Gene rearrangement during the ontogeny of T-and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contain premature terminations codons (ptcs). We performed sequence analysis of reverse transcriptase-polymerase chain reaction products from fetal and adult thymus and found that newly transcribed TCR- pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR- transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR- exons triggered a decrease in mRNA levels. Ptc-bearing TCR- transcripts were selectively depressed in levels in a cell clone that contained both an in-frame and an out-of-frame gene, thus demonstrating the allelic specificity of this down-regulatory response. Protein synthesis inhibitors with different mechanism of action (anisomysin, cycloheximide, emetine, pactamycin, puromycin, and polio virus) all reversed the downregulatory response. Ptc-bearing transcripts were induced within 0.5 h after cycloheximide treatment. The reversal by protein synthesis inhibitors was not restricted to lymphoid cells, as shown with TCR- and -globin constructs transfected in HeLa cells. Collectively, the data suggest that the ptc-mediated mRNA decay pathway requires an unstable protein, a ribosome, or a ribosome-like entity. Protein synthesis inhibitors may be useful tools toward elucidating the molecular mechanism of ptc-mediated mRNA decay, an enigmatic response that can occur in the nuclear fraction of mammalian cells.
T-cell receptor (TCR)1 and immunoglobulin (Ig) genes undergo programmed rearrangement events during lymphocyte ontogeny. During this process, variable (V) elements are juxtaposed to joining (J) elements to create functional genes (1, 2).In some TCR and Ig genes, diversity (D) elements are also included in this rearrangement process. The tremendous combinatorial possibilities afforded by this rearrangement mechanism permit the generation of a wide variety of antigen receptors. Additional variability is provided by the enzyme terminal transferase which introduces random nucleotides at the junctions between V, D, and, J elements (1, 2). Variability is also engendered by the low fidelity of the rearrangement event itself; the borders of each element are not fixed, sometimes leading to small deletions at the junctions between the V, D, and J elements. The collective result of these insertional and deletional events is that a large fraction of rearrangement events will generate out-of-frame (nonproductive) genes that contain premature termination codons (ptcs).Since out-of-frame TCR and Ig genes are commonly generated during normal lymphocyte development, there may exist a mechanism that diminishes the expression of these nonfunctional ptc-containing genes. Consistent with...
In antigen-presenting cells, class II molecules of the major histocompatibility complex (MHC) bind peptides derived from endocytosed proteins. In certain B-lymphoblastoid cell mutants, MHC class II molecule-peptide complex formation is impaired, resulting in deficient antigen-presenting function. MHC deletion mutants with this defect map the responsible gene(s) to the class II region of the MHC. Here we report that multiple independent mutants with the class II presentation defect harbour lesions in HLA-DMB, an MHC-linked gene encoding a class II-like beta-chain. Expression of DMB complementary DNA in mutants lacking DMB messenger RNA restores the wild-type phenotype. These results establish HLA-DM as a critical regulatory molecule in class II-restricted antigen presentation and suggest that it functions at an intracellular site to promote class II molecule-peptide association.
Improved post-harvest cell wall deconstruction of tall fescue leaves has been demonstrated by in-planta co-expression of a constitutively expressed ferulic acid esterase together with a senescence-induced β-1,4 endoxylanase. Tall fescue plants (Festuca arundinacea) constitutively expressing vacuole- or apoplast-targeted ferulic acid esterase from Aspergillus niger were retransformed with a senescence-induced and apoplast-targeted β-1,4 endo-xylanase from Trichoderma reesei. Enzyme activities in co-expressing plants stabilized after repeated vegetative propagation, with xylanase activity in senescent leaves increasing and ferulic acid esterase activity decreasing after tillering. Plants co-expressing both enzymes in the apoplast, with the lowest levels of ferulate monomers and dimers and the lowest levels of cell wall arabinoxylans, released ten times more cell wall hydroxycinnamic acids and five times more arabinoxylan from the cell wall on autodigestion compared to expression of ferulic acid esterase or xylanase alone. These plants also showed a 31 % increase in cellulase-mediated release of reducing sugars, a 5 % point increase in in vitro dry matter digestibility and a 23 % increase in acetyl bromide-soluble lignin. However, plant growth was adversely affected by expressing FAE in the apoplast, giving plants with narrower shorted leaves, and a 71 % decrease in biomass.
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