Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.     

Genetic analysis identifies a function for the queC (ybaX) gene product at an initial step in the queuosine biosynthetic pathway in Escherichia coli

Queuosine (Q), one of the most complex modifications occurring at the wobble position of tRNAs with GUN anticodons, is implicated in a number of biological activities, including accuracy of decoding, virulence, and cellular differentiation. Despite these important implications, its biosynthetic pathway has remained unresolved. Earlier, we observed that a naturally occurring strain of Escherichia coli B105 lacked Q modification in the tRNAs. In the present study, we developed a genetic screen to map the defect in E. coli B105 to a single gene, queC (renamed from ybaX), predicted to code for a 231-amino-acid-long protein with a pI of 5.6. As analyzed by mobility of tRNA(Tyr) on acid urea gels and two-dimensional thin-layer chromatography of the modified nucleosides, expression of QueC from a plasmid-borne copy confers a Q+ phenotype to E. coli B105. Further, analyses of tRNA(Tyr) from E. coli JE10651 (queA mutant), its derivative generated by deletion of chromosomal queC (queA deltaqueC), and E. coli JE7325, deficient in converting preQ0 to preQ1, have provided the first genetic evidence for the involvement of QueC at a step leading to production of preQ0, the first known intermediate in the generally accepted pathway that utilizes GTP as the starting molecule. In addition, we discuss the possibilities of collaboration of QueC with other cellular proteins in the production of preQ0.     

Plasmodium falciparum: characterization of a late asexual stage golgi protein containing both ankyrin and DHHC domains

Proteins containing the DHHC motif have been shown to function as palmitoyl transferases. The palmitoylation of proteins has been shown to play an important role in the trafficking of proteins to the proper subcellular location. Herein, we describe a protein containing both ankyrin domains and a DHHC domain that is present in the Golgi of late schizonts of P. falciparum. The timing of expression as well as the location of this protein suggests that it may play an important role in the sorting of proteins to the apical organelles during the development of the asexual stage of the parasite.     

Role of caveolae in Leishmania chagasi phagocytosis and intracellular survival in macrophages

Caveolae are membrane microdomains enriched in cholesterol, ganglioside M1 (GM1) and caveolin-1. We explored whether caveolae facilitate the entry of Leishmania chagasi into murine macrophages. Transient depletion of macrophage membrane cholesterol by 1 h exposure to methyl-beta-cyclodextrin (MbetaCD) impaired the phagocytosis of non-opsonized and serum-opsonized virulent L. chagasi. In contrast, MbetaCD did not affect the phagocytosis of opsonized attenuated L. chagasi. As early as 5 min after phagocytosis, virulent L. chagasi colocalized with the caveolae markers GM1 and caveolin-1, and colocalization continued for over 48 h. We explored the kinetics of lysosome fusion. Whereas fluorescent-labelled dextran entered macrophage lysosomes by 30 min after addition, localization of L. chagasi in lysosomes was delayed for 24-48 h after phagocytosis. However, after transient depletion of cholesterol from macrophage membrane with MbetaCD, the proportion of L. chagasi-containing phagosomes that fused with lysosomes increased significantly. Furthermore, intracellular replication was impaired in parasites entering after transient cholesterol depletion, even though lipid microdomains were restored by 4 h after treatment. These observations suggest that virulent L. chagasi localize in caveolae during phagocytosis by host macrophages, and that cholesterol-containing macrophage membrane domains, such as caveolae, target parasites to a pathway that promotes delay of lysosome fusion and intracellular survival.     

Prodrug and covalent linker strategies for the solubilization of dual-action antioxidants/iron chelators

Water soluble prodrugs of hybrid free radical scavenger/iron chelating molecules, based on 3,5-disubstituted-4-hydroxyphenyl derivatives and 3-hydroxy-2-methyl-4(1H)-pyridinone (deferiprone), have been prepared. Related hybrid molecules containing a covalent poly(ethylene)glycol or an amine linker were also synthesized.     

Effect of undernutrition on deciduous tooth emergence among Rajput children of Shimla District of Himachal Pradesh, India

This article examines the influence of nutritional status on the emergence of deciduous dentition in a cross-sectional sample of 510 rural Rajput children from the Jubbal and Kotkhai Tehsils, Shimla District, Himachal Pradesh, India. The nutritional status of each child was evaluated using Z-scores of height/supine length-for-age (HAZ), weight-for-age (WAZ), and weight-for-height (WHZ). The effects of sex and side on deciduous dental emergence were not statistically significant. Partial correlation indicates that the number of emerged teeth (T) was more strongly correlated with height than with other anthropometric variables. In most age groups, the stunted boys and girls (HAZ <-2) had fewer emerged teeth than nonstunted age peers (HAZ >-2). The mean T in underweight children was also less than that of the normal children, with a few exceptions. The stunted children have a significantly greater likelihood of delayed emergence of deciduous dentition. Measures of linear growth status are more closely related to dental development than measures of growth in mass. The findings indicate that even moderate undernutrition can delay deciduous tooth emergence.     

Genetics and characterization of an open flower mutant in chickpea

The chickpea (Cicer arietinum L.) is a self-pollinated grain legume with cleistogamous flowers. A spontaneous open-flower mutant, designated OFM-3, was identified in which reproductive organs were not enclosed by the keel petals and thus remained exposed. All 10 stamens in this mutant were free, whereas these are in diadelphous (9 fused + 1 free) condition in normal chickpea flowers. A large number of pods (73%) remained unfilled (empty) in OFM-3, though its pollen fertility was as high as the standard cultivars. The open-flower trait was found to be recessive and controlled by a single gene. OFM-3 was crossed with earlier reported open-flower mutants, ICC 16341 and ICC 16129, to establish trait relationships of genes controlling open flower traits in these mutants. It was found that each of these mutants has a unique gene for open flower trait. The genes controlling open flower trait in ICC 16341, ICC 16129, and OFM-3 were designated ofl-1, ofl-2, and ofl-3, respectively. Breeding lines with open flower trait and higher percentage of filled pods have been developed from the progenies of the crosses of OFM-3 with normal-flowered lines. The open flower trait offers opportunity for exploring hybrid technology in the chickpea.     

Recruitment of memory B cells to lymph nodes remote from the site of immunization requires an inflammatory stimulus

Successful recall Ab responses require recruitment of quiescent memory B cells to secondary lymphoid organs. However, the cellular dynamics of memory cells responding to local antigenic challenge at lymphoid sites distal from the initial Ag encounter are not well understood. We show in this study that memory B cells generated following s.c. immunization in one footpad generate secondary responses to soluble Ag given i.p. but not to Ag given s.c. in the contralateral footpad unless LPS is coadministered. Memory B cells do not express CD62L, and CD62L(-ve) cells cannot enter lymph nodes unless LPS-mediated inflammation is induced there. Functional TLR4 is required on the B cells, as well as on non-B cells, in the lymph node to achieve full recruitment. Furthermore, splenectomized mice fail to respond to such inflammatory s.c. challenge in contralateral footpads, unlike lymphadenectomized mice lacking the original draining lymph nodes. Splenectomized mice also fail to respond to i.p. challenge with soluble Ag. Together, these data indicate that, unlike the central memory pool of T cells, which circulates through resting lymph nodes, the majority of long-lived memory B cells are spleen resident and require inflammatory signals for mounting recall responses at distal challenge sites.