A developmental study of rat sperm and testis selenoproteins

Essentially all of the selenium in the rat spermatozoon is bound to a polypeptide of Mr 15,000-17,000 confined to the capsule that surrounds the sperm mitochondria. Isoelectric focussing of isolated 75Se-labelled, carboxymethylated mitochondrial capsule protein (MCP) reveals the presence of at least four radioactive components, with a predominant charge isomer at pI4.6. The sperm selenoprotein appears to be identical with MCP, as judged by the exact coincidence of radioactivity and protein stain during two-dimensional electrophoresis. The temporal pattern of 75Se-labelling of rat caput epididymal spermatozoa after intratesticular 75Se injection suggests that maximum incorporation of 75Se into MCP occurs in step 7-step 12 spermatids and that 75Se uptake ceases during step 15 of spermiogenesis. The developmental appearance of sperm selenoprotein in rat testis therefore appears to lag several days behind that reported for MCP in mouse testis, suggesting the presence of selenium-free MCP in immature germ cells. SDS gel electrophoretic analysis of testis subcellular fractions 24 h after 75Se injection into rat testis at 21, 28 and 90 days of age indicates that sperm selenoprotein first appears in very low concentration during late meiosis and that its concentration increases sharply during early spermiogenesis. Additional 75Se-labelled polypeptides were detected on the gels, most of them of higher molecular weight than MCP. At least two of these (Mr 47,000 and 54,000) displayed a marked decrease in labelling between 5 and 24 h after injection into adult testis, coincident with a comparable increase in 75Se-labelled MCP, indicating that they may be precursors of MCP.     

Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena acts as a sequence-specific endoribonuclease. Specificity of cleavage is determined by Watson-Crick base pairing between the active site of the RNA enzyme (ribozyme) and its RNA substrate [Zaug, A. J., Been, M. D., & Cech, T. R. (1986) Nature (London) 324, 429-433]. Surprisingly, single-base changes in the substrate RNA 3 nucleotides preceding the cleavage site, giving a mismatched substrate-ribozyme complex, enhance the rate of cleavage. Mismatched substrates show up to a 100-fold increase in kcat and, in some cases, in kcat/Km. A mismatch introduced by changing a nucleotide in the active site of the ribozyme has a similar effect. Addition of 2.5 M urea or 3.8 M formamide or decreasing the divalent metal ion concentration from 10 to 2 mM reverses the substrate specificity, allowing the ribozyme to discriminate against the mismatched substrate. The effect of urea is to decrease kcat and kcat/Km for cleavage of the mismatched substrate; Km is not significantly affected at 0-2.5 M urea. Thus, progressive destabilization of ribozyme-substrate pairing by mismatches or by addition of a denaturant such as urea first increases the rate of cleavage to an optimum value and then decreases the rate.     

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.     

Biocalorimetry- supported analysis of fermentation processes

The close relation between metabolic activity and heat release means that calorimetry can be successfully applied for on-line monitoring of biological processes. Since the use of available calorimeters in biotechnology is difficult because of technical limitations, a new sensitive heat-flux calorimeter working as a laboratory fermenter was developed and tested for different aerobic and anaerobic fermentations with Saccharomyces cerevisiae and Zymommonas mobilis. The aim of the experiments was to demonstrate the abilities of the method for biotechnological purposes. Fermentations as well as the corresponding heat, substrate and product analyses were reproducible. During experiments the heat signal was used as a sensitive and fast indicator for the response of the organisms to changing conditions. One topic was the monitoring of diauxic growth phenomena during batch fermentations, which may affect process productivity. S. cerevisiae was used as the test organism and a protease-excreting Bacillus licheniformis strain as an industrial production system. Other experiments focused on heat measurements in continuous culture under substrate-limiting conditions in order to analyse bacterial nutrient requirements. Again, Z. mobilis was used as the test organism. Ammonium, phosphate, magnesium, biotin and panthothenate, as important substrate compounds, were varied. The results indicate that these nutrients are required in lower amounts for growth than formerly suggested. Thus, a combination of heat measurements and other methods may rapidly improve our knowledge of nutrient requirements even for a well-known microorganism like Z. mobilis. *** DIRECT SUPPORT *** AG903062 00004     

Copy Number Variation in Transcriptionally Active Regions of Sexual and Apomictic Boechera Demonstrates Independently Derived Apomictic Lineages

In asexual (apomictic) plants, the absence of meiosis and sex is expected to lead to mutation accumulation. To compare mutation accumulation in the transcribed genomic regions of sexual and apomictic plants, we performed a double-validated analysis of copy number variation (CNV) on 10 biological replicates each of diploid sexual and diploid apomictic Boechera, using a high-density (>700 K) custom microarray. The Boechera genome demonstrated higher levels of depleted CNV, compared with enriched CNV, irrespective of reproductive mode. Genome-wide patterns of CNV revealed four divergent lineages, three of which contain both sexual and apomictic genotypes. Hence genome-wide CNV reflects at least three independent origins (i.e., expression) of apomixis from different sexual genetic backgrounds. CNV distributions for different families of transposable elements were lineage specific, and the enrichment of LINE/L1 and long term repeat/Copia elements in lineage 3 apomicts is consistent with sex and meiosis being mechanisms for purging genomic parasites. We hypothesize that significant overrepresentation of specific gene ontology classes (e.g., pollen–pistil interaction) in apomicts implies that gene enrichment could be an adaptive mechanism for genome stability in diploid apomicts by providing a polyploid-like system for buffering the effects of deleterious mutations.