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PubMed1997

Susceptibility loci for distinct components of developmental dyslexia on chromosomes 6 and 15.

Six extended dyslexic families with at least four affected individuals were genotyped with markers in three chromosomal regions: 6p23-p21.3, 15pter-qter, and 16pter-qter. Five theoretically derived phenotypes were used in the linkage analyses: (1) phonological awareness; (2) phonological decoding; (3) rapid automatized naming; (4) single-word reading; and (5) discrepancy between intelligence and reading performance, an empirically derived, commonly used phenotype. Two-point and multipoint allele-sharing analyses of chromosome 6 markers revealed significant evidence (P < 10(-6)) for linkage of the phonological awareness phenotype to five adjacent markers (D6S109, D6S461, D6S299, D6S464, and D6S306). The least compelling results were obtained with single-word reading. In contrast, with chromosome 15 markers, a LOD score of 3.15 was obtained for marker D15S143 at theta = 0.0 with single-word reading. Multipoint analyses with markers adjacent to D15S143 (D15S126, D15S132, D15S214, and D15S128) were positive, but none reached acceptable significance levels. Chromosome 15 analyses with the phonological awareness phenotype were negative. Parametric and nonparametric linkage analyses with chromosome 16 markers were negative. The most intriguing aspect of the current findings is that two very distinct reading-related phenotypes, reflecting different levels in the hierarchy of reading-related skills, each contributing to different processes, appear to be linked to two different chromosomal regions.

Adolescent↗
PubMed2025

Decoding the PTTG family's contribution to LUAD pathogenesis: a comprehensive study on expression, epigenetics, and therapeutic interventions.

BACKGROUND: Lung adenocarcinoma (LUAD) stands as a prevalent malignancy, yet its pathology remains incompletely comprehended. METHODS: In this comprehensive study, we explored the roles of the pituitary tumor-transforming gene (PTTG) family, including PTTG1, PTTG2, and the pseudogene PTTG3P in lung adenocarcinoma (LUAD). Employing a multi-faceted approach, we conducted in-depth analyses using clinical samples and expression datasets. RESULTS: Our findings revealed a significant up-regulation of PTTG family genes in LUAD cell lines and tissue samples compared to adjacent normal controls, suggesting their potential as diagnostic biomarkers. Through promoter methylation and mutational analyses, we uncovered regulatory mechanisms influencing PTTG gene expression. The exploration of the PTTG family's impact on LUAD patient survival demonstrated their association with adverse outcomes, emphasizing their potential prognostic relevance. Moreover, functional assays demonstrated that the knockdown of PTTG1 and PTTG2 genes resulted in the reduction of cell proliferation, colony formation, and cell migration abilities in A549 and H1975 LUAD cells. Furthermore, our investigation extended to therapeutic avenues, where we identified Calcitriol as a potential drug within the DrugBank database to down-regulate PTTG genes. Molecular docking analyses provided insights into the strong interaction between Calcitriol and PTTG1/PTTG2 proteins, laying the groundwork for further exploration of Calcitriol in LUAD treatment. CONCLUSION: In conclusion, this study contributes a comprehensive understanding of the PTTG family's involvement in LUAD, shedding light on their diagnostic, prognostic, and therapeutic implications.

Humans↗
PubMed2001

Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction.

Interactions within the decoding center of the 30 S ribosomal subunit have been investigated by constructing all 15 possible mutations at nucleotides C1402 and A1500 in helix 44 of 16 S rRNA. As expected, most of the mutations resulted in highly deleterious phenotypes, consistent with the high degree of conservation of this region and its functional importance. A total of seven mutants were viable under conditions where the mutant ribosomes comprised 100 % of the ribosomal pool. A suppressor mutation specific for the C1402U-A1500G mutant was isolated at position 1520 in helix 45 of 16 S rRNA. In addition, lack of dimethylation of A1518/A1519 caused by mutation of the ksgA methylase enhanced the deleterious effect of many of the 1402/1500 mutations. These data suggest that a higher-order interaction between helices 44 and 45 in 16 S rRNA is important for the proper functioning of the ribosome. This is consistent with the recent high-resolution crystal structures of the 30 S subunit, which show a tertiary interaction between the 1402/1500 region of helix 44 and the dimethyl A stem loop.

Aminoglycosides↗
PubMed1994

Analysis of action of wobble nucleoside modifications on codon-anticodon pairing within the ribosome.

Wobble rules for modified residues in the first anticodon position are derived. All known modifications are considered individually. Stereochemical analysis was made taking into account the interaction between the ribosomal A and P-site bound codon-anticodon duplexes. The wobble base-pair was considered as the right one if its formation did not lead to an uncompensated loss of hydrogen bonds or polar atom-ion bonds. From this requirement it follows that all modifications of U should restrict its translational specificity to purines (with the exception of xo5U, which should decode A, G and U). The restriction is carried out in a unified way: modifications inhibit the large propeller twist resulting from an increase of about 35 degrees in the torsion angle of the anticodon wobble base, interacting with the third codon base via a hydrogen-bonded water molecule. Such a twist is required to avoid a loss of the hydrogen bond of the bonded water molecule. The modifications in S2U, Se2U and Um should weaken their pairing with G, because they deform one of the two hydrogen bonds of the guanine NH2 group. G should be recognized by Se2U better than by S2U for the reason that the hydrogen bond Se...HN is weaker than the hydrogen bond S...HN. Among the modifications of C and G only that in k2C has a pronounced effect on wobble. The nucleoside k2C should pair only with A. The N-2 atom of k2C should be in the pyramidal state. The consequences following from the interduplex interaction are formulated. According to one of them, adenosine in the wobble position of the P-site tRNA should destabilize the A-site duplex. This can serve as an explanation for the fact that adenosine is very rarely observed in the anticodon wobble position.

Adenosine↗
PubMed1992

Expression of argU, the Escherichia coli gene coding for a rare arginine tRNA.

The Escherichia coli argU gene encodes the rare arginine tRNA, tRNA(UCUArg), which decodes the similarly rare AGA codons. The argU promoter is, with two exceptions, a typical, strongly expressed stable RNA gene promoter which is stimulated by an upstream activator sequence. Unlike other tRNA operons, however, argU expression is severely inhibited by sequences downstream of the transcription start point. In vivo, nucleotides +2 to +45 inhibited expression by 25- to 100-fold when measured by fusion of argU promoter regions to the chloramphenicol acetyltransferase reporter gene or by quantitative primer extension analysis. In vitro, linearized argU promoter fragments on which the argU region ended at +1 supported 5- to 10-fold-more transcription than when the argU region ended at +45. This difference in degree of inhibition between in vivo and in vitro conditions suggests that several factors, some of which could be absent in vitro, might limit expression in vivo. Alternatively, one mechanism might limit expression both in vivo and in vitro but function more efficiently in vivo. A second difference from strongly expressed stable RNA promoters is the fact the argU gene is relatively insensitive to growth rate regulation, at least when assayed on a multicopy plasmid.

Base Sequence↗
PubMed1991

Spatial organization of template polynucleotides on the ribosome determined by fluorescence methods.

The spatial organization of template polynucleotides on the ribosome and the dynamics of their interaction with 30 S subunits have been studied by fluorescence spectroscopy. The topography of the mRNA in the ribosome has been determined using singlet-singlet energy transfer. This method has allowed us to estimate distances between donors and acceptors of energy which have been linked to the terminal residues of template polynucleotides (poly- and oligo(U) and oligo(A] and 16 S RNA or to SH-groups of ribosomal proteins S1 and S8. The dynamics of mRNA-ribosome interaction have been investigated by the fluorescence stopped-flow technique. It has been shown that the binding to the 30 S subunit of poly(U) with length much shorter (16 nucleotides) than that covered by the ribosome is greatly enhanced by protein S1. However, the final position of oligo(U)16 on the 30 S subunit, which probably includes the ribosomal decoding site, proves to be quite different from that occupied by oligo(U)16 on a free protein S1. Interaction of oligo- and poly(U) with the 30 S subunit occurs in at least two steps: the first one is as fast as the interaction of poly(U) with free S1, whereas the second step represents a first-order reaction. Therefore, the second step may reflect some rearrangement of the template in the ribosome after its primary binding. It is suggested that protein S1 in some cases may fulfill the role of a transient binding site for mRNA in the course of its interaction with the ribosome. The general shape of the template in the mRNA binding region of the ribosome has been studied using various synthetic ribopolynucleotides and has been shown to be similar. It can be represented by a loop(s) or "U-turn(s)". On the basis of estimation of distances from the ends of poly(U) to some well-localized points on the 30 S ribosomal surface, a tentative model of mRNA path through the ribosome is proposed.

Bacterial Proteins↗
PubMed2026

Decoding Nonlinearities in AAV-Based Gene Therapy Using PBPK Modelling.

The objective of this research was to develop a physiologically based pharmacokinetic (PBPK) model for AAV-based gene therapy, which can capture the nonlinearity observed in both viral vector and transgene product pharmacokinetics (PK) across a wide range of doses, while accounting for the effect of immunogenicity. To develop the PBPK model, previously published PK data generated in mice using AAV8 vector containing the transgene for a non-binding monoclonal antibody was used. Immunocompetent mice were administered with AAV at a wide range of doses (1E8, 1E9, 5E9, 1E10, 2E10, 1E11, 2E11, 1E12, and 1E13vg per mouse), and the PK of transgene and transgene product (i.e., antibody) in plasma and/or tissue was collected. The nonlinearity in transgene product concentrations was characterized using a saturable production process and a concentration-dependent antibody elimination rate was used to characterize the effect of anti-drug antibody (ADA) on transgene product. The model successfully described the PK of both the vector and the transgene product across all dose levels and accurately captured the sigmoidal dose-exposure-response relationship for AAV. Notably, the model described a dose-dependent ADA response, with the high dose group exhibiting an earlier onset and faster rate of transgene product elimination. Lower dose group showed delayed onset and minimal ADA-mediated elimination of transgene product. Overall, the PBPK model presented here effectively characterizes vector and transgene product kinetics in mice and demonstrates utility in preclinical-to-clinical translation and dose optimization of AAV-based gene therapies.

Animals↗
PubMed2004

Codon usage bias from tRNA's point of view: redundancy, specialization, and efficient decoding for translation optimization.

The selection-mutation-drift theory of codon usage plays a major role in the theory of molecular evolution by explaining the co-evolution of codon usage bias and tRNA content in the framework of translation optimization. Because most studies have focused only on codon usage, we analyzed the tRNA gene pool of 102 bacterial species. We show that as minimal generation times get shorter, the genomes contain more tRNA genes, but fewer anticodon species. Surprisingly, despite the wide G+C variation of bacterial genomes these anticodons are the same in most genomes. This suggests an optimization of the translation machinery to use a small subset of optimal codons and anticodons in fast-growing bacteria and in highly expressed genes. As a result, the overrepresented codons in highly expressed genes tend to be the same in very different genomes to match the same most-frequent anticodons. This is particularly important in fast-growing bacteria, which have higher codon usage bias in these genes. Three models were tested to understand the choice of codons recognized by the same anticodons, all providing significant fit, but under different classes of genes and genomes. Thus, co-evolution of tRNA gene composition and codon usage bias in genomes seen from tRNA's point of view agrees with the selection-mutation-drift theory. However, it suggests a much more universal trend in the evolution of anticodon and codon choice than previously thought. It also provides new evidence that a selective force for the optimization of the translation machinery is the maximization of growth.

Anticodon↗
PubMed1984

Human C4 polymorphism: pedigree analysis of qualitative, quantitative, and functional parameters as a basis for phenotype interpretations.

Ten families with 82 members were investigated for C4A- and B polymorphism in a blind trial. Phenotyping was done on neuraminidase treated sera by immunofixation and simultaneously by hemolytic overlay electrophoresis. In addition Rg, Ch, BF, C2, HLA-A, B, C, DR, and GLO were determined. After decoding the samples the reliability of blind typing was found to be 84.4% according to segregation patterns. Inconsistencies occurred mostly when A4, A2, or A92 were present. The detection of silent A*Q0 and B*Q0 alleles was more critical than that of "difficult" allotypes. The quantitation of the C4A/B ratio by densitometry of stained gels or by conventional immunochemical measurements of serum C4 level could not substantially improve the identification of A*Q0 or B*Q0. C4 dependent activity in radial diffusion hemolysis showed satisfactory correspondence with the number of expressed C4B alleles. At least three haplotypes with two C4A genes (duplicated A genes) were observed as ascertained from offspring analysis in accordance with the MHC segregation pattern. Individuals with the duplicated C4A gene (C4A*3, A*2, in the absence of any other expressed A allele or together with C4A*92) showed only partial inhibition of Rodgers antisera. Partial inhibition of Chido antisera was seen in individuals with C4B 2 (in the absence of other B allotypes). The findings support the hypothesis of at least two structural C4 loci. They also demonstrate the inconsistency of quantitative data in the recognition of silent alleles.

Alleles↗
PubMed2026

Decoding context-dependent sirtuin pharmacology in cancer: Metabolic-epigenetic switches and precision therapeutic targeting.

Sirtuins (SIRT1-SIRT7) are a family of NAD+-dependent lysine deacetylases that possess mono-ADP-ribosyltransferase activity and integrate cellular metabolic status with chromatin regulation, genome maintenance, redox homeostasis, immune responses, and adaptation to cancer therapies. Their translational value has been obscured by a recurring paradox: the same isoform may constrain malignant transformation in one setting yet support metastatic competence, stemness, immune evasion, or drug resistance in another. This review reframes that paradox as a measurable problem of context. We define a SIRT context code in which NAD+ availability and compartmentalization, subcellular localization, PTM state, chromatin occupancy, oncogenic genotype, cell lineage, and tumor microenvironment jointly determine sirtuin output. Using recent mechanistic and translational evidence, we summarize how sirtuins regulate metabolic switching, histone acetylation and lactylation, genome stability, cancer-associated fibroblast programs, regulatory T-cell enrichment, cancer stem-cell plasticity, angiogenesis, and resistance to DNA-damaging, targeted, and immune therapies. We further argue that successful sirtuin pharmacology will require context matching rather than indiscriminate activation or inhibition. Priorities include spatial and single-cell biomarker discovery, compartment-specific NAD+ measurements, PTM-resolved activity assays, structure-guided isoform-selective agents, and degrader strategies targeting non-catalytic scaffolding functions. Sirtuins should therefore be viewed as metabolic-epigenetic decision nodes rather than fixed oncogenes or tumor suppressors. However, the evidence remains predominantly preclinical, and our search identified no clinical-stage oncology trials of direct sirtuin modulators using prospective biomarker stratification, underscoring that this framework remains translationally aspirational rather than clinically validated.

Humans↗
PubMed2001

Developmental dyslexia: an update on genes, brains, and environments.

The science of reading and developmental dyslexia has experienced spectacular advances during the last few years. Five aspects of this research are discussed in the article. (1) The holistic phenomenon of reading is complex. Many lower-level psychological processes (e.g., phonemic awareness, phonological decoding, ability to process stimuli rapidly and automatize this process, memory, ability to recognize words) contribute to a single act of reading. Conceptualizing the complex process of reading through its partly overlapping but partly independent components--which contribute to, but do not fully explain, the holistic process of reading--provides an excellent model for understanding complex hierarchies of higher mental functions. Those who master reading skills successfully and those who have difficulties doing so differ in a wide range of reading-related processes. The central deficit experienced by poor readers appears to be related to phonological processing (a complex hierarchy of functions related to processing phonemes), whereas characteristics of automatization processes seem to moderate the reading outcome for people whose phonological skills are weak. (2) There are new data addressing models of dyslexia in languages other than English. The most fascinating finding is that the model implicating phonological deficit as central to dyslexia, and the lack of ability to automatize as leading to troubled reading, appears to be universal, regardless of the specific language. However, there is an interaction effect between the characteristics of a particular language and the developmental model of dyslexia. In phonologically more difficult languages (e.g., English), the most pronounced weakness appears to occur in phonological processing, whereas in phonologically easier languages (e.g., German), the crucial role in the manifestation of dyslexia is played by the lack of the skills needed to achieve automatization. (3) There is abundant evidence that reading (i.e., any single act of reading as well as reading as a holistic process) is "cooked" by the brain. Although no unified brain map of reading has been developed, some specific areas of the brain have been implicated in different reading-related cognitive processes by different laboratories and on different samples. (4) Indisputable evidence has been accumulated suggesting the involvement of the genome in developmental dyslexia. As of now, specific regions of the genome have been identified as being intimately involved with a number of different reading-related processes. Today the field of developmental dyslexia is the only area of genetic studies of human abilities and disabilities in which linkages to the genome have been robustly replicated in independent laboratories. (5) Finally, evidence suggests that developmental dyslexia might be only one of the manifestations of a deep, underlying, anatomical syndrome. The comorbidity of developmental dyslexia with both internalizing and externalizing behavioral disturbances, as well as with other learning disabilities, underscores the need for wide-ranging cognitive and behavioral approaches in the remediation programs offered to dyslexic children.

Adolescent↗
PubMed1991

Cloning and sequence analysis of the Schistosoma mansoni membrane glycoprotein antigen gene GP22.

A family of Schistosoma mansoni proteins (18-22 kDa, pI 5.3-5.8) are biosynthesized in juvenile worms and immunoprecipitated by antibodies uniquely present in protective Fischer rat antiserum. A cDNA clone, lambda gt11-40, expressing epitopes common to this protein family was used to obtain a genomic DNA clone, by hybridization with a lambda gt11-40 oligonucleotide probe. In the 1.37 kb of genomic DNA sequenced, an open reading frame of 182 amino acids was identified on the strand corresponding to lambda gt11-40 coding sequences, and those of identical independently isolated cDNA clones defining a 25-kDa surface membrane glycoprotein. The new S. mansoni gene is termed GP22. There are two candidate promoters, confirmed by primer extension studies with worm RNA. Promoter 1 (P1) is preceded by a G + C-rich region and potential CAAT sequences, and is to the 5'-side of P2. Transcription from P1 is initiated at 2 different sites, apparently producing mRNAs with different translation start sites (ATG). Decoding these mRNAs yields protein products of 182 (P1), 175 (P1), 140 (P2) and 136 (P2) amino acids. The polypeptides share the following features: a hydrophobic segment near the carboxy terminus sufficient to span a lipid bilayer, with a consensus sequence for thio-esterification by a fatty acid; an external domain containing 2 potential N-linked glycosylation sites; and a candidate leucine-zipper motif, suggesting the protein may exist as a dimer on the worm surface. While sharing these common features in their carboxy terminal regions, the three proteins differ in the length and properties of their amino termini. The 140-amino acid protein has a short hydrophobic amino terminus, while the 175- and 182-amino acid proteins have more extensive hydrophobic sequences, each preceded by a hydrophilic amino terminal sequence. The heterogeneity observed in 2-dimensional gels of the antigen may be explained in part by the size and charge differences among the proteins deduced from the sequence and transcription pattern of this gene. The possibility of stage-specific regulated expression of this candidate vaccine antigen family is an attractive concept, potentially accounting for the phenomenon of concomitant immunity observed in the rat and perhaps other schistosome hosts.

Amino Acid Sequence↗
PubMed2004

Multi-species comparative mapping in silico using the COMPASS strategy.

MOTIVATION: The completion of human and mouse genome sequences provides a valuable resource for decoding other mammalian genomes. The comparative mapping by annotation and sequence similarity (COMPASS) strategy takes advantage of the resource and has been used in several genome-mapping projects. It uses existing comparative genome maps based on conserved regions to predict map locations of a sequence. An automated multiple-species COMPASS tool can facilitate in the genome sequencing effort and comparative genomics study of other mammalian species. RESULTS: The prerequisite of COMPASS is a comparative map table between the reference genome and the predicting genome. We have built and collected comparative maps among five species including human, cattle, pig, mouse and rat. Cattle-human and pig-human comparative maps were built based on the positions of orthologous markers and the conserved synteny groups between human and cattle and human and pig genomes, respectively. Mouse-human and rat-human comparative maps were based on the conserved sequence segments between the two genomes. With a match to human genome sequences, the approximate location of a query sequence can be predicted in cattle, pig, mouse and rat genomes based on the position of the match relatively to the orthologous markers or the conserved segments. AVAILABILITY: The COMPASS-tool and databases are available at http://titan.biotec.uiuc.edu/COMPASS/

Algorithms↗
PubMed2003

Common gene polymorphisms and nutrition: emerging links with pathogenesis of multifactorial chronic diseases (review).

Rapid progress in human genome decoding has accelerated search for the role of gene polymorphisms in the pathogenesis of complex multifactorial diseases. This review summarizes the results of recent studies on the associations of common gene variants with multifactorial chronic conditions strongly affected by nutritional factors. Three main individual sections discuss genes related to energy homeostasis regulation and obesity, cardiovascular disease (CVD), and cancer. It is evident that several major chronic diseases are closely related (often through obesity) to deregulation of energy homeostasis. Multiple polymorphic genes encoding central and peripheral determinants of energy intake and expenditure have been revealed over the past decade. Food intake control may be affected by polymorphisms in the genes encoding taste receptors and a number of peripheral signaling peptides such as insulin, leptin, ghrelin, cholecystokinin, and corresponding receptors. Polymorphic central regulators of energy intake include hypothalamic neuropeptide Y, agouti-related protein, melanocortin pathway factors, CART (cocaine- and amphetamine-regulated transcript), some other neuropeptides, and receptors for these molecules. Potentially important polymorphisms in the genes encoding energy expenditure modulators (alpha- and beta- adrenoceptors, uncoupling proteins, and regulators of adipocyte growth and differentiation) are also discussed. CVD-related gene polymorphisms comprising those involved in the pathogenesis of atherosclerosis, blood pressure regulation, hemostasis control, and homocysteine metabolism are considered in a separate section with emphasis on multiple polymorphisms affecting lipid transport and metabolism and their interactions with diet. Cancer-associated polymorphisms are discussed for groups of genes encoding enzymes of xenobiotic metabolism, DNA repair enzymes, factors involved in the cell cycle control, hormonal regulation-associated proteins, enzymes related to DNA methylation through folate metabolism, and angiogenesis-related factors. There is an apparent progress in the field with hundreds of new gene polymorphisms discovered and characterized, however firm evidence consistently linking them with pathogenesis of complex chronic diseases is still limited. Ways of improving the efficiency of candidate gene approach-based studies are discussed in a short separate section. Successful unraveling of interaction between dietary factors, polymorphisms, and pathogenesis of several multifactorial diseases is exemplified by studies of folate metabolism in relation to CVD and cancer. It appears that several new directions emerge as targets of research on the role of genetic variation in relation to diet and complex chronic diseases. Regulation of energy homeostasis is a fundamental problem insufficiently investigated in this context so far. Impacts of genetic variation on systems controlling angiogenesis, inflammatory reactions, and cell growth and differentiation (comprising regulation of the cell cycle, DNA repair, and DNA methylation) are also largely unknown and need thorough analysis. These goals can be achieved by complex simultaneous analysis of multiple polymorphic genes controlling carefully defined and selected elements of relevant metabolic and regulatory pathways in meticulously designed large-scale studies.

Cardiovascular Diseases↗
PubMed1994

Two coding change mutations in the HIS2(2) allele characterize the salivary histatin 3-2 protein variant.

The decoded amino acid sequence of a salivary protein variant, histatin 3-2 (formerly termed Pb c), that is found primarily and in high frequency in Black populations was determined by genomic PCR and direct sequencing of the HIS2(2) allele. Two different mutations that cause coding changes were found in exon 5. The first mutation is a single nucleotide (T-->A) substitution that causes a TAT (Tyr)-->TAA (Stop) change at residue 28. This premature stop mutation results in a 27 amino acid histatin 3-2 protein, which is 5 amino acids smaller than the common histatin 3-1 allelic protein (a product of the HIS2(1) allele). The second mutation, a single nucleotide (G-->A) substitution (located only 19 nucleotides upstream of the first mutation) causes a CGA (Arg)-->CAA (Gln) change at residue 22, which eliminates a proteolytic cleavage site. These two mutations explain the differences in electrophoretic patterns of HIS2(1) versus HIS2(2) coded histatin peptides and may have functional significance. Each mutation alters a different DNA restriction site, and this provides a DNA-based test for the mutations. This test should greatly simplify population and family studies of this protein polymorphism, since the saliva-based test is considerably more problematic. Elucidation here of the derived protein sequence of the variant histatin 3-2 protein may also facilitate functional studies.

Alleles↗
PubMed1992

Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.

Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio-uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio-U residues were activated by UV irradiation and the resulting sites of cross-linking to 16S RNA analysed. Three distinct cross-links were consistently observed: (i) from position '+6' of the mRNA (the 3'-base of the A-site codon) to base 1052 of 16S RNA; (ii) from position '+7' of the mRNA to base 1395; and (iii) from '+11' to base 532. Individual yields of the cross-links were strongly dependent on the particular mRNA sequence in each case. The '+11/532' and '+6/1052' cross-links were always entirely tRNA-dependent, whereas the '+7/1395' cross-link was observed at lower intensity in the absence of tRNA. In the presence of a second (A-site bound) tRNA the +6/1052 cross-link was markedly reduced. A cross-link to the 1050 region was again observed when a message carrying a thio-U at position '+9' was translocated on the ribosome so as to bring the thio-U to position +6. Taken together, the data are incompatible with some current models both for the three-dimensional arrangement of 16S RNA and for the orientation of the tRNA-mRNA complex in the ribosome.

Base Sequence↗
PubMed2006

Mapping the proteome of poplar and application to the discovery of drought-stress responsive proteins.

Poplar is the first forest tree genome to be decoded. As an initial step to the comprehensive analysis of poplar proteome, we described reference 2-D-maps for eight tissues/organs of the plant, and the functional characterization of some proteins. A total of 398 proteins were excised from the gels. About 91.2% were identified by nanospray LC-MS/MS, based on comparison with 260,000 Populus sp. ESTs. In comparison, reliable PMFs were obtained for only 51% of the spots by MALDI-TOF-MS, from which 43% (83 spots) positively matched gene models of the Populus trichocarpa genome sequence. Among these 83 spots, 58% matched with the same proteins as identified by LC-MS/MS, 21.7% with unknown function proteins and 19.3% with completely different functions. In the second phase, we studied the effect of drought stress on poplar root and leaf proteomes. The function of up- and down-regulated proteins is discussed with respect to the physiological response of the plants and compared with transcriptomic data. Some important clues regarding the way poplar copes with water deficit were revealed.

Databases, Protein↗
PubMed2006

The A-site finger in 23 S rRNA acts as a functional attenuator for translocation.

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

Base Sequence↗