Exclusion of 5-HT2A and 5-HT2C receptor genes as candidate genes for migraine.

Several lines of investigation suggest that the serotonergic system may be involved in the pathogenesis of migraine. In particular, drugs which block 5-HT2 receptor subtypes appear to be effective migraine prophylactic agents. Therefore, chromosomal DNA regions overlapping the 5-HT2A (13q14-q22) and 5-HT2C(Xq22-25) receptor loci were analyzed for possible linkage to the clinical diagnosis of migraine. No evidence for linkage to either chromosomal region was found, although a small subset of migrainous families showed positive likelihood of odds (LOD) scores. However, a homogeneity (HOMOG) analysis provided no statistical evidence for locus heterogeneity. The coding region of the 5-HT2A and 5-HT2C receptor genes was also analyzed in migraine patients and unaffected controls using polmerase chain reaction and direct sequencing. No mutations were found in the deduced amino acid sequence of either receptor in the sample of migraineurs tested. These results indicate that DNA-based mutations in the 5-HT2A and 5-HT2C receptors are not generally involved in the pathogenesis of migraine.

Immunochemical and molecular analysis of antigen binding to lipid anchored and soluble forms of an MHC independent human alpha/beta T cell receptor.

We have constructed antigen-specific chimeric human T cell receptor (TCR) molecules deleted of the transmembrane domain and containing the signal sequence for the biosynthesis of the phosphatidyl inositol glycan (GPI) linkage. These membrane-anchored forms of the TCR alpha and beta chains have been expressed in non-T cells, and they are recognized by alpha or beta TCR specific monoclonal antibodies. We have utilized both immunochemical methods and flow cytometry to prove that the enzyme phosphatidylinositol phospholipase C (PI/PLC) is able to cleave the GPI anchored TCR as a heterodimer from the CHO cell surface. We have demonstrated that the alpha/beta TCR heterodimer on the surface of CHO cells will recognize and bind polymers containing fluorescein (FL-polymer), and the binding activity is completely eliminated by the enzyme, PI/PLC. Moreover, soluble forms of the alpha/beta heterodimer will bind tightly to FL substituted sepharose, which demonstrates the retention of biological activity by the TCR after solubilization. Molecular modelling of the putative antigen binding site of the alpha FL beta FL TCR was derived from the known atomic coordinates of eight different hapten or peptide specific antibodies. Mutagenesis of several residues predicted from the model to be important in FL binding gave results consistent with involvement of Ig equivalent CDR2 and CDR3 domains in the antigen binding pocket. Therefore, using a model hapten system in studying recognition of the TCR independent of MHC interactions, we conclude that amino acid residues located in similar positions within CDR domains as compared to the case of MHC restricted TCR recognition are used in the binding of either hapten or peptide antigens.

A fully active variant of dihydrofolate reductase with a circularly permuted sequence.

The amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene. By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved. The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein. Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme. The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant. This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus. Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process.

High-level production of biologically active human alpha 1-antitrypsin in Escherichia coli.

A cDNA clone containing the complete human alpha 1-antitrypsin sequence was isolated from a human liver cDNA bank by screening with a chemically synthesized oligonucleotide probe. DNA sequences encoding the alpha 1-antitrypsin mature polypeptide were inserted into an Escherichia coli expression vector that allows transcription from the efficient leftward promoter of bacteriophage lambda (PL) and initiation of translation at the lambda cII gene ribosome-binding site. This construction resulted in the induction of a 45-kilodalton protein at a level of approximately 15% of total cell protein. The polypeptide produced was recognized by antisera raised against human alpha 1-antitrypsin protein and displayed normal biological activity in an in vitro antielastase assay.

Promoter sequences of eukaryotic protein-coding genes.

In vitro genetic techniques were used to study the sequence requirements for the initiation of specific transcription. Deletion mutants were constructed around the putative promoter of the adenovirus-2 major late and chicken conalbumin genes. Specific transcription in vitro by RNA polymerase B together with a HeLa cell cytoplasmic extract was used as the test for promoter function. With this approach sequences which are essential for the initiation of specific transcription in vitro, were shown to be located between 12 and 32 base pairs upstream from the 5' end of these genes.