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Biomedical subjects

M Mittmann

Publications and source records attributed to M Mittmann.

9 recordsLinked to original sources
PubMed1999

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Culture Media↗
PubMed1999

High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays.

A highly reliable and efficient technology has been developed for high-throughput DNA polymorphism screening and large-scale genotyping. Photolithographic synthesis has been used to generate miniaturized, high-density oligonucleotide arrays. Dedicated instrumentation and software have been developed for array hybridization, fluorescent detection, and data acquisition and analysis. Specific oligonucleotide probe arrays have been designed to rapidly screen human STSs, known genes and full-length cDNAs. This has led to the identification of several thousand biallelic single-nucleotide polymorphisms (SNPs). Meanwhile, a rapid and robust method has been developed for genotyping these SNPs using oligonucleotide arrays. Each allele of an SNP marker is represented on the array by a set of perfect match and mismatch probes. Prototype genotyping chips have been produced to detect 400, 600 and 3000 of these SNPs. Based on the preliminary results, using oligonucleotide arrays to genotype several thousand polymorphic loci simultaneously appears feasible.

Genetic Markers↗
PubMed1998

Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome.

Single-nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome, and they provide powerful tools for a variety of medical genetic studies. In a large-scale survey for SNPs, 2.3 megabases of human genomic DNA was examined by a combination of gel-based sequencing and high-density variation-detection DNA chips. A total of 3241 candidate SNPs were identified. A genetic map was constructed showing the location of 2227 of these SNPs. Prototype genotyping chips were developed that allow simultaneous genotyping of 500 SNPs. The results provide a characterization of human diversity at the nucleotide level and demonstrate the feasibility of large-scale identification of human SNPs.

Algorithms↗
PubMed1997

Expression of retinoid-X receptors (-alpha,-beta,-gamma) and retinoic acid receptors (-alpha,-beta,-gamma) in normal human skin: an immunohistological evaluation.

Increasing evidence suggests that the retinoid-X receptors (RXR-alpha,-beta,-gamma) play a crucial role in regulating the transcriptional activity of several steroid hormone receptors, including the receptors for retinoic acid (RAR-alpha,-beta,-gamma), 1,25-dihydroxyvitamin D3 and thyroid hormone. We investigated the localization of the different types of RXR-alpha,-beta,-gamma and RAR-alpha,-beta,-gamma proteins in frozen sections of normal human skin (n = 12) in situ, applying recently raised corresponding specific monoclonal antibodies and an immunohistochemical technique that we established for the detection of these nuclear receptors. Our findings indicate that RXR-alpha,-beta,-gamma and RAR-alpha,-beta,-gamma proteins can be detected by immunohistochemistry in normal human skin. In contrast to RXR-alpha,-beta,-gamma as well as RAR-alpha and RAR-gamma proteins that were consistently detected in cell layers of the viable epidermis, RAR-beta was only focally demonstrated in single epidermal cells in three out of 12 biopsies analysed. Immunohistochemical labelling of RAR-alpha,-beta,-gamma and RXR-alpha,-beta,-gamma proteins in epidermal nuclei was also pronounced in the stratum granulosum, suggesting a function of RXR and RAR proteins in the transition from proliferation to differatiation in epidermal keratinocytes. Expression of RXRs and RARs in hair follicles, sebaceous glands and endothelial cell points to a biological function from these nuclear receptors to hair growth as well as to the physiology of sebaceous glands and endothelial cells.

Humans↗
PubMed1997

Genome-wide expression monitoring in Saccharomyces cerevisiae.

The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes. This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry. The measurements are quantitative, sensitive, specific, and reproducible. Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media. Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell. Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.

Cell Division↗
PubMed1996

Immunohistochemical detection of retinoic acid receptor alpha in human skin: a comparison of different fixation protocols.

A technique for the immunohistochemical detection of retinoic acid receptor alpha in cryostat sections of normal human skin in situ has been developed. A highly specific mouse monoclonal antibody, directed against the F region of retinoic acid receptor alpha, was used and a panel of 10 fixation protocols investigated. A three-step protocol, consisting of sequential fixation in 3.7% paraformaldehyde, methanol and acetone, revealed strong nuclear immunoreactivity in epidermal keratinocytes and other cell types present in normal human skin. Other fixation protocols, including fixation regimens using formaldehyde or Carnoy's solution, were less suitable or unsuitable for detection of the receptor in cryostat sections of human skin.

Animals↗
PubMed1996

Expression monitoring by hybridization to high-density oligonucleotide arrays.

The human genome encodes approximately 100,000 different genes, and at least partial sequence information for nearly all will be available soon. Sequence information alone, however, is insufficient for a full understanding of gene function, expression, regulation, and splice-site variation. Because cellular processes are governed by the repertoire of expressed genes, and the levels and timing of expression, it is important to have experimental tools for the direct monitoring of large numbers of mRNAs in parallel. We have developed an approach that is based on hybridization to small, high-density arrays containing tens of thousands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide chemistry. RNAs present at a frequency of 1:300,000 are unambiguously detected, and detection is quantitative over more than three orders of magnitude. This approach provides a way to use directly the growing body of sequence information for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize small arrays containing hundreds of thousands of specifically chosen oligonucleotides, the method is readily scalable to the simultaneous monitoring of tens of thousands of genes.

Animals↗
PubMed1996

Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy.

A quantitative and highly parallel method for analysing deletion mutants has been developed to aid in determining the biological function of thousands of newly identified open reading frames (ORFs) in Saccharomyces cerevisiae. This approach uses a PCR targeting strategy to generate large numbers of deletion strains. Each deletion strain is labelled with a unique 20-base tag sequence that can be detected by hybridization to a high-density oligonucleotide array. The tags serve as unique identifiers (molecular bar codes) that allow analysis of large numbers of deletion strains simultaneously through selective growth conditions. Hybridization experiments show that the arrays are specific, sensitive and quantitative. A pilot study with 11 known yeast genes suggests that the method can be extended to include all of the ORFs in the yeast genome, allowing whole genome analysis with a single selective growth condition and a single hybridization.

DNA↗