Biomedical subjects

V Aidinis

Publications and source records attributed to V Aidinis.

9 recordsLinked to original sources

PubMed2006

Operational criteria for selecting a cDNA microarray data normalization algorithm.

Microarray technology allows gene expression profiling at a global level. Many algorithms for the normalization of raw microarray data have been proposed, but no attempt has yet been made to propose operationally verifiable criteria for their comparative evaluation, which is necessary for the selection of the most appropriate method for a given dataset. This study develops a set of operational criteria for assessing the impact of various normalization algorithms in terms of accuracy (bias), precision (variance) and over-fitting (information reduction). The use of these criteria is illustrated by applying the three most widely used algorithms (global median normalization, spiked-in based normalization and lowess) on a specifically designed, multiply-controlled dataset.

Algorithms↗

PubMed2005

The transcriptional landscape of the mammalian genome.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

3' Untranslated Regions↗

PubMed2000

Definition of minimal domains of interaction within the recombination-activating genes 1 and 2 recombinase complex.

During V(D)J recombination, recognition and cleavage of the recombination signal sequences (RSSs) requires the coordinated action of the recombination-activating genes 1 and 2 (RAG1/RAG2) recombinase complex. In this report, we use deletion mapping and site-directed mutagenesis to determine the minimal domains critical for interaction between RAG1 and RAG2. We define the active core of RAG2 required for RSS cleavage as aa 1-371 and demonstrate that the C-terminal 57 aa of this core provide a dominant surface for RAG1 interaction. This region corresponds to the last of six predicted kelch repeat motifs that have been proposed by sequence analysis to fold RAG2 into a six-bladed beta-propeller structure. Residue W317 within this sixth repeat is shown to be critical for mediating contact with RAG1 and concurrently for stabilizing binding and directing cleavage of the RSS. We also show that zinc finger B (aa 727-750) of RAG1 provides a dominant interaction domain for recruiting RAG2. In all, the data support a model of RAG2 as a multimodular protein that utilizes one of its six faces for establishing productive contacts with RAG1.

Amino Acid Sequence↗

PubMed1999

The RAG1 homeodomain recruits HMG1 and HMG2 to facilitate recombination signal sequence binding and to enhance the intrinsic DNA-bending activity of RAG1-RAG2.

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

Binding Sites↗

PubMed1998

The effect of Me2+ cofactors at the initial stages of V(D)J recombination.

V(D)J site-specific recombination mediates the somatic assembly of the antigen receptor gene segments. This process is initiated by the recombination activating proteins RAG1 and RAG2, which recognize the recombination signal sequences (RSS) and cleave the DNA at the coding/RSS junction. In this study, we show that RAG1 and RAG2 have the ability to directly interact in solution before binding to the DNA. RAG1 forms a homodimer, which leads to the appearance of two distinct RAG1.RAG2 complexes bound to DNA. To investigate the properties of the two RAG1.RAG2 complexes in the presence of different Me2+ cofactors, we established an in vitro Mg2+-based cleavage reaction on a single RSS. Using this system, we found that Mg2+ confers a specific pattern of DNA binding and cleavage. In contrast, Mn2+ allows aberrant binding of RAG1.RAG2 to single-stranded RSS and permits cleavage independent of binding to the nonamer. To determine the contribution of Me2+ ions at the early stages of V(D)J recombination, we analyzed specific DNA recognition and cleavage by RAG1.RAG2 on phosphorothioated substrates. These experiments revealed that Me2+ ions directly coordinate the binding of RAG1.RAG2 to the RSS DNA.

Animals↗

PubMed1995

Two immunologically related polypeptides of 72/74 kDa specify a novel 70-100S heterogeneous nuclear RNP.

Evidence suggesting the presence in rat liver nuclear extracts of a new RNP complex of 70-110S has been provided [Hatzoglou, M., Adamtziki, E., Margaritis, L. and Sekeris, C.E (1985) Exp. Cell. Res. 157, 227-241]. Biochemical features unique to this RNP were its stability to salt and RNase digestion and the presence of a pair of polypeptides of 72/74 kDa. By producing antibodies against the 72/74 kDa polypeptides these proteins have been defined as integral components of the 70-110S RNP complex. They comprise two immunologically related polypeptides with an exclusively nucleoplasmic localization, giving a speckled pattern in a diffuse background, similar, but not identical, to the Sm antigen. The 70-110S RNP complex, referred to as large heterogeneous nuclear RNP (LH-nRNP), has a simple protein pattern that includes, in addition to the 72/74 kDa proteins, three stably associated polypeptides of apparent molecular size 110, 61 and 59 kDa. The bulk of its RNA component represents a discrete RNA population of 10-20S, belonging to a subset of the RNA detected within immunopurified HeLa hnRNP complexes. These RNA species are RNA polymerase II transcripts of greater stability relative to the bulk of hnRNA, containing oligo(A) or poly(A) sequences. Immunodepletion and/or antibody addition studies in HeLa splicing extracts using antibodies with specificity for the 72/74 kDa proteins revealed a rather strong inhibition of splicing activity, suggesting participation of the LH-nRNP complex in in vitro splicing.

Animals↗

PubMed1995

Detection of human-specific anti-la(SSB) antibodies in patients with rheumatoid arthritis.

Sera from anti-Ro(SSA) positive and negative patients with rheumatoid arthritis (RA) were compared with those from patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE) with regard to anti-La(SSB) antibodies. Several assays for anti-La(SSB) (RNA precipitation, counterimmunoelectrophoresis, and immunoblotting) demonstrated the presence of such antibodies in selected anti-Ro(SSA) positive (10/19 in RA and 18/37 in pSS and SLE) but not in anti-Ro(SSA) negative sera. In agreement with previous reports, anti-La(SSB) antibodies from pSS and SLE patients uniformly reacted with various cellular extracts used as sources of La(SSB) antigen, including extracts from human cultured cells (HeLa), calf thymus and rabbit thymus. In contrast, while all 10 anti-La(SSB) sera from RA patients reacted with the human HeLa cell extracts, only three of them also reacted with calf and rabbit thymus extracts. These results were further supported by the analysis of a cohort of 70 consecutively selected sera (displaying monospecific anti-Ro(SSA) reactivity against calf thymus extract) from patients with various rheumatic disorders, where human-specific anti-La(SSB) were detected in two out of the five of RA patients studied.

Animals↗

PubMed1994

Anti-5S RNA/protein (RNP) antibody levels correlate with disease activity in a patient with systemic lupus erythematosus (SLE) nephritis.

A patient with systemic lupus erythematosus (SLE) and nephritis without antibodies to dsDNA but with antibodies to a 5S RNA/protein (RNP) complex is presented. Combined RNA precipitation and Western blotting experiments strongly suggested that these newly identified autoantibodies recognized a distinct epitope on the L5 ribosomal protein of the L5/5S RNP complex first described by Steitz et al. [1]. Quantification of the anti-5S RNP antibody levels was done by hybridizing Northern blots of immunoprecipitated RNA from serial serum samples with a 32P-labelled oligoprobe specific for the 5S ribosomal RNA. These studies revealed a strong association between anti-5S RNP autoantibody titre and severity of SLE nephritis over a 3-year prospective study. Our results indicate that the L5/5S RNP can be a target of autoimmune response, and and may serve, in some cases, as marker of SLE severity and response to therapy.

Antibody Specificity↗

PubMed1993

Detection of anti-Ro(SSA) antibodies in autoimmune diseases: comparison of five methods.

Counterimmunoelectrophoresis (CIE), RNA precipitation, ELISA and immunoblotting against cytoplasmic HeLa cell extract (IB-HeLa) and erythrocyte extract (IB-RBC) were applied to detect anti-Ro(SSA) antibodies in 93 sera selected from patients with various autoimmune diseases [47 were anti-Ro(SSA) positive by CIE]. The RNA precipitation assay, which demonstrated the highest sensitivity was selected as the reference method. CIE was found to be reliable with a specificity of 100% and a sensitivity of 89%. ELISA showed a comparable specificity (95%) but somewhat lower sensitivity (72%). Antibodies to 52 or 60 kDa Ro(SSA) proteins by IB-HeLa demonstrated a high specificity (95 and 97% respectively) but a low overall sensitivity (36 and 17% respectively). Anti-Ro(SSA) antibodies to 52, 54 and 60 kDa erythrocyte proteins by IB-RBC, had a variable overall specificity (95, 97 and 57%) and sensitivity (51, 13 and 34%). The anti-52 kDa antibodies detected by IB-HeLa correlated to those found by IB-RBC (P < 0.001) and occurred predominantly in primary Sjögren's syndrome (P < 0.001, sensitivity: 71 and 77%) as well as in sera with anti-Ro(SSA) and anti-La(SSB) antibodies (P < 0.001). These findings confirm that RNA precipitation assay has the highest sensitivity and specificity for anti-Ro(SSA) antibody detection. However, until a more sensitive ELISA is available, CIE because of its reliability appears to be the method of choice. Finally IB-RBC was found to be more sensitive than IB-HeLa for the detection of anti-Ro52 kDa antibodies.

Adult↗