In antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen. We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared. Using the polymerase chain reaction, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin. From these libraries, VH domains were expressed and secreted from Escherichia coli. Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range. Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies. We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands.

We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.

We show that PCRI can be p med diretly2 on bacterial plaques or colonies, circumventing all DNA preparation. This allows the presence, size and orientation of inserts to be determined rapidly by amplificaton with flanking primers% and (for orietion) by including a singie intemal primer. For Ml3mp and pUC-based vectors, inserts of up to 3.7 kb are readily amplified using primers (A and B in upper figure) flanking the insert. The orientation of the insert may be screened using a primer (C) within the insert. The amplified materal can be used directy for restrcton digestion, cloning3, and sequencing4. Amplification reactions. 20 1tJ aliquots of 10mM Trs/HCI pH 8.3 at 250C, 50mM KCI, 1.5 mM MgCI2, 0.1 mg/ml gelatine, 0.25 mM dNTP, 10 pmol each oligonucleotide, 2.5 U Taq polymerase (Cetus), and template DNA were overlaid with paraffin oil, and subjected to 30 rounds of temperature cycling: 940C 1 min, 550C 1 min, 720C 2 min (3 min for inserts > 2.5 kb) and a final 5 min 550C step, on a PHC-1 programmable heating block (Techne). 5 1 of the reaction mixture was analysed on a 1-2% agarose gel containing 1 pg/ml EtBr. PCR on colonies. Colonies were resuspended in 0.5 ml water (live bacteria could be rescued at this stage) and boiled in a water bath for 5 minutes. After centrifugation for 2 min at 13000 rpm, 5 p1 of the supernatant was used in the PCR. PCR on plaques. Phage were transferred from a single plaque to a 20gi PCR reaction mix using a fresh © IRL Press

Diacylglycerol (DG) kinase attenuates the level of the second messenger DG in signal transduction, and therefore possibly modulates protein kinase C (PKC). DG kinase was purified to homogeneity from human white blood cells, showing an M 1 of 86 kDa as determined by SDS‐PAGE and gel filtration. Two amino acid sequences of tryptic peptides from DG kinase were determined and degenerate oligonucleotides were prepared and used in the polymerase chain reaction. An amplified DNA fragment was subsequently used to clone the full‐length human DG kinase cDNA. This sequence is the human homolog of porcine DG kinase cDNA sequence reported recently [1]. The sequence contains a double EF‐ hand structure typical for Ca2+ binding proteins. DG kinase further contains a double cysteine repeat that is present in all PKC isoforms, where it constitutes the phorbol ester (and most likely diacylglycerol) binding site. Therefore we speculate that the double cysteine repeat in DG kinase is involved in DG binding. DG kinase is transcribed as a single mRNA of 3.2 kb, that is highly expressed in T‐lymphocytes. The human DG kinase cDNA when transfected in mammalian cells (COS‐7) results in a 6–7‐fold increase of DG kinase activity.

The molecular mechanisms of alphavbeta3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human alphavbeta3 (r-alphavbeta3) and compared the activation state of these with alphavbeta3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-alphavbeta3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alphavbeta3 and the receptor in situ on the cell surface. r-alphavbeta3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-alphavbeta3. r-alphavbeta3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native alphavbeta3. On M21-L4 melanoma cells, alphavbeta3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated alphaIIbbeta3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified alphavbeta3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of alphaIIbbeta3 in situ, intracellular controls lower the affinity of alphavbeta3, and the cytoplasmic domains may act as a target for negative regulators of alphavbeta3 activity.