A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene.

A plasmid vector with a multiple cloning site (MCS) for positive selection of cloned inserts in Escherichia coli (Ec) has been devised, based on the expression plasmid (pMT416) for the bacterial ribonuclease barnase (Barn). The host is protected from the lethal effect of moderate expression of barn by expression of the gene bars, encoding its inhibitor, barstar (Bars), placed on the same plasmid. Full expression, however, is lethal. Induction is also lethal with the derived plasmid, pMT440, which has the pUC19 MCS inserted into barn. Under inducing conditions, transformation by the vector is lethal unless the product of the modified barn is inactivated by insertion of cloned DNA fragments into the MCS. Plasmid pMT440 is, therefore, a generally useful selective cloning vector not requiring any special strain of Ec.

Recombinant barnase as a label in ELISA.

Recombinant barnase was proposed as a label in the enzyme-linked immunosorbent assay (ELISA). Barnase-conjugated pig transferrin was prepared by the periodate oxidation procedure. Solid-phase bound barnase activity was determined from the change in RNA-ethidium bromide complex fluorescence upon RNA hydrolysis. The sensitivity of transferrin-barnase conjugate determination in ELISA was no less than 5 ng per well. The conjugate was applied in competition ELISA for free transferrin determination.

A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene.

A new phagemid cloning vector for positive selection of recombinants, pBa-7, was constructed which contains an active barnase gene encoding the cytotoxic ribonuclease from Bacillus amyloliquefaciens, under control of the lac promoter. PBa-7 is a derivative of the high-copy number pBluescript II KS+ phagemid in which the modified barnase killer gene has been fused downstream from the lac promoter of the pBluescript II KS+ multiple restriction site. When a lacIq-negative Escherichia coli strain is transformed by this vector, the active barnase blocks bacterial growth by massive RNA destruction [1]. However, if barnase is inactivated by insertion of a foreign DNA fragment into the multirestriction site of the vector, this recombinant plasmid no longer interferes with the host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are considerably simplified. When E. coli transformants are plated out on rich medium with ampicillin, only cells containing recombinant plasmids give rise to colonies. In a lacIq-positive host, the positive selection is IPTG-dependent. Therefore, pBa-7 phagemid can be amplified and prepared in large quantities from lacIq-positive E. coli hosts. Hence, pBa-7 seems to be suitable for most genetic engineering manipulations.

Group-selective immunoassay.

A new solid-phase immunoassay technique has been applied for anti-digoxin monoclonal antibodies (Mabs) detection. At the first assay step, anti-digoxin Mabs (IgG1, Kaff = 9.2 x 10(9) M-1) were bound to a specially prepared immunosorbent, the microtiter plates coated with digoxin-human serum albumin conjugate (Dig-HSA), in which free amino groups were protected by a glutaraldehyde cross-linking modification. The modification did not essentially influence the antibody-binding capacity of the immunosorbent. After antigen-antibody reaction, free amino groups were located only on the anti-digoxin Mabs, bound to chemically modified immunosorbent. At the second assay step, free amino groups of anti-digoxin Mabs were biotinylated by N-hydroxysuccinimide-biotin ester. Then the biotin residues were detected by the streptavidin-peroxidase conjugate. The method does not require second labeled antibodies and may be used for anti-hapten hybridoma screening.

Fast filtration enzyme immunoassay for haptens.

An immunometric method for determination of hapten concentration in fluids has been developed. High-affinity hapten-specific enzyme-labeled monoclonal antibodies are mixed with a sample containing hapten, then the mixture is filtered through a membrane with immobilized hapten. The level of enzyme activity retained by the membrane is inversely proportional to the concentration of hapten in a sample. The assay has been developed for theophylline, digoxin and phenobarbital. The coefficient of variation is less than 5% and the test takes about 2 min.

Genetic analysis of the maternally induced affinity enhancement in the non-Ox1 idiotypic antibody repertoire of the primary immune response to 2-phenyloxazolone.

The early phases of ontogeny are decisive for the development of the B-cell repertoire. Here, we demonstrate that maternal tertiary immunization of BALB/c mice with 2-phenyloxazolone (phOx) caused a drastic alteration of the primary antigen-specific repertoire of the offspring. Maternal tertiary immunization or quaternary antibodies, which exhibited an extremely weak cross-reactivity with the major Ox1 idiotype (IdOx1), induced a change in the proportion of IdOx1/non-IdOx1 antiphOx antibodies in the F1 and F2 primary repertoire. The observed variability in the level of IdOx1 expression (10-90%) exceeded even the seemingly genetically based differences between various mouse strains. In comparison with the non-IdOx1 of control mice, half of the non-IdOx1 antibodies showed a 5-100-fold enhanced affinity. Sixty per cent of these antibodies exhibited an affinity identical to that of IdOx1 antibodies, which are normally of the highest affinity, while the remaining 40% exceeded even that of IdOx1 by a factor of 10. The non-IdOx1 were encoded by VH/VL genes and/or combinations thereof which are either new, hitherto unobserved in the antiphOx response, or typical of memory responses in normal mice. The significance of these data is discussed with respect to the possibility that maternal antibodies, which are acquired through multiple immune maturation processes, may have an epigenetic (non-Mendelian) inheritable potential for the offspring.

Ribonuclease-charged vector for facile direct cloning with positive selection.

Plasmid vectors for positive selection of cloned inserts in Escherichia coli were devised, based on an expression plasmid (pMT416) for the bacterial ribonuclease barnase. In addition to the barnase gene under control of a synthetic tac promoter, these plasmids carry the gene for the barnase inhibitor, barstar, the constitutive expression of which protects the bacterium from the detrimental effects of moderate barnase production. Full expression of the barnase gene overcomes protection by barstar and becomes lethal. Having a unique SmaI/XmaI site in the barnase structural gene, pMT416 itself can be used as a selective vector: uncut or religated pMT416 will preclude growth while plasmids with inserts in the barnase gene will allow the cells to survive. The entire pUC polylinker was inserted into the barnase gene in place of the Val-36 codon. This insert of nineteen largely hydrophilic amino acids does not prevent the lethal effect of full expression of the gene. The resulting plasmid, pMT440, is a generally useful selective cloning vector representing the "kill-the-rest" approach.