Comparison of tRNA conformation during different phases of reproduction

The present study is a comparison of tRNA conformation from ovary of Heteropneustes fossilis in its active phase of reproduction (when it is highly engaged in protein synthesis i.e. previtellogenic phase) with inactive phase (when tRNA is mainly stored in mature ovary i.e. spawning phase). Transfer RNA of active phase is shown to be compact, flexible and susceptible towards nuclease. Compact tRNA structure is evidenced by higher hyperchromicity and presence of relatively less Gm modifications thereby allowing adequate hydrogen bonding between D loop and T loop. Higher sensitivity of tRNA towards Mg++ reflects its higher flexibility towards internal environment. This structure of tRNA may be required for active protein synthesis. On the other hand tRNA of inactive phase is shown to be relaxed but resistant towards nuclease which may be favoured for storage in mature ova of a teleost as maternal carry over

Escherichia coli rpoS gene has an internal secondary translation initiation region

Sigma S (σ s) encoded by rpoS in Escherichia coli is a stationary phase specific sigma subunit of the RNA polymerase holoenzyme. Widespread among the E. coli K12 strains is an amber mutation that prematurely terminates σ s. These rpoSAm mutants would be expected to show no σ s activity. However, suppressor free rpoSAm mutants retain an intermediate catalase activity, a sigma S controlled function. By analyzing the sequence of the rpoS gene we hypothesize that a 277 amino acids long Δ1-53σ s of about 30 kDa can be translated from an internal secondary translation initiation region (STIR, AGGGAGN 11GUG) that is located downstream of the amber codon. By cloning this rpoSAm gene, following the expression, function, and N-terminal sequence of this mutant protein, we report the presence of a functional internal STIR in E. coli rpoS, from where a truncated but nevertheless functional form of σ s can be synthesized

A stop codon-dependent internal secondary translation initiation region in Escherichia coli rpoS

Sigma S (σ(s)) encoded by rpoS is a stationary phase-specific σ subunit of the Escherichia coli RNA polymerase holoenzyme. In many E. coli strains, rpoS has an amber stop as codon 33 (rpoSAm), resulting in a 32-amino-acid-long peptide. Nevertheless, suppressor-free rpoSAm strains have functional σ(S). This led us to hypothesize the presence of an intracistronic secondary translational initiation region (STIR) in the E. coli rpoS gene. Here, we demonstrate that the STIR is functional and is controlled by the upstream amber stop codon 33. Removal of the primary translational initiation region did not abolish translation from STIR, ruling out translational coupling. Importantly, the functional STIR conferred survival advantage. Taken together, our results reveal a hitherto unknown physiologically significant post-transcriptional process in E. coli rpoSAm strains

Arabinosylcytosine downregulates thymidine kinase and induces cross-resistance to zidovudine in T-lymphoid cells

The aim of this study was to determine molecular mechanism(s) responsible for the reduced thymidine kinase activity (TK) observed earlier in an arabinosylcytosine (araC) resistant lymphoid cell line (H9-araC cells), which was obtained following continuous cultivation of H9 cells in the presence of 0.5 μM araC. Compared to H9 cells, in H9-araC cells TK1 and TK2 gene expressions were reduced to 17.7% and 2.5%, respectively, and the cellular AZT accumulation was diminished to 35.8%. These cells were also found cross-resistant to azidothymidine (>42-fold). There was no significant difference in the expression of MDR1, MRP4 or TK protein. The lack of correlation between the expressions of TK protein and TK1 and TK2 suggests that post-translational factors may also play a role in the reduced TK activity in H9-araC cells. These findings suggest that araC affects TK expression at the genetic level

2′, 3′-Dideoxycytidine represses thymidine kinases 1 and 2 expression in T-lymphoid cells

In vitro culture of H9 human lymphoid cells in the presence of 5.0 μM dideoxycytidine (ddC), for about 40–45 days, selected cells (H9-ddC cells), which were resistant to the drug and cross-resistant to AZT (zidovudine) and 5-fluoro-2′-deoxyuridine (FdUR). The major mechanism of cross-resistance to AZT and FdUR in these cells was low cellular activity of thymidine kinase (TK). To explore molecular mechanisms of the reduced TK activity in H9-ddC cells, the mRNA expression of TK1 and TK2 and western blot analysis of TK1 protein were performed. RT-PCR analysis revealed that in H9-ddC cells the expression of both TK1 and TK2 mRNA was reduced to 27.1% and 79.4%, respectively. The reduced TK1 gene expression was confirmed by an absence of a detectable TK1 protein band in western blot of H9-ddC cells. These results demonstrate that long-term treatment of H9 cells in the presence of ddC down-regulated TK1 and TK2 gene expression and reduced the expression and activity of TK in the resistant cells