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1.
Rabbits were immunised againstEscherichia coli ribosomes and the partially purified immunoglobulin G fraction had maximum ability to precipitate the ribosomes as well as
the extracted ribosomal proteins. By digestion of immuno-globulin G with papain, monovalent Fab fragments were produced. The
70 S ribosome and its subunits (50 S and 30 S) were separately treated with Fab and then tested in the kinetic assay of degradation
of ribosomes by ribonuclease I at various Mg2+ concentrations. Treated ribosomes and their subunits were degraded at faster rates than the nontreated ones; the rates in
both the control and the treated cases were dependent on the concentration of Mg2+. These results indicate the unfolding of the structure of the ribosome on treatment with antibody fragments, which may be
due to the weakening of the interaction between rRNAs and ribosomal proteins. 相似文献
2.
ATM phosphorylates histone H2AX in response to DNA double-strand breaks 总被引:38,自引:0,他引:38
Burma S Chen BP Murphy M Kurimasa A Chen DJ 《The Journal of biological chemistry》2001,276(45):42462-42467
A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks. 相似文献
3.
4.
Functional analysis of cauliflower mosaic virus 35S promoter: re-evaluation of the role of subdomains B5, B4 and B2 in promoter activity 总被引:3,自引:0,他引:3
Bhullar S Datta S Advani S Chakravarthy S Gautam T Pental D Burma PK 《Plant biotechnology journal》2007,5(6):696-708
The cauliflower mosaic virus 35S (35S) promoter is used extensively for transgene expression in plants. The promoter has been delineated into different subdomains based on deletion analysis and gain-of-function studies. However, cis -elements important for promoter activity have been identified only in the domains B1 ( as-2 element), A1 ( as-1 element) and minimal promoter (TATA box). No cis -elements have been described in subdomains B2–B5, although these are reported to be important for the overall activity of the 35S promoter. We have re-evaluated the contribution of three of these subdomains, namely B5, B4 and B2, to 35S promoter activity by developing several modified promoters. The analysis of β-glucuronidase gene expression driven by the modified promoters in different tissues of primary transgenic tobacco lines, as well as in seedlings of the T1 generation, revealed new facets about the functional organization of the 35S promoter. This study suggests that: (i) the 35S promoter truncated up to –301 functions in a similar manner to the –343 (full-length) 35S promoter; (ii) the Dof core and I-box core observed in the subdomain B4 are important for 35S promoter activity; and (iii) the subdomain B2 is essential for maintaining an appropriate distance between the proximal and distal regions of the 35S promoter. These observations will aid in the development of functional synthetic 35S promoters with decreased sequence homology. Such promoters can be used to drive multiple transgenes without evoking promoter homology-based gene silencing when attempting gene stacking. 相似文献
5.
6.
The mechanism of protein synthesis is still unknown due to inability to detect the so-called enzyme "peptidyl transferase" even after elucidation of high-resolution crystal structure of ribosome. We have recently shown by model building and semi-empirical energy calculation that the tRNA molecule at P-site of ribosome may act as peptidyl transferase (Das et al. (1999) J. Theor. Biol. 200, 193-205). We proposed that the tetrahedral intermediate formed from nucleophylic attack of CO of P-site amino-acylated tRNA by NH2 of A-site amino-acylated tRNA is converted to a six-member ring intermediate by conformational change. This ring intermediate produces a free tRNA and a tRNA covalently linked to a peptide. However, energy of the six-member ring intermediate was calculated to be quite high. We show here that the energy values of all the reactants, intermediates and products are within the expected range when they are calculated using high level ab initio quantum chemical methods. 相似文献
7.
A study of bacterial surface oligosaccharides were investigated among
different strains of Neisseria gonorrhoeae to correlate structural features
essential for binding to the MAb 2C7. This epitope is widely expressed and
conserved in gonococcal isolates, characteristics essential to an effective
candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared
by a modification of the hot phenol-water method from which de-O-acetylated
LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and
ES-MSnin a triple quadrupole and an ion trap mass spectrometer,
respectively. Previously documented natural heterogeneity was apparent from
both LOS and OS preparations which was admixed with fragments induced by
hydrazine and mild acid treatment. Natural heterogeneity was limited to
phosphorylation and antenni extensions to the alpha-chain. Mild acid
hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic
linkage of lipid A. OS structures were determined by collisional and
resonance excitation combined with MS and multistep MSn which provided
sequence information from both neutral loss, and nonreducing terminal
fragments. A comparison of OS structures, with earlier knowledge of MAb
binding, enzyme treatment, and partial acid hydrolysis indicates a generic
overlapping domain for 2C7 binding. Reoccurring structural features include
a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the
nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc
(gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the
central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain),
moiety is required although extensions to this residue appear unnecessary.
相似文献
8.
The binding of the nonintercalating dye berenil to the 70S ribosome of Escherichia coli has been demonstrated by spectrophotometric measurements and gel filtration through Biogel P100 column. The berenil spectrum is gradually shifted towards the red region with the increasing amount of ribosome added, the isosbestic point being at 375 nm. There is positive cooperativity in the binding of berenil to the ribosome as demonstrated by the equilibrium dialysis. On binding with berenil, the ribosome is degraded faster by RNase I especially at low Mg++ concentration and its capacity to inhibit RNase I catalysed hydrolysis of ribopolymers is decreased. These indicate the unfolding of the structure of the ribosome. 相似文献
9.
We report in this study, an improved method for identifying male sterile–restorer combinations using the barnase–barstar system of pollination control for heterosis breeding in crop plants, as an alternative to the conventional line × tester
cross method. In this strategy, a transgenic male sterile barnase line was retransformed with appropriate barstar constructs. Double transformants carrying both the barnase and barstar genes were identified and screened for their male fertility status. Using this strategy, 66–90% of fertile retransformants
(restored events) were obtained in Brassica juncea using two different barstar constructs. Restored events were analysed for their pollen viability and copy number of the barstar gene. Around 90% of the restored events showed high pollen viability and ∼30% contained single copy integrations of the barstar gene. These observations were significantly different from those made in our earlier studies using line (barnase) × tester (barstar) crosses, wherein only two viable male sterile–restorer combinations were identified by screening 88 different cross-combinations.
The retransformation strategy not only generated several independent restorers for a given male sterile line from a single
transformation experiment but also identified potential restorers in the T0 generation itself leading to significant savings in time, cost and labour. Single copy restored plants with high pollen viability
were selfed to segregate male sterile (barnase) and restorer (barstar) lines in the T1 progeny which could subsequently be diversified into appropriate combiners for heterosis breeding. This strategy will be
particularly useful for crop plants where poor transformation frequencies and/or lengthy transformation protocols are a major
limitation. 相似文献
10.
Jagannath A Sodhi YS Gupta V Mukhopadhyay A Arumugam N Singh I Rohatgi S Burma PK Pradhan AK Pental D 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2011,122(6):1091-1103
Oil content and oil quality fractions (viz., oleic, linoleic and linolenic acid) are strongly influenced by the erucic acid
pathway in oilseed Brassicas. Low levels of erucic acid in seed oil increases oleic acid content to nutritionally desirable
levels, but also increases the linoleic and linolenic acid fractions and reduces oil content in Indian mustard (Brassica juncea). Analysis of phenotypic variability for oil quality fractions among a high-erucic Indian variety (Varuna), a low-erucic east-European
variety (Heera) and a zero-erucic Indian variety (ZE-Varuna) developed by backcross breeding in this study indicated that
lower levels of linoleic and linolenic acid in Varuna are due to substrate limitation caused by an active erucic acid pathway
and not due to weaker alleles or enzyme limitation. To identify compensatory loci that could be used to increase oil content
and maintain desirable levels of oil quality fractions under zero-erucic conditions, we performed Quantitative Trait Loci
(QTL) mapping for the above traits on two independent F1 doubled haploid (F1DH) mapping populations developed from a cross
between Varuna and Heera. One of the populations comprised plants segregating for erucic acid content (SE) and was used earlier
for construction of a linkage map and QTL mapping of several yield-influencing traits in B. juncea. The second population consisted of zero-erucic acid individuals (ZE) for which, an Amplified Fragment Length Polymorphism
(AFLP)-based framework linkage map was constructed in the present study. By QTL mapping for oil quality fractions and oil
content in the ZE population, we detected novel loci contributing to the above traits. These loci did not co-localize with
mapped locations of the fatty acid desaturase 2 (FAD2),
fatty acid desaturase 3 (FAD3) or fatty acid elongase (FAE) genes unlike those of the SE population wherein major QTL were found to coincide with mapped locations of the FAE genes. Some of the new loci identified in the ZE population could be detected as ‘weak’ contributors (with LOD < 2.5) in
the SE population in which their contribution to the traits was “masked” due to pleiotropic effects of erucic acid genes.
The novel loci identified in this study could now be used to improve oil quality parameters and oil content in B. juncea under zero-erucic conditions. 相似文献