Biomedical subjects

Michael B Stadler

Publications and source records attributed to Michael B Stadler.

7 recordsLinked to original sources

PubMed2026

A practical guide to studying genome function using single-molecule genomics.

Single-molecule genomics (SMG) has transformed our ability to study the mechanisms that regulate the genome by enabling profiling of the activity of regulatory factors on individual DNA molecules genome-wide. SMG is able to quantify molecular heterogeneity and the co-occurrence of regulatory events, including epigenetic modifications, transcription factor binding and chromatin organization on single DNA molecules. SMG reveals dynamics of chromatin interactions that cannot be measured by conventional genomics assays. Therefore, SMG offers a unique platform to study how regulatory events combine to control genome activity. In this Expert Recommendation article, we provide a practical guide for adopting SMG and outline best practices.

Journal Article↗

PubMed2006

Inference of splicing regulatory activities by sequence neighborhood analysis.

Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI) that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE) and silencer (ESS) hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences.

Binding Sites↗

PubMed2005

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha.

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.

Amino Acid Sequence↗

PubMed2005

Detection of one VH antibody sequence in both healthy donors and urticaria patients.

We have previously isolated anti-FcepsilonRIalpha autoantibodies from phage libraries of healthy donors and urticaria patients. Strikingly, the same antibody, LTMalpha15, was isolated from both libraries. Sequence analysis revealed a germline configuration of the LTMalpha15 variable heavy (V(H)) chain with a slightly mutated variable light (V(L)) chain supporting its classification as a natural autoantibody. Distribution analysis of anti-FcepsilonRIalpha autoantibodies by functional or serological tests delivered conflicting data. For this reason we have developed a new real-time PCR to analyse the distribution of LTMalpha15V(H) in healthy donors and urticaria patients. Our new bioinformatic program permitted the design of a minor groove binder (MGB) TaqMan probe that specifically detected the LTMalpha15V(H). We were able to demonstrate a broad range of rearranged V(H) gene copy number without any correlation to the state of health. Monitoring LTMalpha15V(H) gene copy number in a single donor over a period of 70 days revealed a time-related fluctuation of circulating B cells carrying LTMalpha15V(H). We propose that our real-time PCR may serve as a model for the quantification of natural antibody sequences at a monoclonal level.

Autoantibodies↗

PubMed2005

Single nucleotide polymorphisms as a prerequisite for autoantigens.

It is still elusive why certain self proteins induce an autoimmune response. One immunological hypothesis is that only modified or altered self-proteins may become a target. Thus, we asked whether such alterations may actually be genetic polymorphisms that can be revealed by analyzing sequence variability in the known human autoantigens. Indeed, we found autoantigens to contain significantly more single nucleotide polymorphisms (SNP) than other human genes do. Our finding may offer an explanation for autoimmune responses through allogeneic exposure. Besides other contributing factors in autoimmunity, SNP may represent an essential prerequisite for the primary induction of an autoimmune response.

Amino Acid Sequence↗

PubMed2004

A highly conserved interspecies V(H) in the human genome.

Idiotype conservation between human and mouse antibodies has been observed in association with various infectious and autoimmune diseases. We have isolated a human anti-idiotypic antibody to a mouse monoclonal anti-IgE antibody (BSW17) suggesting a conserved interspecies idiotype associated with an anti-IgE response. To find the homologue of BSW17 in the human genome we applied the guided selection strategy. Combining V(H) of BSW17 with a human V(L) repertoire resulted in three light chains. The three V(L) chains were then combined with a human V(H) repertoire resulting in three clones specific for human IgE. Surprisingly, one clone, Hu41, had the same epitope specificity and functional in vitro activity as BSW17 and V(H) complementarity-determining regions identical with that of BSW17. Real-time PCR analysis confirmed the presence of the Hu41 V(H) sequence in the human genome. These data document the first example of the isolation of a human antibody where high sequence similarity to the original murine V(H) sequence is associated with common antigen and epitope specificity.

Amino Acid Sequence↗

PubMed2003

Allergenicity prediction by protein sequence.

Potential allergenicity of transgenic proteins for consumption must be investigated before their introduction into the food chain. A prerequisite is sequence analysis. We have critically reviewed the performance of the current guidelines proposed by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) for allergenicity prediction based on protein sequence and show that its precision is very low. To improve prediction, we propose a new strategy based on sequence motifs identified from a new allergen database. If tested on random test sequences and known allergens, both methods are apparently very sensitive. However, the precision of our motif-based prediction (95.5%) is superior to the current method (36.6%). We conclude that the proposed motif-based prediction is a superior alternative to the current method for use in the decision-tree approach for allergenicity assessment.

Allergens↗