Applications of CRISPR/Cas9 technology for modification of the plant genome

Genetica - The CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) system was discovered in bacteria and archea as an acquired immune response to...     

Evolution of Escherichia coli in different carbon environments for 2,000 generations

Cellular energetics is thought to have played a key role in dictating all major evolutionary transitions in the history of life on Earth. However, how exactly cellular energetics and metabolism come together to shape evolutionary paths is not well understood. In particular, when an organism is evolved in different energy environments, what are the phenomenological differences in the chosen evolutionary trajectories, is a question that is not well understood. In this context, starting from an Escherichia coli K‐12 strain, we evolve the bacterium in five different carbon environments—glucose, arabinose, xylose, rhamnose and a mixture of these four sugars (in a predefined ratio) for approximately 2,000 generations. At the end of the adaptation period, we quantify and compare the growth dynamics of the strains in a variety of environments. The evolved strains show no specialized adaptation towards growth in the carbon medium in which they were evolved. Rather, in all environments, the evolved strains exhibited a reduced lag phase and an increased growth rate. Sequencing results reveal that these dynamical properties are not introduced via mutations in the precise loci associated with utilization of the sugar in which the bacterium evolved. These phenotypic changes are rather likely introduced via mutations elsewhere on the genome. Data from our experiments indicate that evolution in a defined environment does not alter hierarchy in mixed‐sugar utilization in bacteria.     

Genomewide computational analysis of nitrate response elements in rice and Arabidopsis

Nitrate response element (NRE) was originally reported to be comprised of an Ag/cTCA core sequence motif preceded by a 7-bp AT rich region, based on promoter deletion analyses in nitrate and nitrite reductases from Arabidopsis thaliana and birch. In view of hundreds of new nitrate responsive genes discovered recently, we sought to computationally verify whether the above motif indeed qualifies to be the cis-acting NRE for all the responsive genes. We searched for the specific occurrence of at least two copies of the above motif in and around the nitrate responsive genes and elsewhere in the Arabidopsis and rice (Oryza sativa) genomes, with respect to their positional, orientational and strand-specific bias. This is the first comprehensive analysis of NREs for 625 nitrate responsive genes of Arabidopsis and their rice homologs, representing dicots and monocots, respectively. We report that the above motifs are present almost randomly throughout these genomes and do not reveal any specificity or bias towards nitrate responsive genes. This also seems to be true for smaller subsets of nitrate responsive genes in Arabidopsis, such as the 21 early responsive genes, 261 and 90 genes for root-specific and shoot-specific response, respectively, and 25 housekeeping genes. This necessitates a fresh search for candidate sequences that qualify to be NREs in these and other plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of different moieties from the lipooligosaccharide molecule in biological activities of the Moraxella catarrhalis outer membrane

Lipooligosaccharide (LOS), a major component of the outer membrane of Moraxella catarrhalis, consists of two major moieties: a lipid A and a core oligosaccharide (OS). The core OS can be dissected into a linker and three OS chains. To gain an insight into the biological activities of the LOS molecules of M. catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a serotype A O35Elgt3 LOS mutant. MALDI-TOF-MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted of a Glc-Kdo(2) (linker)-lipid A structure. Phenotypic analysis revealed that the mutant was similar to the wild-type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents. However, the mutant was sensitive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells. These data, combined with our previous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, whereas the linker moiety is critical for maintenance of the outer membrane integrity and stability to preserve normal cell growth. Both the lipid A and linker moieties contribute to the LOS toxicity.     

Extent of mismatch between the period of circadian clocks and light/dark cycles determines time‐to‐emergence in fruit flies

Circadian clocks time developmental stages of fruit flies Drosophila melanogaster, while light/dark (LD) cycles delimit emergence of adults, conceding only during the “allowed gate.” Previous studies have revealed that time‐to‐emergence can be altered by mutations in the core clock gene period (per), or by altering the length of LD cycles. Since this evidence came from studies on genetically manipulated flies, or on flies maintained under LD cycles with limited range of periods, inferences that can be drawn are limited. Moreover, the extent of shortening or lengthening of time‐to‐emergence remains yet unknown. In order to pursue this further, we assayed time‐to‐emergence of D. melanogaster under 12 different LD cycles as well as in constant light (LL) and constant dark conditions (DD). Time‐to‐emergence in flies occurred earlier under LL than in LD cycles and DD. Among the LD cycles, time‐to‐emergence occurred earlier under T4–T8, followed by T36–T48, and then T12–T32, suggesting that egg‐to‐emergence duration in flies becomes shorter when the length of LD cycles deviates from 24 h, bearing a strong positive and a marginally negative correlation with day length, for values shorter and longer than 24 h, respectively. These results suggest that the extent of mismatch between the period of circadian clocks and environmental cycles determines the time‐to‐emergence in Drosophila.     

Regulation of hindbrain Pyy expression by acute food deprivation, prolonged caloric restriction, and weight loss surgery in mice

PYY is a gut-derived putative satiety signal released in response to nutrient ingestion and is implicated in the regulation of energy homeostasis. Pyy-expressing neurons have been identified in the hindbrain of river lamprey, rodents, and primates. Despite this high evolutionary conservation, little is known about central PYY neurons. Using in situ hybridization, PYY-Cre;ROSA-EYFP mice, and immunohistochemistry, we identified PYY cell bodies in the gigantocellular reticular nucleus region of the hindbrain. PYY projections were present in the dorsal vagal complex and hypoglossal nucleus. In the hindbrain, Pyy mRNA was present at E9.5, and expression peaked at P2 and then decreased significantly by 70% at adulthood. We found that, in contrast to the circulation, PYY-(1-36) is the predominant isoform in mouse brainstem extracts in the ad libitum-fed state. However, following a 24-h fast, the relative amounts of PYY-(1-36) and PYY-(3-36) isoforms were similar. Interestingly, central Pyy expression showed nutritional regulation and decreased significantly by acute starvation, prolonged caloric restriction, and bariatric surgery (enterogastroanastomosis). Central Pyy expression correlated with body weight loss and circulating leptin and PYY concentrations. Central regulation of energy metabolism is not limited to the hypothalamus but also includes the midbrain and the brainstem. Our findings suggest a role for hindbrain PYY in the regulation of energy homeostasis and provide a starting point for further research on gigantocellular reticular nucleus PYY neurons, which will increase our understanding of the brain stem pathways in the integrated control of appetite and energy metabolism.     

A novel role for RAD54: this host protein modulates geminiviral DNA replication

Geminiviruses primarily encode only few factors, such as replication initiator protein (Rep), and need various host cellular machineries for rolling-circle replication (RCR) and/or recombination-dependent replication (RDR). We have identified a host factor, RAD54, in a screen for Rep-interacting partners and observed its role in DNA replication of the geminivirus mungbean yellow mosaic India virus (MYMIV). We identified the interacting domains ScRAD54 and MYMIV-Rep and observed that ScRAD54 enhanced MYMIV-Rep nicking, ATPase, and helicase activities. An in vitro replication assay demonstrated that the geminiviral DNA replication reaction depends on the viral Rep protein, viral origin of replication sequences, and host cell-cycle proteins. Rad54-deficient yeast nuclear extract did not support in vitro viral DNA replication, while exogenous addition of the purified ScRAD54 protein enhanced replication. The role of RAD54 in in planta replication was confirmed by the transient replication assay; i.e., agroinoculation studies. RAD54 is a well-known recombination/repair protein that uses its DNA-dependent ATPase activity in conjunction with several other host factors. However, this study demonstrates for the first time that the eukaryotic rolling-circle replicon depends on the RAD54 protein.     

The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase

Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).