Development of intron targeted amplified polymorphic markers of metal homeostasis genes for monitoring their introgression from <Emphasis Type="Italic">Aegilops</Emphasis> species to wheat

The identification of transfers of useful alien genes for metal homeostasis from non-progenitor Aegilops species using the widely available anchored wheat SSR markers is difficult due to their lower polymorphism with the distant related wild species and the lack of locus specificity further restricts their application. The present study deals with the development of intron targeted amplified polymorphic (ITAP) markers for the metal homeostasis genes present on chromosomes of groups 2 and 7 of Triticeae. The mRNA sequences of 27 metal homeostasis genes were retrieved from different plant species using NCBI database and their BLASTn was performed against the wheat draft genome sequences in Ensemblplants to get exonic and intronic sequences of the corresponding metal homeostasis genes in wheat. The ITAP primers were developed in such a way that they would anneal to the conserved flanking exonic regions of the genes and amplify across highly variable introns within the PCR limits. The primers led to the amplification of variable intronic sequences of genes with polymorphism between non-progenitor Aegilops species and the recipient wheat cultivars. Further, the polymorphic ITAP markers were used to characterize the transfers of metal homeostasis genes from the non-progenitor Aegilops species to the BC₂F₅ wheat-Aegilops derivatives, developed through induced homoeologous pairing. The derivatives with significant percent increase in grain Fe and Zn content over the elite cultivar PBW343 LrP showed the introgression of some of the useful Aegilops alleles of the metal homeostasis genes. The use of different metal homeostasis genes using this approach is the first report of the direct contribution of the genes for increasing the grain micronutrient content for developing biofortified wheat lines with reduced linkage drag.     

Phosphorylation of the pro-apoptotic protein BIK: mapping of phosphorylation sites and effect on apoptosis

BIK is a pro-apoptotic BCL-2 family member and is the founding member of a subfamily of pro-apoptotic proteins known as "BH3-alone" proteins. Ectopic expression of BIK induces apoptosis in variety of mammalian cells. BIK complexes with various anti-apoptotic BCL-2 family proteins such as adenovirus E1B-19K and BCL-2 via the BH3 domain. However, the heterodimerization activity of BIK alone is insufficient for its apoptotic activity. Previous studies have shown that phosphorylation regulates the functional activity of both anti-apoptotic and pro-apoptotic members of the BCL-2 family. Here, we have examined phosphorylation of BIK and its effect on the apoptotic activity of BIK. We show that BIK exists as a phosphoprotein and is phosphorylated at residues 33 (threonine) and 35 (serine). Mutation of the phosphorylation sites, in which the Thr and Ser residues were changed to alanine residues, reduced the apoptotic activity of BIK without significantly affecting its ability to heterodimerize with BCL-2. Our results suggest that phosphorylation of BIK is required for eliciting efficient apoptotic activity. Partial purification of the protein kinase from HeLa cell cytoplasmic extracts suggest that BIK may be phosphorylated by a casein kinase II-related enzyme.     

Restriction endonuclease resistant chromatin in human chromosomes

Summary The recent addition of restriction endonucleases in obtaining selective bands in the human genome has added a new dimension to molecular genetics. However, a considerable discrepancy exists in banding patterns produced by AluI in chromosomes 19 and 20, by MboI in chromosomes 4, 5, 8, 21 and 22 and by RsaI in chromosomes 12, 21 and 22. The principal causes of these differences are highlighted.     

Influence of carbohydrates on quantitative aspects of growth and embryo formation in wild carrot suspension cultures

The rate of kaurene biosynthesis from mevalonate in a cell-free enzyme preparation from the endosperm of immature seeds of Marah macrocarpus is regulated by adenylate energy charge. The response curve is typical of a biosynthetic energy-utilizing sequence in which the rate of biosynthesis increases sharply as the energy charge is increased above 0.80. ADP proved to be an effective inhibitor of this process. AMP gave no inhibition at concentrations up to 2 mm and orthophosphate gave no inhibition up to 15 mm. Measurement of the pool sizes of intermediates in the sequence showed that the presence of ADP caused an increase in the levels of 5-phosphomevalonate and 5-pyrophosphomevalonate and a decrease in the levels of isopentenyl pyrophosphate and kaurene. These results indicate that pyrophosphomevalonate decarboxylase is the enzyme most subject to regulation by adenylate energy charge. The rate of conversion of isopentenyl pyrophosphate to kaurene and the rate of utilization of mevalonate by mevalonate kinase were not influenced by variations in the adenylate energy charge.     

Detection of fos protein during osteogenesis by monoclonal antibodies.

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.     

Identification and characterization of a UDP-D-glucuronate 4-epimerase in Arabidopsis

One of the major sugars present in the plant cell wall is d-galacturonate, the dominant monosaccharide in pectic polysaccharides. Previous work indicated that one of the activated precursors necessary for the synthesis of pectins is UDP-d-galacturonate, which is synthesized from UDP-d-glucuronate by a UDP-d-glucuronate 4-epimerase (GAE). Here, we report the identification, cloning and characterization of a GAE6 from Arabidopsis thaliana. Functional analysis revealed that this enzyme converts UDP-d-glucuronate to UDP-d-galacturonate in vitro. An expression analysis of this epimerase and its five homologs in the Arabidopsis genome by quantitative RT-PCR and promoter::GUS fusions indicated differential expression of the family members in plant tissues and expression of all isoforms in the developing pollen of A. thaliana.     

Influenza virus H1N1pdm09 infections in the young and old: evidence of greater antibody diversity and affinity for the hemagglutinin globular head domain (HA1 Domain) in the elderly than in young adults and children

The H1N1 2009 influenza virus (H1N1pdm09) pandemic had several unexpected features, including low morbidity and mortality in older populations. We performed in-depth evaluation of antibody responses generated following H1N1pdm09 infection of naïve ferrets and of 130 humans ranging from the very young (0 to 9 years old) to the very old (70 to 89 years old). In addition to hemagglutination inhibition (HI) titers, we used H1N1pdm09 whole-genome-fragment phage display libraries (GFPDL) to evaluate the antibody repertoires against internal genes, hemagglutinin (HA), and neuraminidase (NA) and also measured antibody affinity for antigenic domains within HA. GFPDL analyses of H1N1pdm09-infected ferrets demonstrated gradual development of antibody repertoires with a focus on M1 and HA1 by day 21 postinfection. In humans, H1N1pdm09 infection in the elderly (>70 years old) induced antibodies with broader epitope recognition in both the internal genes and the HA1 receptor binding domain (RBD) than for the younger age groups (0 to 69 years). Importantly, post-H1N1 infection serum antibodies from the elderly demonstrated substantially higher avidity for recombinant HA1 (rHA1) (but not HA2) than those from younger subjects (50% versus <22% 7 M urea resistance, respectively) and lower antibody dissociation rates using surface plasmon resonance. This is the first study in humans that provides evidence for a qualitatively superior antibody response in the elderly following H1N1pdm09 infection, indicative of recall of long-term memory B cells or long-lived plasma cells. These findings may help explain the age-related morbidity and mortality pattern observed during the H1N1pdm09 pandemic.