Current advances and future directions in genetic enhancement of a climate resilient food legume crop,cowpea (Vigna unguiculata L. Walp.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Cowpea (Vigna unguiculata (L.) Walp.) is a warm-season legume crop which is widely grown by resource-poor small and marginal farmers of Sub-Saharan...     

Genetic identity and diversity of Nigerian cacao genebank collections verified by single nucleotide polymorphisms (SNPs): a guide to field genebank management and utilization

Nigeria is the sixth largest cacao producer in the world. Field performance and quality of cacao hybrid families is largely dependent on the genetic integrity of parental clones obtained in field genebank collections. However, information on the impact of mislabeling on seed garden output in Nigeria is lacking. Using 63 single nucleotide polymorphism (SNP) markers, we analyzed 1457 cacao trees sampled from seven major field genebank plots in Nigeria to assess the genetic integrity in Nigerian cacao germplasm. The procedure of multilocus matching with known reference clones revealed up to 78% mislabeling in recently introduced international germplasm. A high rate of mislabeling was also revealed in the West African local selections and breeding lines, using Bayesian assignment test. The problem of mislabeling has been attributed to errors from the sources of introduction, pre-planting labeling errors, and rootstocks overtaking budded scions due to poor field management. The analysis of genetic diversity revealed a good representation of the available cacao germplasm groups in Nigerian field genebanks, indicating that the genetic base of Nigeria cacao germplasm has been significantly widened through germplasm introductions. However, only a small proportion of the available germplasm in the genebank have been utilized for variety development. This study proved the utility of SNP markers for cleaning up the genebanks and reducing offtypes; thereby providing a strong basis for improving the accuracy and efficiency in cacao genebank management and breeding, as well as for mobilizing improved varieties to cacao farmers in Nigeria.     

Microsatellite loci to assess genetic variation in Tor putitora

Seven polymorphic microsatellite DNA loci were identified in golden mahseer, Tor putitora, through cross‐species amplification. Thirty‐two primers developed for three cyprinid fishes were tested in the study. The genetic variation detected at each microsatellite locus in T. putitora specimens (n = 107), collected from three different rivers and one lake was assessed. The allele frequencies deviated significantly from that expected under Hardy–Weinberg equilibrium. The mean observed heterozygosity values ranged from 0.29 to 0.40. Significant genotype heterogeneity indicated that the samples were not drawn from the same gene pool. The results indicate that the identified microsatellite loci exhibit promise for use in fine scale population structure analyses of T. putitora.     

Current advances and future prospects in production of recombinant insulin and other proteins to treat diabetes mellitus

Biotechnology Letters - Diabetes mellitus is the most prevalent deadly disease caused by the destruction and dysfunction of pancreatic β cells that consequentially increased blood glucose...     

Changes in Water Soluble Protein Profile of Antarctic Cyanobacterium Nostoc Commune under Temperature Downshift from 25° to 5 °C

The cyanobacterium Nostoc commune (Nostocales) is an isolate from the Schirmacher Oasis Antarctica. The cyanobacterium is psychrotropic in nature; and maintained in laboratory at 25?°C temperature, in unialgal form. Here, we studied the change in protein profile of water soluble proteins from exponentially growing N. commune upon downshift from its optimum growth temperature (25?°C) to a low temperature (5?°C). Experimental set up used to analyze the proteome were- a sudden shift to low temperature (i.e., cold shock), after short- (8?days) and long-term acclimation (7?weeks) to low temperature (5?°C). Cold-shock resulted in an increase in Low molecular weight proteins (LMWPs) with clouding of diffused proteins. Further increase in the duration of incubation period (short- and long-term acclimation) caused dissociation of proteins, indicated by NaCl (50–600mM) induced dissociation of proteins. That is, high molecular weight proteins (HMWPs) dissociated into LMWPs resulting in an increased number of protein bands. This was further confirmed by addition of LMWPs (≤10KDa) resulting in re-association of proteins into HMWPs. Hence, we report that the cold-induced synthesis of LMWPs (≤10kDa) is a strategy adopted by the N. commune to survive at low temperature of Antarctica.     

Differential expression of DAHP synthase and chorismate mutase in various organs of Brassica juncea and the effect of external factors on enzyme activity

The multibranched shikimic acid pathway was discovered as the biosynthetic route to the aromatic amino acids phenylalanine, tyrosine and tryptophan and a host of other secondary metabolites. An extensive body of work is available on the characterization of various enzymes of this pathway in order to understand the underlying mechanisms of aromatic amino acid biosynthesis and secondary metabolism in higher plants. In the present investigation, selective assays, based on feedback regulation patterns and divalent cation requirements, were used to monitor the isozyme profiles of two of the key regulatory enzymes of this pathway. 3-Deoxy- d -arabino heptulosonate-7-phosphate synthase (DAHP synthase/DS) (EC 4.1.2.15) and chorismate mutase (CM) (EC 5.4.99.4) have been characterized from different vegetative and reproductive organs of Brassica juncea cv. Pusa Bold. An attempt has also been made to investigate the effect of external factors, such as light and wounding on the regulation of these enzymes. The results reveal differential expression of DAHP synthase and CM in various organs of Brassica and an adaptability of plants to various stresses by up or down regulation of these enzymes.     

Population genetic structure and phylogeography of cyprinid fish, <Emphasis Type="Italic">Labeo dero</Emphasis> (Hamilton, 1822) inferred from allozyme and microsatellite DNA marker analysis

We examined population structure of Labeo dero (Hamilton, 1822) from different riverine locations in India using 10 polymorphic allozyme and eight microsatellite loci. For analysis, 591 different tissue samples were obtained from commercial catches covering a wide geographic range. Allozyme variability (An = 1.28–1.43, Ho = 0.029–0.071) was much lower than for microsatellites (An = 4.625–6.125, Ho = 0.538–0.633). Existence of rare alleles was found at three allozyme (MDH-2*, GPI* and PGDH*) and at two microsatellite loci (R-3* and MFW-15*). Deviation from Hardy–Weinberg equilibrium (P < 0.05, after the critical probability levels were adjusted for sequential Bonferroni adjustment) could be detected at three loci (EST-1*, -2* and XDH*) whereas, after correction for null alleles, two microsatellite loci (MFW-1*,-15*) deviated from HWE in the river Yamuna. Fst for all the samples combined over all allozyme loci was found to be 0.059 suggesting that 5.9% of the total variation was due to genetic differentiation while microsatellite analysis yielded 0.019 which was concordant to mean Rst (0.02). Hierarchical partition of genetic diversity (AMOVA) showed that greater variability (approx. 95%) was due to within population component than between geographical regions. Based on distribution of genetic differentiation detected by both markers, at least five different genetic stocks of L. dero across its natural distribution could be identified. These results are useful for the evaluation and conservation of L. dero in natural water bodies.     

Human plasma high-density lipoproteins are stabilized by kinetic factors

High-density lipoproteins (HDL) are heterogeneous complexes of proteins and lipids that mediate cholesterol removal from the body. Our thermal and chemical denaturation studies of mature spherical HDL isolated from human plasma show that, contrary to the widely held assumption, the particle stability has a kinetic rather than thermodynamic origin. Guanidinum hydrochloride (GdmHCl) concentration jumps at 25 degrees C monitored by circular dichroism (CD) at 222 nm reveal two dominant irreversible kinetic phases in HDL denaturation. The slower phase (relaxation time tau(1) approximately 2 x 10(4) seconds) is observed in 1-6 M GdmHCl, and the faster phase (tau(2) approximately 2 x 10(3) seconds) is detected in 3-6 M GdmHCl. Comparison of the free energy barriers associated with these phases, deltaG* = 16-17 kcal mol(-1), with the near-zero apparent thermodynamic stability inferred from the spectroscopic measurements after prolonged incubation in 0-6 M GdmHCl at 22 degrees C indicates the kinetic origin for HDL stabilization. Electron microscopic analysis of HDL incubated in 0-6 M GdmHCl suggests that the slower kinetic phase involves HDL fusion, while the faster phase involves particle rupture and release of the apolar lipid core. Thermal denaturation experiments indicate high enthalpic barriers for the particle rupture that may arise from the transient disruption of lipid and/or protein packing interactions. These results corroborate our earlier analysis of model discoidal HDL and indicate that a kinetic mechanism provides a universal natural strategy for lipoprotein stabilization. Such a mechanism may facilitate structural integrity of the heterogeneous lipoprotein particles, slow their spontaneous interconversions, and thereby modulate lipoprotein lifetime and functions.     

Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin,HbN in Escherichia coli

Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.