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Z Chrzanowska-Lightowlers

Publications and source records attributed to Z Chrzanowska-Lightowlers.

6 recordsLinked to original sources
PubMed2004

Isolation and identification of a protein binding to the localization element of Metallothionein-1 mRNA.

mRNA localization provides a mechanism for localized protein synthesis. mRNAs encoding certain proteins, including c-MYC, c-FOS, MT-1 (Metallothionein-1) and vimentin, are localized around the nuclei of mammalian cells and are associated with the cytoskeleton. Targeting of these mRNAs to the perinuclear cytoplasm is mediated by elements within their 3'-UTRs (3'-untranslated regions), but many of the trans-acting proteins remain unidentified. UV cross-linking assays using radiolabelled transcripts indicated that a protein of approx. 50 kDa (from the Chinese-hamster ovary cell extracts) bound to the MT-1 3'-UTR sequence. Competition experiments using unlabelled mutant 3'-UTR RNAs revealed that the binding of this protein is specific to localization-positive mutants. Isolation of a 50 kDa protein was achieved by an RNA affinity-based method in which biotinylated MT-1 3'-UTR RNA was anchored to paramagnetic beads. Bound proteins were eluted and analysed by SDS/PAGE. The 50 kDa protein was extracted from the gel, subjected to trypsin digestion and identified by matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry as eukaryote elongation factor 1alpha.

3' Untranslated Regions↗
PubMed2001

Fending off decay: a combinatorial approach in intact cells for identifying mRNA stability elements.

The strategy of systematic evolution, whereby nucleic acid sequences or conformers can be selected and amplified from a randomized population, has been exploited by many research groups for numerous purposes. It is, however, a technique largely performed in vitro, under nonphysiological conditions. We have now modified this in vitro approach to accomplish selection in growing cells. Here, we report that this new methodology has been used in vivo to select RNA elements that confer increased transcript stability. A randomized cassette was embedded in a 3'-untranslated region (UTR), downstream from the luciferase reporter open reading frame. A heterogeneous population of capped luciferase mRNA was then generated by in vitro transcription. Human liver Hep G2 cells were electroporated with this population of luciferase mRNA and total cytoplasmic RNA was isolated after varying lengths of incubation. Following RT-PCR, the 3' UTR was used to reconstruct a new population of luciferase templates, permitting subsequent cycles of in vitro transcription, electroporation, RNA isolation, and RT-PCR. Increasing the incubation time at each cycle before RNA isolation imposed selection for stable transcripts. The functional half-life of the luciferase mRNA population increased from 55 to 140 min after four cycles. Subsequent sequencing of the selected 3' UTRs revealed G-U rich elements in clones with extended chemical and functional half-lives.

3' Untranslated Regions↗
PubMed1999

Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets.

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.

Animals↗
PubMed1997

Intracellular mitochondrial triplasmy in a patient with two heteroplasmic base changes.

We report the clinical, biochemical, and genetic investigation of a patient with a severe mitochondrial encephalomyopathy. Genetic studies identified a novel, heteroplasmic tRNA mutation at nt 10010. This T-->C transition is located in the DHU loop of mitochondrial tRNA(Gly). In skeletal muscle, it was present at lower levels in cytochrome c oxidase (COX)-normal (87.2% +/- 11%) compared with COX-deficient fibers (97.3% +/- 2.6%); it was found in skin fibroblasts and blood cells, but at lower levels of heteroplasmy (15% +/- 6% and 17% +/- 10%, respectively). A second, heteroplasmic transition (A-->G), at nt 5656, showed a different distribution than the tRNA(Gly) mutation, with very low levels in skeletal muscle (< 3%) but higher levels in blood (22.7% +/- 3%) and skin fibroblasts (21% +/- 2%). These transitions were followed both in vivo, by repeat biopsy and blood sampling, and in vitro, by establishing primary cultures of myoblasts and skin fibroblasts. Repeat muscle biopsy showed a dramatic increase in COX-deficient fibers, but not of the tRNAGly mutation. Indeed, no significant change in heteroplasmy was measured for either substitution in muscle or blood. In vitro analysis gave very different results. The T10010C was not found in cultured myoblasts, even at early passage. In uncloned fibroblasts, the T10010C was stable (approximately 10%) for several passages but then gradually was lost. In contrast, the A5656G rose progressively from 27% to 91%. In cloned fibroblasts, different combinations of both base-pair changes and wild type could be identified, confirming the presence of clonal, intracellular triplasmy.

Adult↗
PubMed1991

Subunit function in eukaryote cytochrome c oxidase. A mutation in the nuclear-coded subunit IV allows assembly but alters the function and stability of yeast cytochrome c oxidase.

Strains of the yeast Saccharomyces cerevisiae disrupted in YCOX4, the nuclear gene encoding cytochrome c oxidase subunit IV, do not assemble a functional or spectrally visible oxidase. We report the characterization of a yeast strain, RM1, expressing a mutated YCOX4 gene which is temperature sensitive for respiration at 37 degrees C, but incorporates cytochrome aa3 over all growth temperatures. The mutant enzyme is less stable than the wild type, with subunit IV readily proteolyzed without gross denaturation of the complex but with a concomitant loss of oxidase activity. When grown fermentatively at 37 degrees C, cytochrome c oxidase from the mutant strain had a turnover number of less than 3% of the normal complex, while Km values and subunit levels were comparable to normal. Thus alterations in subunit IV can perturb the enzyme structure and alter its catalytic rate, implying a role for this subunit in cytochrome c oxidase function as distinct from assembly.

Base Sequence↗