Cloning of ribosomal RNA genes from an Indian isolate of Giardia lamblia and the use of intergenic nontranscribing spacer regions in the differentiation of Giardia from other enteric pathogens.

The ribosomal RNA genes from an Indian isolate of Giardia lamblia have been cloned and characterized with respect to size, composition and copy number. Southern blotting and rDNA cloning of Giardia lamblia revealed that genes coding for ribosomal RNA (rRNA) are exceptionally small and are encoded within a 5.6 kb genome fragment repeat. The rDNA repeat unit of this isolate was found to be highly G-C rich like other human isolates and the physical map showed several SmaI sites. There are 132 copies of the rDNA repeat unit per cell in a head to tail arrangement. Two fragments corresponding to intergenic (0.2 kb and 0.3 kb) region and one (0.8 kb) containing both an intergenic region and a small part of the small subunit ribosomal RNA (SS rRNA) have been identified within the rDNA. These were used in heterogeneity studies of Giardia isolated from two geographic locations as well as in the analysis of cross reactivity with other enteric organisms. In Southern blots, all the three fragments were found to be highly specific for the differential diagnosis of Giardia spp. from the other enteric pathogens. These findings should help in developing a sensitive and more specific method for the diagnosis of giardiasis over currently available techniques.

Mutagenesis study to ameliorate the bacterial expression of phosphoprotein P of vesicular stomatitis virus.

The phosphoprotein gene of vesicular stomatitis virus, a Rhabdovirus, has been inserted into bacterial expression plasmids containing the Escherichia coli tac promoter and ribosome binding site (RBS). A low level of expression of the protein was detected. Sequence analysis showed the presence of 15 nucleotides in the spacer region i.e., between the Shine- Dalgarno sequence and ATG. Alteration of the distance and the sequence in the spacer region by oligonucleotide-directed mutagenesis revealed a correlation among the expression levels, accessibility of the RBS and requirement for a minimum spacing of at least 7 nucleotides between the Shine-Dalgarno sequence and ATG for optimal gene expression.

Effect of proteolytic digestion on the function of vesicular stomatitis virus ribonucleoprotein complex.

The nucleocapsid protein (49 Kd) of vesicular stomatitis virus is tightly bound to the genome rendering the latter transcriptionally competent. Controlled digestion with chymotrypsin removed a 12 Kd peptide from the complex. The resulting complex failed to serve as template for genome transcription in vitro when the polymerase components L and NS proteins were added. A template-associated protein kinase activity was also lost upon chymotrypsin treatment. However, the cleaved nucleocapsid protein (37 Kd) was still capable of binding tightly with the genome template and retained the epitope recognized by a monoclonal antibody. These results suggest that the nucleocapsid protein possesses separate domains that mediate binding to polymerase complex and maintain the structural integrity of the template.