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1.
Eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at or very near origins in G1 phase, which licenses origin firing in S phase. The archaeal DNA replication machinery broadly resembles the eukaryal apparatus, though simpler in form. The eukaryotic replication initiator origin recognition complex (ORC), which serially recruits Cdc6 and other pre-RC proteins, comprises six components, Orc1-6. In archaea, a single gene encodes a protein similar to both the eukaryotic Cdc6 and the Orc1 subunit of the eukaryotic ORC, with most archaea possessing one to three Orc1/Cdc6 orthologs. Genome sequence analysis of the extreme acidophile Picrophilus torridus revealed a single Orc1/Cdc6 (PtOrc1/Cdc6). Biochemical analyses show MBP-tagged PtOrc1/Cdc6 to preferentially bind ORB (origin recognition box) sequences. The protein hydrolyzes ATP in a DNA-independent manner, though DNA inhibits MBP-PtOrc1/Cdc6-mediated ATP hydrolysis. PtOrc1/Cdc6 exists in stable complex with PCNA in Picrophilus extracts, and MBP-PtOrc1/Cdc6 interacts directly with PCNA through a PIP box near its C terminus. Furthermore, PCNA stimulates MBP-PtOrc1/Cdc6-mediated ATP hydrolysis in a DNA-dependent manner. This is the first study reporting a direct interaction between Orc1/Cdc6 and PCNA in archaea. The bacterial initiator DnaA is converted from an active to an inactive form by ATP hydrolysis, a process greatly facilitated by the bacterial ortholog of PCNA, the β subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNA-interacting protein motifs in several archaeal PCNAs suggest the possibility of a similar mechanism of regulation existing in archaea. This mechanism may involve other yet to be identified archaeal proteins. 相似文献
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Ratul Saha Robert S. Donofrio Susan T. Bagley 《Journal of industrial microbiology & biotechnology》2010,37(8):843-848
A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of
three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms
of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since
they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but
are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both
whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 101 colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining
concentrations from used MWF samples, indicating levels between 2.9 × 103 and 3.9 × 106 CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 101 to 1.4 × 105 CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently
used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs. 相似文献
4.
Zabardast T. Buriev Sukumar Saha Ibrokhim Y. Abdurakhmonov Johnie N. Jenkins Abdusattor Abdukarimov Brian E. Scheffler David M. Stelly 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2010,120(3):587-606
MIC-3 is a recently identified gene family shown to exhibit increased root-specific expression following nematode infection of
cotton plants that are resistant to root-knot nematode. Here, we cloned and sequenced MIC-3 genes from selected diploid and tetraploid cotton species to reveal sequence differences at the molecular level and identify
chromosomal locations of MIC-3 genes in Gossypium species. Detailed sequence analysis and phylogenetic clustering of MIC-3 genes indicated the presence of multiple MIC-3 gene members in Gossypium species. Haplotypes of a MIC-3 gene family member were discovered by comparative analysis among consensus sequences across genotypes within an individual
clade in the phylogram to overcome the problem of duplicated loci in the tetraploid cotton. Deficiency tests of the SNPs delimited
six At-genome members of the MIC-3 family clustered to chromosome arm 4sh, and one Dt-genome member to chromosome 19. Clustering was confirmed by long-PCR amplification of the intergenic regions using At-genome-specific MIC-3 primer pairs. The clustered distribution may have been favored by selection for responsiveness to evolving disease and/or
pest pressures, because large variants of the MIC-3 gene family may have been recovered from small physical areas by recombination. This could give a buffer against selection
pressure from a broad range of pest and pathogens in the future. To our knowledge, these are the first results on the evolution
of clustering and genome-specific haplotype members of a unique cotton gene family associated with resistant response against
a major pathogen. 相似文献
5.
Dipnarayan Saha Vajinder Kumar Shripad Ramachandra Bhat Ramamurthy Srinivasan 《Plant Molecular Biology Reporter》2011,29(2):265-277
Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop
plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing
the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion
analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal
promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the
LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region
of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present
study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering. 相似文献
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Sayan Chowdhury Rupkatha Mukhopadhyay Sourav Saha Amartya Mishra Souvik Sengupta Syamal Roy Hemanta K. Majumder 《The Journal of biological chemistry》2014,289(23):16129-16147
In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB25R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB25R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance. 相似文献
8.
Adaptation to hypoxia, defined as a condition of inadequate oxygen supply, has enabled humans to successfully colonize high altitude regions. The mechanisms attempted by organisms to cope with short-term hypoxia include increased ATP production via anaerobic respiration and stabilization of Hypoxia Inducible Factor 1α (HIF-1α). However, less is known about the means through which populations adapt to chronic hypoxia during the process of development within a life time or over generations. Here we show that signaling via the highly conserved Wnt pathway impacts the ability of Drosophila melanogaster to complete its life cycle under hypoxia. We identify this pathway through analyses of genome sequencing and gene expression of a Drosophila melanogaster population adapted over >180 generations to tolerate a concentration of 3.5–4% O2 in air. We then show that genetic activation of the Wnt canonical pathway leads to increased rates of adult eclosion in low O2. Our results indicate that a previously unsuspected major developmental pathway, Wnt, plays a significant role in hypoxia tolerance. 相似文献
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Ghotas Evindar Sylvie G. Bernier Malcolm J. Kavarana Elisabeth Doyle Jeanine Lorusso Michael S. Kelley Keith Halley Amy Hutchings Albion D. Wright Ashis K. Saha Gerhard Hannig Barry A. Morgan William F. Westlin 《Bioorganic & medicinal chemistry letters》2009,19(2):369-372
In the design of potent and selective sphingosine-1-phosphate receptor agonists, we were able to identify two series of molecules based on phenylamide and phenylimidazole analogs of FTY-720. Several designed molecules in these scaffolds have demonstrated selectivity for S1P receptor subtype 1 versus 3 and excellent in vivo activity in mouse. Two molecules PPI-4621 (4b) and PPI-4691 (10a), demonstrated dose responsive lymphopenia, when administered orally. 相似文献