Testing of newly developed glycophospholipid antigen for the detection of P. falciparum malaria by laser light immunoassay in endemic and non-endemic areas.

A glycophospholipid (GPL) antigen isolated from Plasmodium falciparum culture supernatant has been tested for its antigenicity. Detection of malaria positive known blood samples and unknown field samples from endemic and non-endemic areas were compared. In this study laser light scattering immunoassay (LIA) was used for the detection of P. falciparum malaria. Test results of control (malaria negative samples from Surat) were compared with known positive samples and unknown malaria positive field samples. A positive correlation has been observed (97%) in falciparum positive samples from laboratory and unknown samples from endemic area (Haldwani) by LIA method using GPL antigen. From the results of the study it was found that GPL antigen has a better antigenic property and can detect almost all the cases of Pf malaria by LIA method.

An alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system.

The type II restriction endonuclease, Bam HI, has been overexpressed in E. coli by cloning the Bam HI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of Bam HI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK14. Our overexpressing clone, pAABRw in BL21 cells in presence of Bam HI methylase in pMAP6 following induction with IPTG yields about 9.2 x 10(6) units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH139 cells even in the absence of T7 polymerase and cognate Bam HI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.

DNA recognition and structural specificities.

How a short DNA sequence interacts in a sequence specific manner with appropriate protein is understood only in certain systems for which high resolution crystal structures of the protein-DNA complexes are available. The base sequence of DNA is sensed directly (read-out) by the protein through the major or minor groove, while DNA shape also is sensed through multiple interactions with the sugar phosphate backbone. Several repressors, activators and restriction endonucleases complexed with their cognate DNA oligomers are now known and reviewed here. If the binding site on DNA has two fold symmetry, the protein interacts as dimer and uses a variety of structural motifs for specific interaction. The level of specificity of interaction is enhanced by flexibility and/or distortion in either the DNA or protein tertiary structure.

Cloning of ferredoxin I gene from Azotobacter vinelandii using synthetic oligonucleotide probes.

Two synthetic oligonucleotide probe mixtures, whose sequences were inferred from two separate stretches of amino acids, one closer to the carboxy terminal and the other closer to the amino terminal, of ferredoxin I protein of Azotobacter vinelandii, were used to select ferredoxin I gene clones from a cosmid gene library of Azotobacter vinelandii. Restriction analysis revealed that 7 out of 10 selected clones were of the same type. All these clones were found to hybridize with fixABCX genes of Rhizobium meliloti.