Biomedical subjects

Mark R Flory

Publications and source records attributed to Mark R Flory.

6 recordsLinked to original sources

PubMed2004

An SMC-domain protein in fission yeast links telomeres to the meiotic centrosome.

Abnormal centrosomal structures similar to those occurring in human cancers are induced in fission yeast by overexpression of the pericentrin homolog Pcp1p. Analysis of abnormal Pcp1p-containing structures with quantitative mass spectrometry and isotope-coded affinity tags identified a coiled-coil, structural maintenance of chromosomes (SMC) domain protein. This protein, termed Ccq1p (coiled-coil protein quantitatively enriched), localizes with Taz1p to telomeres in normal vegetative cells. Fluorescence resonance energy transfer (FRET) measurements indicate that Ccq1p also interacts with centrosomal Pcp1p in mating pheromone-stimulated cells containing centrosomally clustered telomeres. We provide evidence that the Ccq1p-Pcp1p interaction, while essential for meiosis, is deleterious when forced to occur during vegetative growth. Cells lacking one ccq1 allele exhibit a loss-of-function phenotype including abnormally long cell length, chromosome segregation failure, telomeric shortening, and defective telomeric clustering during meiotic prophase. Our data indicate a mechanism underlying meiotic chromosomal bouquet formation and suggest a recruitment model for supernumerary centrosome toxicity.

Alleles↗

PubMed2004

Gene expression analyzed by high-resolution state array analysis and quantitative proteomics: response of yeast to mating pheromone.

The transcriptome provides the database from which a cell assembles its collection of proteins. Translation of individual mRNA species into their encoded proteins is regulated, producing discrepancies between mRNA and protein levels. Using a new modeling approach to data analysis, a striking diversity is revealed in association of the transcriptome with the translational machinery. Each mRNA has its own pattern of ribosome loading, a circumstance that provides an extraordinary dynamic range of regulation, above and beyond actual transcript levels. Using this approach together with quantitative proteomics, we explored the immediate changes in gene expression in response to activation of a mitogen-activated protein kinase pathway in yeast by mating pheromone. Interestingly, in 26% of those transcripts where the predicted protein synthesis rate changed by at least 3-fold, more than half of these changes resulted from altered translational efficiencies. These observations underscore that analysis of transcript level, albeit extremely important, is insufficient by itself to describe completely the phenotypes of cells under different conditions.

Computational Biology↗

PubMed2003

The centrosomal proteins pericentrin and kendrin are encoded by alternatively spliced products of one gene.

Pericentrin, a critical centrosome component first identified in mouse, recruits factors required for assembly of the mitotic spindle apparatus. A similar yet larger human protein named kendrin was recently identified, but its relationship to pericentrin was not clear. Extensive sequence homology between the mouse chromosome 10 region encoding pericentrin and the human chromosome 21 region encoding kendrin indicates that these proteins are encoded by syntenic loci. However, comparison of the published mouse pericentrin cDNA sequence to mouse genomic DNA sequences revealed two important differences: the stop codon present in the published mouse pericentrin cDNA is not found in the mouse genomic sequence, and the 3' end of the published mouse pericentrin cDNA is a fragment from a different mouse chromosome. To resolve these discrepancies, we sequenced a mouse expressed sequence tag (EST) that corresponds to the 3' end for a 7.1-kb mouse pericentrin RNA encoded on chromosome 10. Extensive northern blot analysis revealed that the pericentrin gene displays a complex expression pattern in both mouse and human: a 10-kb kendrin transcript is found in most tissues, whereas smaller transcripts are detected in a limited subset of tissues. These analyses demonstrate that pericentrin and kendrin are encoded by one gene, correct the previously published pericentrin cDNA sequence, and describe the complex expression pattern for a gene important for centrosome function in normal and transformed cells.

Alternative Splicing↗

PubMed2003

Proteomic approaches for the identification of cell cycle-related drug targets.

Drugs affecting the cell cycle provide insights into mechanisms underlying cancer and suggest strategies for ablating uncontrolled growth. Essential to an understanding of the activity of such compounds is the identification of the set of proteins affected, either directly or indirectly, by the drug. The combination of novel technologies for stable isotope protein tagging, chromatographic separation, tandem mass spectrometry, and data processing is an extremely powerful means for providing such identifications and, in addition, for establishing a proteome-wide profile of all proteins whose abundance levels or phosphorylation state are affected by the drug.

Animals↗

PubMed2002

Advances in quantitative proteomics using stable isotope tags.

A great deal of current biological and clinical research is directed at the interpretation of the information contained in the human genome sequence in terms of the structure, function and control of biological systems and processes. Proteomics, the systematic analysis of proteins, is becoming a critical component in this endeavor because proteomic measurements are carried out directly on proteins--the catalysts and effectors of essentially all biological functions. To detect changes in protein profiles that might provide important diagnostic or functional insights, proteomic analyses necessarily have to be quantitative. This article summarizes recent technological advances in quantitative proteomics.

Animals↗

PubMed2002

Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe.

In the budding yeast Saccharomyces cerevisiae, the calmodulin-binding protein Spc110p/Nuf1p facilitates mitotic spindle formation from the fungal centrosome or spindle pole body (SPB). The human Spc110p orthologue kendrin is a centrosomal, calmodulin-binding pericentrin isoform that is specifically overexpressed in carcinoma cells. Here we establish an evolutionary and functional link between Spc110p and kendrin through identification and analysis of similar calmodulin-binding proteins in the fission yeast Schizosaccharomyces pombe (Pcp1p, pole target of calmodulin in S. pombe) and the filamentous fungus Aspergillus nidulans. Like Spc110p and kendrin, Pcp1p and the A. nidulans protein contain predicted coiled-coil secondary structure and a COOH-terminal calmodulin-binding region. Green fluorescent protein fusions of Pcp1p localize to the SPB as analyzed by fluorescence and immunoelectron microscopy. Pcp1p overexpression causes chromosome missegregation, multiple mitotic spindle fragments, and multiple abnormal SPB-like structures, a phenotype remarkably similar to that of many human carcinoma lines, which exhibit chromosome and spindle defects, and supernumerary centrosomes.

Amino Acid Sequence↗