portion of the DNA molecules. Examination of molecularly cloned fragments of virus DNA containing these variable sites allowed a more complete analysis of this phenomenon.