Biomedical subjects

James P Kastenmayer

Publications and source records attributed to James P Kastenmayer.

5 recordsLinked to original sources

PubMed2006

Functional genomics of genes with small open reading frames (sORFs) in S. cerevisiae.

Genes with small open reading frames (sORFs; <100 amino acids) represent an untapped source of important biology. sORFs largely escaped analysis because they were difficult to predict computationally and less likely to be targeted by genetic screens. Thus, the substantial number of sORFs and their potential importance have only recently become clear. To investigate sORF function, we undertook the first functional studies of sORFs in any system, using the model eukaryote Saccharomyces cerevisiae. Based on independent experimental approaches and computational analyses, evidence exists for 299 sORFs in the S. cerevisiae genome, representing approximately 5% of the annotated ORFs. We determined that a similar percentage of sORFs are annotated in other eukaryotes, including humans, and 184 of the S. cerevisiae sORFs exhibit similarity with ORFs in other organisms. To investigate sORF function, we constructed a collection of gene-deletion mutants of 140 newly identified sORFs, each of which contains a strain-specific "molecular barcode," bringing the total number of sORF deletion strains to 247. Phenotypic analyses of the new gene-deletion strains identified 22 sORFs required for haploid growth, growth at high temperature, growth in the presence of a nonfermentable carbon source, or growth in the presence of DNA damage and replication-arrest agents. We provide a collection of sORF deletion strains that can be integrated into the existing deletion collection as a resource for the yeast community for elucidating gene function. Moreover, our analyses of the S. cerevisiae sORFs establish that sORFs are conserved across eukaryotes and have important biological functions.

Conserved Sequence↗

PubMed2005

The C-terminal half of Saccharomyces cerevisiae Mad1p mediates spindle checkpoint function, chromosome transmission fidelity and CEN association.

The evolutionarily conserved spindle checkpoint is a key mechanism ensuring high-fidelity chromosome transmission. The checkpoint monitors attachment between kinetochores and mitotic spindles and the tension between sister kinetochores. In the absence of proper attachment or tension, the spindle checkpoint mediates cell cycle arrest prior to anaphase. Saccharomyces cerevisiae Mad1p is required for the spindle checkpoint and for chromosome transmission fidelity. Moreover, Mad1p associates with the nuclear pore complex (NPC) and is enriched at kinetochores upon checkpoint activation. Using partial mad1 deletion alleles we determined that the C-terminal half of Mad1p is necessary and sufficient for checkpoint activation in response to microtubule depolymerizing agents, high-fidelity transmission of a reporter chromosome fragment, and in vivo association with centromeres, but not for robust NPC association. Thus, spindle checkpoint activation and chromosome transmission fidelity correlate and these Mad1p functions likely involve kinetochore association but not robust NPC association. These studies are the basis for elucidating the role of protein complexes containing Mad1p in the spindle checkpoint pathway and in maintaining genome stability in S. cerevisiae and other systems.

Alleles↗

PubMed2004

AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets.

Messenger RNA degradation is an essential step in gene expression that can be regulated by siRNAs or miRNAs. However, most of our knowledge of in vivo eukaryotic mRNA degradation mechanisms derives from Saccharomyces cerevisiae, which lacks miRNAs and RNAi capability. Using reverse genetic and microarray analyses, we have identified multiple substrates of AtXRN4, the Arabidopsis homolog of the major yeast mRNA degrading exoribonuclease, Xrn1p. Insertional mutation of AtXRN4 leads to accumulation of the 3' end of several mRNAs, in a manner that correlates with increased stability of the 3' end, and is reversed following complementation with AtXRN4. Moreover, 3' products of miRNA-mediated cleavage of SCARECROW-LIKE transcripts and several other miRNA target transcripts are among those that accumulate in xrn4 mutants. The demonstration that an Xrn1p homolog degrades mRNA in a multicellular eukaryote and contributes to the miRNA-mediated decay pathway of selected targets has implications for XRNs in other organisms.

Arabidopsis↗

PubMed2003

Plant snoRNA database.

The Plant snoRNA database (http://www.scri.sari.ac.uk/plant_snoRNA/) provides information on small nucleolar RNAs from Arabidopsis and eighteen other plant species. Information includes sequences, expression data, methylation and pseudouridylation target modification sites, initial gene organization (polycistronic, single gene and intronic) and the number of gene variants. The Arabidopsis information is divided into box C/D and box H/ACA snoRNAs, and within each of these groups, by target sites in rRNA, snRNA or unknown. Alignments of orthologous genes and gene variants from different plant species are available for many snoRNA genes. Plant snoRNA genes have been given a standard nomenclature, designed wherever possible, to provide a consistent identity with yeast and human orthologues.

Arabidopsis↗

PubMed2002

Experimental RNomics: identification of 140 candidates for small non-messenger RNAs in the plant Arabidopsis thaliana.

BACKGROUND: Genomes from all organisms known to date express two types of RNA molecules: messenger RNAs (mRNAs), which are translated into proteins, and non-messenger RNAs, which function at the RNA level and do not serve as templates for translation. RESULTS: We have generated a specialized cDNA library from Arabidopsis thaliana to investigate the population of small non-messenger RNAs (snmRNAs) sized 50-500 nt in a plant. From this library, we identified 140 candidates for novel snmRNAs and investigated their expression, abundance, and developmental regulation. Based on conserved sequence and structure motifs, 104 snmRNA species can be assigned to novel members of known classes of RNAs (designated Class I snmRNAs), namely, small nucleolar RNAs (snoRNAs), 7SL RNA, U snRNAs, as well as a tRNA-like RNA. For the first time, 39 novel members of H/ACA box snoRNAs could be identified in a plant species. Of the remaining 36 snmRNA candidates (designated Class II snmRNAs), no sequence or structure motifs were present that would enable an assignment to a known class of RNAs. These RNAs were classified based on their location on the A. thaliana genome. From these, 29 snmRNA species located to intergenic regions, 3 located to intronic sequences of protein coding genes, and 4 snmRNA candidates were derived from annotated open reading frames. Surprisingly, 15 of the Class II snmRNA candidates were shown to be tissue-specifically expressed, while 12 are encoded by the mitochondrial or chloroplast genome. CONCLUSIONS: Our study has identified 140 novel candidates for small non-messenger RNA species in the plant A. thaliana and thereby sets the stage for their functional analysis.

Arabidopsis↗