Genetic diversity and relationships among 6 Amaranthus species from 8 phytogeographic regions of the Indo-Gangetic plains were analyzed using a random amplified polymorphic DNA (RAPD) marker. RAPD primers yielded a total of 262 amplicons, ranging from approximately 250 to approximately 3000 bp in size with an average of 13.1 amplicons per primer, of which 254 amplicons (96.94%) were polymorphic. The genetic similarity coefficient among all the Amaranthus species ranged from 0.16 to 0.97 with a mean similarity coefficient of 0.56, indicating that variation existed in the genetic diversity of different populations. In the unweighted pair group method with arithmetic average dendrogram, populations of the same species clustered together. A unique 1371-bp RAPD band specific for Amaranthus gangeticus (syn. tricolor) of a particular phytogeographic region was converted to a sequenced characterized amplified region (SCAR) marker. The translated marker sequence showed homology with hemagglutinin protein. This SCAR marker is potentially useful for germplasm conservation and identification of amaranth ecotype.

Hemp (Cannabis sativa L.) cultivation and utilization is an ancient practice to human civilization. There are some controversies on the origin and subsequent spread of this species. Ancient plant DNA has proven to be a powerful tool to solve phylogenetic problems. In this study, ancient DNA was extracted from an archaeological specimen of Cannabis sativa associated with archaeological human remains from China. Ribosomal and Cannabis specific chloroplast DNA regions were PCR amplified. Sequencing of a species-specific region and subsequent comparison with published sequences were performed. Successful amplification, sequencing and sequence comparison with published data suggested the presence of hemp specific DNA in the archeological specimen. The role of Humulus japonicus Sieb. et Zucc. in the evolution of Cannabis is also indicated. The identification of ancient DNA of 2500 years old C. sativa sample showed that C. sativa races might have been introduced into China from the European-Siberian center of diversity.

Papain is a plant cysteine protease of industrial importance having a two-domain structure with its catalytic cleft located at the domain interface. A structure-based rational design approach has been used to improve the thermostability of papain, without perturbing its enzymatic activity, by introducing three mutations at its interdomain region. A thermostable homologue in papain family, Ervatamin C, has been used as a template for this purpose. A single (K174R), a double (K174RV32S) and a triple (K174RV32SG36S) mutant of papain have been generated, of which the triple mutant shows maximum thermostability with the half-life (t(1/2)) extended by 94 min at 60 degrees C and 45 min at 65 degrees C compared to the wild type (WT). The temperature of maximum enzymatic activity (T(max)) and 50% maximal activity (T(50)) for the triple mutant increased by 15 and 4 degrees C, respectively. Moreover, the triple mutant exhibits a faster inactivation rate beyond T(max) which may be a desirable feature for an industrial enzyme. The values of t(1/2) and T(max) for the double mutant lie between those of the WT and the triple mutant. The single mutant however turns out to be unstable for biochemical characterization. These results have been substantiated by molecular modeling studies which also indicate highest stability for the triple mutant based on higher number of interdomain H-bonds/salt-bridges, less interdomain flexibility and lower stability free-energy compared to the WT. In silico studies also explain the unstable behavior of the single mutant.

Papain is the archetype of a broad class of cysteine proteases (clan C1A) that contain a pro-peptide in the zymogen form which is required for correct folding and spatio-temporal regulation of proteolytic activity in the initial stages after expression. This study reports the X-ray structure of the zymogen of a thermostable mutant of papain at 2.6 Å resolution. The overall structure, in particular that of the mature part of the protease, is similar to those of other members of the family. The structure provides an explanation for the molecular basis of the maintenance of latency of the proteolytic activity of the zymogen by its pro-segment at neutral pH. The structural analysis, together with biochemical and biophysical studies, demonstrated that the pro-segment of the zymogen undergoes a rearrangement in the form of a structural loosening at acidic pH which triggers the proteolytic activation cascade. This study further explains the bimolecular stepwise autocatalytic activation mechanism by limited proteolysis of the zymogen of papain at the molecular level. The possible factors responsible for the higher thermal stability of the papain mutant have also been analyzed.