Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.

Acute leukemia with promyelocytic features in PML/RARα transgenic mice

Acute promyelocytic leukemia (APL) is associated with reciprocal chromosomal translocations involving the retinoic acid receptor α (RARα) locus on chromosome 17. In the majority of cases, RARα translocates and fuses with the promyelocytic leukemia (PML) gene located on chromosome 15. The resulting fusion genes encode the two structurally unique PML/RARα and RARα/PML fusion proteins as well as aberrant PML gene products, the respective pathogenetic roles of which have not been elucidated. We have generated transgenic mice in which the PML/RARα fusion protein is specifically expressed in the myeloid-promyelocytic lineage. During their first year of life, all the PML/RARα transgenic mice have an abnormal hematopoiesis that can best be described as a myeloproliferative disorder. Between 12 and 14 months of age, 10% of them develop a form of acute leukemia with a differentiation block at the promyelocytic stage that closely mimics human APL even in its response to retinoic acid. Our results are conclusive in vivo evidence that PML/RARα plays a crucial role in the pathogenesis of APL.

Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo

Both caspase-1- and caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. We assessed the contributions of these caspases to Fas signaling in hepatocyte cell death in vitro. Although wild-type, caspase-1−/−, and caspase-3−/− hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3−/− hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1−/− apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation were seen within 6 hr, but neither event was observed for caspase-3−/− hepatocytes. We extended these studies to thymocytes and found that apoptotic caspase-3−/− thymocytes exhibited similar “abnormal” morphological changes and delayed DNA fragmentation observed in hepatocytes. Furthermore, the cleavage of various caspase substrates implicated in mediating apoptotic events, including gelsolin, fodrin, laminB, and DFF45/ICAD, was delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.

Hormone-regulatable neoplastic transformation induced by a Jun-estrogen receptor chimera

The v-jun oncogene encodes a nuclear DNA binding protein that functions as a transcription factor and is part of the activator protein 1 complex. Oncogenic transformation by v-jun is thought to be mediated by the aberrant expression of specific target genes. To identify such Jun-regulated genes and to explore the mechanisms by which Jun affects their expression, we have fused the full-length v-Jun and an amino-terminally truncated form of v-Jun to the hormone-binding domain of the human estrogen receptor. The two chimeric proteins function as ligand-inducible transactivators. Expression of the fusion proteins in chicken embryo fibroblasts causes estrogen-dependent transformation.

Meis1 and pKnox1 bind DNA cooperatively with Pbx1 utilizing an interaction surface disrupted in oncoprotein E2a-Pbx1

E2a-Pbx1 is a chimeric transcription factor oncoprotein produced by the t(1;19) translocation in human pre-B cell leukemia. Class I Hox proteins bind DNA cooperatively with both Pbx proteins and oncoprotein E2a-Pbx1, suggesting that leukemogenesis by E2a-Pbx1 and Hox proteins may alter transcription of cellular genes regulated by Pbx–Hox motifs. Likewise, in murine myeloid leukemia, transcriptional coactivation of Meis1 with HoxA7/A9 suggests that Meis1–HoxA7/9 heterodimers may evoke aberrant gene transcription. Here, we demonstrate that both Meis1 and its relative, pKnox1, dimerize with Pbx1 on the same TGATTGAC motif selected by dimers of Pbx proteins and unidentified partner(s) in nuclear extracts, including those from t(1;19) pre-B cells. Outside their homeodomains, Meis1 and pKnox1 were highly conserved only in two motifs required for cooperativity with Pbx1. Like the unidentified endogenous partner(s), both Meis1 and pKnox1 failed to dimerize significantly with E2a-Pbx1. The Meis1/pKnox1-interaction domain in Pbx1 resided predominantly in a conserved N-terminal Pbx domain deleted in E2a-Pbx1. Thus, the leukemic potential of E2a-Pbx1 may require abrogation of its interaction with members of the Meis and pKnox families of transcription factors, permitting selective targeting of genes regulated by Pbx–Hox complexes. In addition, because most motifs bound by Pbx–Meis1/pKnox1 were not bound by Pbx1–Hox complexes, the leukemic potential of Meis1 in myeloid leukemias may involve shifting Pbx proteins from promoters containing Pbx–Hox motifs to those containing Pbx–Meis motifs.

Identification of semaphorin E as a non-MDR drug resistance gene of human cancers

To improve cancer chemotherapy, a better understanding of the molecular mechanisms of drug resistance is essential. To identify the molecules responsible for drug resistance that is unrelated to MDR1 or MRP gene products, a eukaryotic expression cDNA library of cis-diamminedichloroplatinum(II) (CDDP)-resistant ovarian cancer TYKnuR cells was introduced into Cos-7 cells. After repeated CDDP selection, cDNA homologous to murine semaphorin E was isolated from surviving cells. Human semaphorin E (H-sema E) was overexpressed in CDDP-resistant cell lines and was readily induced not only by diverse chemotherapeutic drugs but also by x-ray and UV irradiation. Transfection of H-sema E conferred a drug-resistant phenotype to CDDP-sensitive cells. In addition, the aberrant expression of H-sema E protein was detected immunohistochemically in 14 of 42 (33.3%) recurrent squamous cell carcinomas removed at autopsy after extensive radiochemotherapy. Recently, another member of the semaphorin family, CD100, was shown to significantly improve the viability of B lymphocytes. These results suggest the involvement of semaphorins in diverse cell survival mechanisms.

Liz1p, a Novel Fission Yeast Membrane Protein, Is Required for Normal Cell Division When Ribonucleotide Reductase Is Inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1+, which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1− cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1+ encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

Cytoplasmic Dynein Heavy Chain 1b Is Required for Flagellar Assembly in Chlamydomonas

A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. The Chlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1b gene have been deleted. The two mutants assemble short flagellar stumps (<1–2 μm) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas.

A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Mutational Effect of Fission Yeast Polα on Cell Cycle Events

Polα is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations in polα+. Germinating spores carrying either a deletion of polα+ (polαΔ) or a structurally intact but catalytically dead polα mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polα, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with a polαts allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polαts in cdc20 (polε mutant) background arrested with a cdc phenoytpe, not a polαts-like phenotype. At 25°C, replication perturbation caused by polαts alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36°C, replication disruption caused by polαts alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polα is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polαts mutations induce differential downstream kinase responses.

The Doa4 Deubiquitinating Enzyme Is Required for Ubiquitin Homeostasis in Yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Δ mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Δ cells. This loss of viability and several other defects of doa4Δ cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.

The Cdc6 Nucleotide–Binding Site Regulates Its Activity in DNA Replication in Human Cells

The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication. A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form. The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective. The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides. Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.

Rad18 Is Required for DNA Repair and Checkpoint Responses in Fission Yeast

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1 is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1 are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage.

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

BIM1 Encodes a Microtubule-binding Protein in Yeast

A previously uncharacterized yeast gene (YER016w) that we have named BIM1 (binding to microtubules) was obtained from a two-hybrid screen of a yeast cDNA library using as bait the entire coding sequence of TUB1 (encoding α-tubulin). Deletion of BIM1 results in a strong bilateral karyogamy defect, hypersensitivity to benomyl, and aberrant spindle behavior, all phenotypes associated with mutations affecting microtubules in yeast, and inviability at extreme temperatures (i.e., ≥37°C or ≤14°C). Overexpression of BIM1 in wild-type cells is lethal. A fusion of Bim1p with green fluorescent protein that complements the bim1Δ phenotypes allows visualization in vivo of both intranuclear spindles and extranuclear microtubules in otherwise wild-type cells. A bim1 deletion displays synthetic lethality with deletion alleles of bik1, num1, and bub3 as well as a limited subset of tub1 conditional-lethal alleles. A systematic study of 51 tub1 alleles suggests a correlation between specific failure to interact with Bim1p in the two-hybrid assay and synthetic lethality with the bim1Δ allele. The sequence of BIM1 shows substantial similarity to sequences from organisms across the evolutionary spectrum. One of the human homologues, EB1, has been reported previously as binding APC, itself a microtubule-binding protein and the product of a gene implicated in the etiology of human colon cancer.