1,720,972 research outputs found
In rice, Oryzalin and abscisic acid differentially affect tubulin mRNA and protein levels
No full textThe effect of the anti-microtubular drug Oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide) on growth and elongation of rice (Oryza sativa L. cv. Arborio) roots and coleoptiles was investigated. At 100 nM, Oryzalin strongly reduced primary root elongation, caused loss of cell anisotropy and the disappearance of the cortical microtubule array. Under these conditions the amounts of alpha- and beta-tubulin protein, but not mRNA, were heavily reduced. Similar data were also obtained in coleoptile segments treated with different concentrations of Oryzalin. However, when coleoptile elongation was inhibited by cis-abscisic acid, remarkable decreases in alpha- and beta-tubulin accumulation were observed to occur at the mRNA level but not at the protein level. The transcriptional decreases could be reversed by re-addition of 3-indole acetic acid. Altogether, these data indicate that rice tubulin accumulation can be controlled at different levels, mRNA or protein, in response to Oryzalin or abscisic acid treatments
Replication pattern of human repeated DNA sequences
No full textEither aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the Eco RI 340 bp family (alpha RI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [3H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [3H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S
Functional analysis of DNA sequences controlling the expression of the rice OsCDPK2 gene
No full textPlant calcium-dependent protein kinases (CDPKs) are involved in calcium-mediated signal transduction pathways. Their expression is finely tuned in different tissues and in response to specific signals, but the mechanism of such a regulation is still largely unknown. OsCDPK2 gene expression is modulated in vivo during rice (Oryza sativa L.) flower development and is downregulated by white light in leaves. In order to identify OsCDPK2 regulatory sequences, we amplified and cloned both the 5' and 3'-flanking regions of the gene. Sequence analysis revealed that the leader sequence is interrupted by an intron, whose regulatory role was investigated. Different beta-gucuronidase (GUS) expression vectors, carrying combinations of the putative OsCDPK2 regulatory regions, were generated and GUS expression was analyzed both in transient assays and in transgenic rice plants. The whole 5'-flanking sequence was able to drive GUS expression in rice calli and leaves transiently transformed with the biolistic technique. Analysis of the GUS expression pattern in transgenic plants revealed strong activity in root tips, leaf veins and mesophyll cells, in flower reproductive organs and in mature pollen grains. Expression was also shown to be subject to an intron-mediated enhancement (IME) mechanism, since the deletion of the leader intron sequence from chimeric OsCDPK2::GUS plasmids almost completely abolished GUS activity. Furthermore, in transiently transformed leaves, GUS expression driven by the OsCDPK2 promoter-leader region was constitutively observed regardless of light or dark exposure. Light-regulated expression was restored by inserting the OsCDPK2 3' untranslated region (3'UTR) downstream of the chimeric OsCDPK2::GUS transcription unit, suggesting that light down-regulation is mediated by a mechanism driven by the 3'UTR
Acquisition of deoxyguanosine resistance by TPA-induced T lymphoid lines
No full textThe human leukemic T cell line 8402, which contains terminal deoxynucleotidyl transferase (TdT) and phenotypically resembles precursor thymocytes, when exposed to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) undergoes in vitro T maturation. TdT disappears from virtually all the cells and a fraction of TdT- -cells express specific T surface markers, such as the T3 determinant. Like T lymphocytes from the thymus, 8402 cells are extremely sensitive to the cytotoxic effect of deoxyguanosine (dGuo). As a consequence of TPA treatment, resistance to dGuo is observed in 8402, as well as in two other TdT+ lymphoid T cell lines, Molt-4 and CEM-10. These results suggest the occurrence of changes in deoxynucleoside metabolism in TPA-treated cells related to the in vitro maturation process. Maturation of 8402 cells, once started, progresses in the presence of additional physiologic stimuli provided by conditioned medium from lymphocyte culture, because a portion of cells display the T8+/T4- phenotype characteristic of cytotoxic/suppressor T cells. This in vitro lymphoid system may thus be used to study the relationships of molecular differentiation between precursor thymocytes and cytotoxic/suppressor T cells
Polypeptide structure of human terminal transferase
No full textThe polypeptide structure of terminal transferase purified from human lymphoblasts was examined with an immunoblot procedure using rabbit anti-calf thymus terminal transferase antibodies. Two doublets of bands of Mr 58-56,000 and Mr 44-42,000 are the major immunoreactive polypeptides. Only the Mr 44-42,000 polypeptides can be efficiently renatured in situ after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Controlled degradation with trypsin produces fully active enzyme containing the α and β polypeptides typical of the low molecular weight terminal transferase, suggesting that the different forms of purified terminal transferase may arise by proteolysis of the Mr 58,000 polypeptide
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