Epitopes of human chorionic gonadotropin and their relationship to immunogenicity and cross-reactivity of beta-chain mutants.

PROBLEM: Human chrionic gonadotropin (hCG) is a placental glycoprotein hormone, a heterodimeric molecule, consisting of alpha and beta chains. It induces the synthesis of progesterone, which is essential for the maintenance of the fertilized egg. Antibodies directed against hCG can, therefore, prevent pregnancy and serve as a vaccine. hCG belongs to the glycoprotein hormone family and shares the alpha chain with the other members. The beta chain is a hormone-specific subunit that is unique to hCG, but still possesses 85% amino acid homology with the beta chain of luteinizing hormone (LH), which means that prolonged immunization with hCG produces antibodies that cross-react with LH. METHOD OF STUDY: We have taken an approach involving the mutation of beta hCG to eliminate cross-reactive epitopes without affecting the natural folding of the polypeptide chain and thus the unique beta hCG-specific epitopes. RESULTS: Several mutants have been constructed that have maintained the binding to hCG-specific monoclonal antibodies (mAbs) but have lost the ability to bind to a panel of LH cross-reactive mAbs. To investigate the immunogenicity of selected mutants, mice were immunized with expression plasmid DNA, containing the gene for wild-type beta hCG and two mutants: mutant 3, with four amino acid substitutions (68 Arg-->Glu; 74 Arg-->Ser; 75 Gly-->His; 79 Val-->His), and mutant 7, with a single amino acid substitution (68 Arg-->Glu). CONCLUSIONS: Although both mutants were able to elicit antibody responses in at least some animals, the levels were less than those seen with the wild-type beta hCG DNA, and there seems still to be a residual cross-reactivity with LH. Attempts to improve the immunogenicity of the mutants and to further modify the sequence to remove the cross-reactivity are currently underway.

Reduced turnover of the D1 polypeptide and photoactivation of electron transfer in novel herbicide resistant mutants of Synechocystis sp. PCC 6803.

Two missense mutants, A263P and S264P, and a deletion mutant des-Ala263, Ser264, have been constructed in the D1 protein of the cyanobacterium Synechocystis sp PCC 6803. All were expected to induce a significant conformational change in the QB-binding region of photosystem II (PSII). Although the des-Ala263, Ser264-D1 mutant accumulated some D1 protein in the thylakoid membrane it was unable to grow photoautotrophically or evolve oxygen. Thermoluminescence and chlorophyll fluorescence studies confirmed that this deletion mutant did not show any functional PSII activity. In contrast, [S264P]D1 was able to grow photoautotrophically and give light-saturated rates of oxygen evolution at 60% of the rate of the wild-type control strain, TC31. The A263P missense mutant was also able to evolve oxygen at 50% of TC31 rates although it did not readily grow photoautotrophically. Thermoluminescence, flash oxygen yield and chlorophyll fluorescence measurements indicated that in both missense mutants electron transfer from QA to QB was significantly impaired in dark adapted cells. However, QA to QB electron transfer could be photoactivated in the mutants by background illumination. Both the A263P and S264P mutants also showed an increase in resistance to the s-triazine family of herbicides although this feature did not hold for the phenolic herbicide, ioxynil. Of particular interest was that the two missense mutants, especially S264P, possessed a slower rate of turnover of the D1 protein compared with TC31 and in vivo contained detectable levels of a 41-kDa adduct consisting of D1 and the alpha subunit of cytochrome b559. When protein synthesis was blocked by the addition of lincomycin, D1 degradation was again slower in S264P than TC31. The results are discussed in terms of structural changes in the QB-binding region which affect herbicide and plastoquinone binding and perturb the normal regulatory factors that control the degradation of the D1 protein and its synchronisation with the synthesis of a replacement D1 protein.

An aroA homologue from Synechocystis sp. PCC 6803.

In this study, we report the entire nucleotide sequence of an aroA homologue encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), isolated from the cyanobacterium Synechocystis sp. PCC 6803. The proposed coding region is an open reading frame of 447 amino acids. The deduced sequence of the gene product is particularly similar to the Gram+ EPSPS sequences available to date, in particular to that in Bacillus subtilis. Analysis of the Synechocystis putative EPSPS sequence does not lead to an obvious explanation for the natural tolerance of this cyanobacterium to glyphosate.

The genes aroA and trnQ are located upstream of psbO in the chromosome of Synechocystis 6803.

We have identified the existence of two genes, trnQ and aroA, located upstream of the psbO gene in Synechocystis sp. PCC 6803. The trnQ gene encodes a glutamine-specific transfer RNA (tRNA(Gln)) and the sequence given is the first reported for any cyanobacterium. The gene seems to exist as a single copy since its deletion results in non-viable mutation. The aroA gene encodes for 5-enolpyruvylshikimate 3-phosphate synthase and its discovery in the genome of Synechocystis 6803 is the first genetic evidence for the existence of the shikimate biosynthetic pathway in cyanobacteria. Interestingly, the partial sequence shares close homologies with the sequences of aroA from Gram-positive bacteria.