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P Hieter

Publications and source records attributed to P Hieter.

At least 19 recordsLinked to original sources
PubMed2001

Novel role for a Saccharomyces cerevisiae nucleoporin, Nup170p, in chromosome segregation.

We determined that a mutation in the nucleoporin gene NUP170 leads to defects in chromosome transmission fidelity (ctf) and kinetochore integrity in Saccharomyces cerevisiae. A ctf mutant strain, termed s141, shows a transcription readthrough phenotype and stabilizes a dicentric chromosome fragment in two assays for kinetochore integrity. Previously, these assays led to the identification of two essential kinetochore components, Ctf13p and Ctf14p. Thus, s141 represents another ctf mutant involved in the maintenance of kinetochore integrity. We cloned and mapped the gene complementing the ctf mutation of s141 and showed that it is identical to the S. cerevisiae NUP170 gene. A deletion strain of NUP170 (nup170 Delta::HIS3) has a Ctf(-) phenotype similar to the s141 mutant (nup170-141) and also exhibits a kinetochore integrity defect. We identified a second nucleoporin, NUP157, a homologue of NUP170, as a suppressor of the Ctf(-) phenotype of nup170-141 and nup170 Delta::HIS3 strains. However, a deletion of NUP157 or several other nucleoporins did not affect chromosome segregation. Our data suggest that NUP170 encodes a specialized nucleoporin with a unique role in chromosome segregation and possibly kinetochore function.

Chromosome Segregation↗
PubMed2000

Pescadillo, a novel cell cycle regulatory protein abnormally expressed in malignant cells.

Using a culture model of glial tumorigenesis, we identified a novel gene that was up-regulated in malignant mouse astrocytes following the loss of p53. The gene represents the murine homologue of pescadillo, an uncharacterized gene that is essential for embryonic development in zebrafish. Pescadillo is a strongly conserved gene containing unique structural motifs such as a BRCA1 C-terminal domain, clusters of acidic amino acids and consensus motifs for post-translational modification by SUMO-1. Pescadillo displayed a distinct spatial and temporal pattern of gene expression during brain development, being detected in neural progenitor cells and postmitotic neurons. Although it is not expressed in differentiated astrocytes in vivo, the pescadillo protein is dramatically elevated in malignant human astrocytomas. Yeast strains harboring temperature-sensitive mutations in the pescadillo gene were arrested in either G(1) or G(2) when grown in nonpermissive conditions, demonstrating that pescadillo is an essential gene in yeast and is required for cell cycle progression. Consistent with the latter finding, DNA synthesis was only observed in mammalian cells expressing the pescadillo protein. These results suggest that pescadillo plays a crucial role in cell proliferation and may be necessary for oncogenic transformation and tumor progression.

Amino Acid Sequence↗
PubMed2000

A putative oncogenic role for MPP11 in head and neck squamous cell cancer.

Genetic alterations of chromosome 7 are common in human cancer. Furthermore, previous studies have supported the presence of a gene important in a broad range of cancers at 7q22-31.1. There is evidence that supports an oncogenic function for this putative gene, as well as evidence that supports a tumor suppressive role. In this study, we used a cross-species candidate gene approach in combination with physical mapping to identify MPP11 as a candidate for the putative cancer-related activity at 7q22-31.1. We then analyzed primary head and neck squamous cell tumors (HNSCCs) for loss of heterozygosity/allelic imbalance (LOH/AI) at the MPP11 genomic locus. Thirty-eight percent of tumors examined displayed LOH/AI involving the MPP11 genomic locus. Mutation analysis of MPP11 in the latter samples did not identify any inactivating mutations. However, immunohistochemical staining of primary tumor sections and Western blot analysis of HNSCC cell lines revealed a tumor-specific high level of expression of MPP11p. Fluorescence in situ hybridization analysis done on the cell lines identified increased chromosome 7 copy number with a concomitant increase in MPP11 copy number. These results suggest an oncogenic role for MPP11 in HNSCC.

Alleles↗
PubMed2000

XREFdb: cross-referencing the genetics and genes of mammals and model organisms.

XREFdb supports the investigation of protein function in the context of information available through work in multiple organisms. In addition to facilitating the association of functional data among known genes from multiple organisms, XREFdb has developed strategies that provide access to information associated with as-yet unstudied genes. The database organizes protein similarity and genetic map positional information from diverse sources in the public domain to facilitate investigator evaluation of potential functional significance. XREFdb is found at URL www.ncbi.nlm.nih.gov/XREFdb

Animals↗
PubMed2000

The APC11 RING-H2 finger mediates E2-dependent ubiquitination.

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

Amino Acid Sequence↗
PubMed1999

Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle.

A genetic synthetic dosage lethality (SDL) screen using CTF13 encoding a known kinetochore protein as the overexpressed reference gene identified two chromosome transmission fidelity (ctf) mutants, YCTF58 and YCTF26. These mutant strains carry independent alleles of a novel gene, which we have designated CTF19. In light of its potential role in kinetochore function, we have cloned and characterized the CTF19 gene in detail. CTF19 encodes a nonessential 369-amino acid protein. ctf19 mutant strains display a severe chromosome missegregation phenotype, are hypersensitive to benomyl, and accumulate at G2/M in cycling cells. CTF19 genetically interacts with kinetochore structural mutants and mitotic checkpoint mutants. In addition, ctf19 mutants show a defect in the ability of centromeres on minichromosomes to bind microtubules in an in vitro assay. In vivo cross-linking and chromatin immunoprecipitation demonstrates that Ctf19p specifically interacts with CEN DNA. Furthermore, Ctf19-HAp localizes to the nuclear face of the spindle pole body and genetically interacts with a spindle-associated protein. We propose that Ctf19p is part of a macromolecular kinetochore complex, which may function as a link between the kinetochore and the mitotic spindle.

Anaphase↗
PubMed1999

Detecting patterns of protein distribution and gene expression in silico.

Most biological information is contained within gene and genome sequences. However, current methods for analyzing these data are limited primarily to the prediction of coding regions and identification of sequence similarities. We have developed a computer algorithm, CoSMoS (for context sensitive motif searches), which adds context sensitivity to sequence motif searches. CoSMoS was challenged to identify genes encoding peroxisome-associated and oleate-induced genes in the yeast Saccharomyces cerevisiae. Specifically, we searched for genes capable of encoding proteins with a type 1 or type 2 peroxisomal targeting signal and for genes containing the oleate-response element, a cis-acting element common to fatty acid-regulated genes. CoSMoS successfully identified 7 of 8 known PTS-containing peroxisomal proteins and 13 of 14 known oleate-regulated genes. More importantly, CoSMoS identified an additional 18 candidate peroxisomal proteins and 300 candidate oleate-regulated genes. Preliminary localization studies suggest that these include at least 10 previously unknown peroxisomal proteins. Phenotypic studies of selected gene disruption mutants suggests that several of these new peroxisomal proteins play roles in growth on fatty acids, one is involved in peroxisome biogenesis and at least two are required for synthesis of lysine, a heretofore unrecognized role for peroxisomes. These results expand our understanding of peroxisome content and function, demonstrate the utility of CoSMoS for context-sensitive motif scanning, and point to the benefits of improved in silico genome analysis.

Gene Expression Regulation, Fungal↗
PubMed1999

Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery.

CTF7 (chromosome transmission fidelity) gene in budding yeast encodes an essential protein that is required for high-fidelity chromosome transmission and contains regions of identity conserved from yeast to man. ctf7 mutant cells arrested prior to anaphase onset contain separated sister chromatids. Thus, Ctf7p is essential for cohesion. Cohesion is established during S phase and then maintained until mitosis. However, Ctf7p activity is required only during S phase, suggesting that Ctf7p functions in the establishment of cohesion. In addition, ctf7 genetically interacts with DNA metabolism mutations pol30 (PCNA) and ctf18 (an RF-C like protein) and ctf7 temperature sensitivity and chromosome loss are rescued by high levels of POL30. These findings provide the first evidence that links the establishment of sister chromatid cohesion to the DNA replication machinery and suggest that the assembly of cohesion (and possibly condensation) complexes are coupled to PCNA-dependent DNA replication. The analysis of Ctf7p also reveals an important connection between sister chromatid cohesion, spindle integrity and the spindle assembly checkpoint.

Acetyltransferases↗
PubMed1999

High-resolution comparative physical mapping of mouse chromosome 10 in the region of homology with human chromosome 21.

Comparative mapping of human and mouse chromosomes can be used to predict locations of homologous loci between the species, provides the substrate to examine the process of chromosomal evolution, and facilitates the continuing development of mouse genetic models for human disorders. A YAC contig of the region of mouse Chromosome (Chr) 10 (MMU10) that demonstrates conserved linkage with the distal portion of human Chr 21 (HSA21) has been constructed. The contig contains all known genes mapped in both species, defines the proximal region of homology between MMU10 and HSA22, and contains the evolutionary junction between HSA21 and HSA22 on MMU10. It consists of 23 YACs and 2 PACs, and covers 3.2 Mb of MMU10. The average marker density for this region is 1 marker/69 kb. Nine of 22 expressed sequences are mapped here for the first time in mouse, and two are newly characterized expressed sequences. The contig also contains 12 simple sequence repeats (SSRs) and 16 YAC and PAC endclone markers. YAC fragmentation analysis was used to create a physical map for the proximal 2.2 Mb of the contig. Cloning of the corresponding region of HSA21 has proven difficult, and the mouse contig includes segments absent from previously described sequence ready maps of HSA21.

Animals↗
PubMed1999

SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex.

We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function. Sgt1p physically associates with Skp1p in vivo and in vitro. SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content. Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function. Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p. Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase. sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro. Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution.

Amino Acid Sequence↗
PubMed1999

NORF5/HUG1 is a component of the MEC1-mediated checkpoint response to DNA damage and replication arrest in Saccharomyces cerevisiae.

Analysis of global gene expression in Saccharomyces cerevisiae by the serial analysis of gene expression technique has permitted the identification of at least 302 previously unidentified transcripts from nonannotated open reading frames (NORFs). Transcription of one of these, NORF5/HUG1 (hydroxyurea and UV and gamma radiation induced), is induced by DNA damage, and this induction requires MEC1, a homolog of the ataxia telangiectasia mutated (ATM) gene. DNA damage-specific induction of HUG1, which is independent of the cell cycle stage, is due to the alleviation of repression by the Crt1p-Ssn6p-Tup1p complex. Overexpression of HUG1 is lethal in combination with a mec1 mutation in the presence of DNA damage or replication arrest, whereas a deletion of HUG1 rescues the lethality due to a mec1 null allele. HUG1 is the first example of a NORF with important biological functional properties and defines a novel component of the MEC1 checkpoint pathway.

Cell Cycle↗
PubMed1999

Increased instability of human CTG repeat tracts on yeast artificial chromosomes during gametogenesis.

Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable. The instability is 6 to 10 times more pronounced in meiosis than during mitotic division. The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present. Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats. The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions. In our experiments, expansions were found almost exclusively during gametogenesis. Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability. These unique patterns have novel implications for possible mechanisms of repeat instability.

Chromosomes, Artificial, Yeast↗
PubMed1999

The anaphase-promoting complex: new subunits and regulators.

Ubiquitin-mediated proteolysis of cell cycle regulators is a crucial process during the cell cycle. The anaphase-promoting complex (APC) is a large, multiprotein complex whose E3-ubiquitin ligase activity is required for the ubiquitination of mitotic cyclins and other regulatory proteins that are targeted for destruction during cell division. The recent identification of new APC subunits and regulatory proteins has begun to reveal some of the intricate mechanisms that govern APC regulation. One mechanism is the use of specificity factors to impose temporal control over substrate degradation. A second mechanism is the APC-mediated proteolysis of specific APC regulators. Finally, components of both the APC and the SCF E3 ubiquitin-ligase complex contain several conserved sequence motifs, including WD-40 repeats and cullin homology domains, which suggest that both complexes may use a similar mechanism for substrate ubiquitination.

Anaphase↗
PubMed1998

Identification of a cullin homology region in a subunit of the anaphase-promoting complex.

The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8). The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned. APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27. Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G1 phase cyclins and cyclin-dependent kinase inhibitors. The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p. APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation.

Amino Acid Sequence↗