Basuthkar J. Rao

APOPTOTIC-LIKE CELL DEATH PATHWAY IS INDUCED IN UNICELLULAR CHLOROPHYTE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELLS FOLLOWING UV IRRADIATION: DETECTION AND FUNCTIONAL ANALYSES1

Chlamydomonas reinhardtii (Ehrenberg) cells exhibited cell death process akin to that of apoptosis when exposed to ultraviolet (UV)‐C irradiation (1–100 J/m2). We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes, flipping of phosphatidylserine, and DNA fragmentation detected by the terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling assay and oligonucleosomal DNA laddering assay. Interestingly, fluorescence imaging of DNA changes in UV‐C exposed cells, following PicoGreen staining, revealed that extra‐nuclear DNA disintegrates before that of nuclear changes, where the latter extensively diffuses out of the nuclear compartment, spreading into the whole cell and reaching the periphery of dying cells. Antibodies against a mammalian caspase‐3 shared epitopes with a protein of 28 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, growth experiments indicate that spent medium recovered from UV‐C exposed cells exhibit a protective effect against cell killing of fresh cultures of C. reinhardtii cells by UV irradiation. The protective effect of UV‐spent medium is not a general growth promotional response on normal cells, but rather, is specific to UV‐exposed cells. We propose a model that C. reinhardtii cells exposed to UV elicit apoptotic‐like changes, which in turn lead to an adaptive response in neighboring cells against fresh rounds of UV exposure, thereby promoting survival of the cell population.

A homologue of the defender against the apoptotic death gene (dad1) in UV-exposed Chlamydomonas cells is downregulated with the onset of programmed cell death

We report here the isolation of a homologue of the potential anti-apoptotic gene, defender against apoptotic death (dad1) from Chlamydomonas reinhardtii cells. Using polymerase chain reaction (PCR), we investigated its expression in the execution process of programmed cell death (PCD) in UV-C exposed dying C. reinhardtii cells. Reverse-transcriptase (RT)-PCR showed that C. reinhardtii dad1 amplification was drastically reduced in UV-C exposed dying C. reinhardtii cells. We connect the downregulation of dad1 with the upregulation of apoptosis protease activating factor-1 (APAF-1) and the physiological changes that occur in C. reinhardtii cells upon exposure to 12 J/m2 UV-C in order to show a reciprocal relationship between proapoptotic and inhibitor of apoptosis factors. The temporal changes indicate a correlation between the onset of cell death and dad1 downregulation. The sequence of the PCR product of the cDNA encoding the dad1 homologue was aligned with the annotated dad1 (C_20215) from the Chlamydomonas database (http://genome.jgi-psf.org:8080/annotator/servlet/jgi.annotation.Annotation?pDb=chlre2); Annotation?pDb=chlre2); this sequence was found to show 100% identity, both at the nucleotide and amino acid level. The 327 bp transcript showed an open reading frame of 87 amino acid residues. The deduced amino acid sequence of the putative C. reinhardtii DAD1 homologue showed 54% identity with Oryza sativa, 56% identity with Drosophila melanogaster, 66% identity with Xenopus laevis, and 64% identity with Homo sapiens, Sus scrofa, Gallus gallus, Rattus norvegicus and Mus musculus.

Chromosome territories reposition during DNA damage-repair response

BackgroundLocal higher-order chromatin structure, dynamics and composition of the DNA are known to determine double-strand break frequencies and the efficiency of repair. However, how DNA damage response affects the spatial organization of chromosome territories is still unexplored.ResultsOur report investigates the effect of DNA damage on the spatial organization of chromosome territories within interphase nuclei of human cells. We show that DNA damage induces a large-scale spatial repositioning of chromosome territories that are relatively gene dense. This response is dose dependent, and involves territories moving from the nuclear interior to the periphery and vice versa. Furthermore, we have found that chromosome territory repositioning is contingent upon double-strand break recognition and damage sensing. Importantly, our results suggest that this is a reversible process where, following repair, chromosome territories re-occupy positions similar to those in undamaged control cells.ConclusionsThus, our report for the first time highlights DNA damage-dependent spatial reorganization of whole chromosomes, which might be an integral aspect of cellular damage response.

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

Pierce’s disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

Transition metal saccharide chemistry and biology: Synthesis, characterization, electrochemistry and EPR studies of oxovanadium(IV) complexes of saccharides and their derivatives and in vitro interaction of some of these with ribonuclease and deoxyribonuclease

Low molecular weight, water-soluble saccharide complexes of oxovanadium(IV) have been synthesized and characterized by analytical, spectroscopic and electrochemical techniques. All the complexes were found to be mononuclear, possessing the VO2+ moiety. These are shown to be hydrolytically and oxidatively stable over a wide range of pH (1-12) and have been extensively characterized by absorption and EPR spectroscopy and by electrochemistry. Several correlations have been drawn from the data generated. Some of these complexes have been demonstrated to possess in vitro RNase inhibition activity with no effect on DNase. This suggests that these molecules closely mimic the substrate portion of the RNase-catalysed RNA hydrolysis and can act as transition-state analogues to RNase.

Inhibition of a cold-active alkaline phosphatase by imipenem revealed by in silico modeling of metallo-β-lactamase active sites

We demonstrate the inhibition of the native phosphatase activity of a cold active alkaline phosphatase from Vibrio (VAP) (IC50 of 44 ± 4 (n = 4) μM at pH 7.0 after a 30 min preincubation) by a specific β‐lactam compound (only by imipenem, and not by ertapenem, meropenem, ampicillin or penicillin G). The homologous scaffold was detected by an in silico analysis that established the spatial and electrostatic congruence of the active site of a Class B2 CphA metallo‐β‐lactamase from Aeromonas hydrophila to the active site of VAP. The tested β‐lactam compounds did not inhibit Escherichia coli or shrimp alkaline phosphatase, which could be ascribed to the lower congruence indicated by CLASP. There was no discernible β‐lactamase activity in the tested alkaline phosphatases. This is the first time a scaffold recognizing imipenem in an alkaline phosphatase (VAP) has been demonstrated.

Co-expressed recombinant human Translin-Trax complex binds DNA

Trax, expressed alone aggregates into insoluble complexes, whereas upon co‐expression with Translin becomes readily soluble and forms a stable heteromeric complex (∼430 kDa) containing both proteins at nearly equimolar ratio. Based on the subunit molecular weights, estimated by MALDI‐TOF‐MS, the purified complex appears to comprise of either an octameric Translin plus a hexameric Trax (calculated MW 420 kDa) or a heptamer each of Trax and Translin (calculated MW 425 kDa) or a hexameric Translin plus an octameric Trax (calculated MW 431 kDa). The complex binds single‐stranded/double‐stranded DNA. ssDNA gel‐shifted complex shows both proteins at nearly equimolar ratio, suggesting that Translin “chaperones” Trax and forms heteromeric complex that is DNA binding competent.

DNA strand exchange activity of rice recombinase OsDmc1 monitored by fluorescence resonance energy transfer and the role of ATP hydrolysis.

Rad51 and disrupted meiotic cDNA1 (Dmc1) are the two eukaryotic DNA recombinases that participate in homology search and strand exchange reactions during homologous recombination mediated DNA repair. Rad51 expresses in both mitotic and meiotic tissues whereas Dmc1 is confined to meiosis. DNA binding and pairing activities of Oryza sativa disrupted meiotic cDNA1 (OsDmc1) from rice have been reported earlier. In the present study, DNA renaturation and strand exchange activities of OsDmc1 have been studied, in real time and without the steps of deproteinization, using fluorescence resonance energy transfer (FRET). The extent as well as the rate of renaturation is the highest in conditions that contain ATP, but significantly less when ATP is replaced by slowly hydrolysable analogues of ATP, namely adenosine 5′‐(β,γ‐imido) triphosphate (AMP‐PNP) or adenosine 5′‐O‐(3‐thio triphosphate) (ATP‐γ‐S), where the former was substantially poorer than the latter in facilitating the renaturation function. FRET assay results also revealed OsDmc1 protein concentration dependent strand exchange function, where the activity was the fastest in the presence of ATP, whereas in the absence of a nucleotide cofactor it was several fold (≈ 15‐fold) slower. Interestingly, strand exchange, in reactions where ATP was replaced with AMP‐PNP or ATP‐γ‐S, was somewhat slower than that of even minus nucleotide cofactor control. Notwithstanding the slow rates, the reactions with no nucleotide cofactor or with ATP‐analogues did reach the same steady state level as seen in ATP reaction. FRET changes were unaffected by the steps of deproteinization following OsDmc1 reaction, suggesting that the assay results reflected stable events involving exchanges of homologous DNA strands. All these results, put together, suggest that OsDmc1 catalyses homologous renaturation as well as strand exchange events where ATP hydrolysis seems to critically decide the rates of the reaction system. These studies open up new facets of a plant recombinase function in relation to the role of ATP hydrolysis.

MutS recognition: multiple mismatches and sequence context effects.

Escherichia coli MutS is a versatile repair protein that specifically recognizes not only various types of mismatches but also single stranded loops of up to 4 nucleotides in length. Specific binding, followed by the next step of tracking the DNA helix that locates hemi-methylated sites, is regulated by the conformational state of the protein as a function of ATP binding/hydrolysis. Here, we study how various molecular determinants of a heteroduplex regulate mismatch recognition by MutS, the critical first step of mismatch repair. Using classical DNase I footprinting assays, we demonstrate that the hierarchy of MutS binding to various types of mismatches is identical whether the mismatches are present singly or in multiples. Moreover, this unique hierarchy is indifferent both to the differential level of DNA helical flexibility and to the unpaired status of the mismatched bases in a heteroduplex. Surprisingly, multiple mismatches exhibit reduced affinity of binding to MutS, compared to that of a similar single mismatch. Such a reduction in the affinity might be due to sequence context effects, which we established more directly by studying two identical single mismatches in an altered sequence background. A mismatch, upon simply being flipped at the same location, elicits changes in MutS specific contacts, thereby underscoring the importance of sequence context in modulating MutS binding to mismatches.

Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications

BackgroundSynonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems.ResultsWe found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in Psilotum nudum and Nephroselmis olivacea mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: r-strategists minimize mutation costs in large genes; K-strategists do the opposite.ConclusionMrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure a priori biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for r- and K- strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of K-strategists, perhaps because domestication increases reproductive output.