A genetic screen for modifiers of drosophila Src42A identifies mutations in Egfr, rolled and a novel signaling gene

Drosophila Src42A, a close relative of the vertebrate c-Src, has been implicated in the Ras-Mapk signaling cascade. An allele of Src42A, Su(Raf)1, dominantly suppresses the lethality of partial loss-of-function Raf mutations. To isolate genes involved in the same pathway where Src42A functions, we carried out genetic screens for dominant suppressor mutations that prevented Su(Raf)1 from suppressing Raf. Thirty-six mutations representing at least five genetic loci were recovered from the second chromosome. These are Drosophila EGF Receptor (Egfr), rolled, Src42A, and two other new loci, one of which was named semang (sag). During embryogenesis, sag affects the development of the head, tail, and tracheal branches, suggesting that it participates in the pathways of Torso and DFGF-R1 receptor tyrosine kinases. sag also disrupts the embryonic peripheral nervous system. During the development of imaginal discs, sag affects two processes known to require Egfr signaling: the recruitment of photoreceptor cells and wing vein formation. Thus sag functions in several receptor tyrosine kinase (RTK)-mediated processes. In addition, sag dominantly enhances the phenotypes associated with loss-of-function Raf and rl, but suppresses those of activated Ras1(V12) mutation. This work provides the first genetic evidence that both Src42A and sag are modulators of RTK signaling.     

A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene

We employed the constitutive BCK1-20 allele of the gene for the MAP kinase kinase kinase (MAP-KKK) in the yeast Pkc signal transduction pathway to develop a genetic screen for mutants in genes encoding upstream components. Transposon mutagenesis yielded a mutant that was completely dependent on the active allele in the absence of osmotic stabilization. The transposon had integrated at the yeast SLG1 (HCS77) locus. This gene encodes a putative membrane protein. Haploid slg1 deletion strains are sensitive to caffeine, as expected for mutants in the Pkc pathway, as well as a variety of other drugs. The response to elevated temperatures and the dependence on osmotic stabilization depends on the genetic background. Thus, in the strain used for mutagenesis, disruption of SLG1 causes the cells to become non-viable in the absence of osmotic stabilization at both 30 degrees C and 37 degrees C. In a different genetic background this phenotype was not observed. Sensitivity of the haploid deletion mutants to caffeine can be partially suppressed by overexpression of genes for other components of the Pkc pathway, such as PKC1, SLT2, ROM2, and STE20. In addition, a SLG1-lacZ reporter construct shows higher expression in the presence of caffeine or magnesium chloride in a wild-type diploid background.     

Fission yeast wee1 protein kinase is not required for DNA damage-dependent mitotic arrest

Checkpoints maintain the dependency relationships between discrete events in the cell cycle (for example, ensuring mitosis does not occur before DNA replication is complete). In Schizosaccharomyces pombe, mitotic checkpoints monitor DNA synthesis and the presence of DNA damage. The replication-dependent mitotic checkpoint prevents mitosis by inactivating p34cdc2 kinase. The mechanism by which the DNA damage checkpoint interacts with the mitotic machinery is distinct from that used by the replication checkpoint. The activity of p34cdc2 is controlled, in part, by the wee1 protein kinase, which inactivates cdc2 through phosphorylation at tyrosine-15 (ref. 7). Here we report normal mitotic arrest after DNA damage in S. pombe cells in which the wee1 gene is defective or missing. We suggest why these findings contradict a recent report which suggested that the wee1 gene product was required for DNA damage-dependent mitotic arrest.     

A novel esterase from Saccharomyces carlsbergensis, a possible function for the yeast TIP1 gene

An extracellular esterase was isolated from the brewer's yeast, Saccharomyces carlsbergensis. Inhibition by diisopropyl fluorophosphate shows that the enzyme has a serine active site. By mass spectrometry, the molecular weight of the enzyme was 16.9 kDa. The optimal pH for activity was in the range of four to five. Esterase activity was found in beer before pasteurization, and a low level of activity was still present after pasteurization. Caprylic acid, which is present in beer, competitively inhibited the esterase. The substrate preference towards esters of p-nitrophenol indicated that the enzyme prefers esters of fatty acids from four to 16 carbon atoms. The esterase has lipolytical activity; olive oil (C-18:1), which is a classical substrate for lipase, was hydrolysed. N-terminal sequence analysis of the esterase yielded a sequence which was identical to the deduced amino acid sequence of the S. cerevisiae TIP1 gene. The esterase preparation did not appear to contain significant amounts of other proteins than Tip1p, indicating that the TIP1 gene is the structural gene for the esterase.     

Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function

Recent evidence suggests that the cost as well as the morbidity associated with the maintenance of hemodialysis access is increasing rapidly; currently, the cost exceeds 1 billion dollars and access related hospitalization accounts for 25% of all hospital admissions in the U.S.A. This increase in cost and morbidity has been associated with several epidemiological trends that may contribute to access failure. These include late patient referral to nephrologists and surgeons, late planning of vascular access as well as a shift from A-V fistulaes to PTFE grafts and temporary catheters, which have a higher failure rate. The reasons for this shift in the types of access is multifactorial and is not explained by changes in the co-morbidities of patients presenting to dialysis. Surgical preference and training also appear to play an important role in the large regional variation and patency rate of these PTFE grafts. We propose a program for early placement of A-V fistulae, a continuous quality improvement, multidisciplinary program to monitor access outcome, the development of new biomaterials, and a research plan to investigate pharmacological intervention to reduce development of stenosis and clinical interventions to treat those that do develop, prior to thrombosis.     

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 degreesC growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36 degreesC on either carbon source. Microscopic observation of cells growing on glucose at 24 degreesC 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 degrees] 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.     

A genetic screen for aminophospholipid transport mutants identifies the phosphatidylinositol 4-kinase, STT4p, as an essential component in phosphatidylserine metabolism

In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae. A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed. We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity. The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport. A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase. The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant. Creation of an STT4 null allele (stt4Delta::HIS3) demonstrates the gene is essential. These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.     

Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 enzymes was severely diminished. Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased Km value for acetyl-CoA, suggesting that the Phe142 and Tyr143 residues are essential for the catalysis.     

A novel fission yeast gene, kms1+, is required for the formation of meiotic prophase-specific nuclear architecture

From archival records, we assessed outcomes achieved by preschoolers with both severe mental retardation and autistic features: (a) an experimental group (n = 11), which received intensive behavioral treatment, and (b) a comparison group (n = 10), which received minimal treatment. At intake (mean CA = 3.08 years), the groups did not differ significantly on any variable. At follow-up children in the experimental group obtained a higher mean IQ and evinced more expressive speech than did those in the comparison group. Behavior problems diminished in both groups. Results indicate that intensively treated children achieved clinically meaningful gains relative to the comparison group but remained quite delayed.     

CMAP: a novel cystatin-like gene involved in liver metastasis

The distribution and ultrastructure of class II major histocompatibility complex (MHC)-positive cells were investigated in human dental pulp, employing immunohistochemistry using an anti-human leukocyte antigen (HLA)-DR-monoclonal antibody. HLA-DR-immunopositive cells, appearing spindle-like or dendritic in profile, were densely distributed throughout the dental pulp. Under the electron microscope, these cells exhibited various sizes of vesicles containing clear or opaque contents, multivesicular bodies and characteristic fine tubulovesicular structures in their cytoplasm. Some reactive cells possessed coated pits and vesicles including electron-dense materials, indicating an active endocytosis. At the periphery of the pulp tissue, the HLA-DR-immunopositive cells were predominantly situated in the subodontoblastic layer, with some located in the odontoblast layer and/or predentin and extending their cytoplasmic processes into the dentinal tubules. Cell processes of these cells occasionally made contact with several odontoblast processes in the same way as the nerve fibers in the predentin. These cells never contained the typical phagosomes frequently observed in the HLA-DR-immunoreactive macrophages in the subodontoblastic layer and the pulp core. The results suggest that the HLA-DR-immunopositive cells in the odontoblast layer and/or predentin have some regulatory function on the odontoblasts under physiological conditions, in addition to their involvement in the initial defense reaction after tooth injury.     

A novel candidate oncogene, MCT-1, is involved in cell cycle progression

Using the arbitrarily primed-PCR (AP-PCR) assay to detect genetic abnormalities that occur in a panel of lymphoid cell lines, we identified an amplified stretch of genomic DNA that contained a putative open reading frame. Northern blot analysis with this genomic clone revealed widespread low level expression in normal human tissue. The full cDNA sequence was obtained with no significant homology to any known genes in the genome database. We termed this novel gene with multiple copies in a T-cell malignancy as MCT-1. MCT-1 was localized to the long arm of chromosome Xq22-24 by flourescence in situ hybridization analysis. Although there was no significant homology at the primary sequence level, there was a limited degree of amino acid homology with a domain of cyclin H that appears to specify protein-protein complexes. This relationship between MCT-1 and cyclin H implied a potential role for MCT-1 in cell cycle regulation. Overexpression of MCT-1 increased the proliferative rate of cells by decreasing the length of the G1 phase without a reciprocal increase in the S and G2-M phases. Recent work has established the role of cell cycle regulatory molecules in the development of certain human malignancies. Therefore, we investigated the transforming ability of MCT-1 overexpression using soft agar growth assays and demonstrated that only MCT-1-overexpressing cells were able to establish colonies. Taken together, MCT-1 is a novel candidate oncogene with homology to a protein-protein binding domain of cyclin H.     

Std1, a gene involved in glucose transport in Schizosaccharomyces pombe

A wild-type strain, Sp972 h-, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [14C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S. pombe, unlike in Saccharomyces cerevisiae. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617-2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.     

Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase

Mutations in the COX17 gene of Saccharomyces cerevisiae cause a respiratory deficiency due to a block in the production of a functional cytochrome oxidase complex. Because cox17 mutants are able to express both the mitochondrially and nuclearly encoded subunits of cytochrome oxidase, the Cox17p most likely affects some late posttranslational step of the assembly pathway. A fragment of yeast nuclear DNA capable of complementing the mutation has been cloned by transformation of the cox17 mutant with a library of genomic DNA. Subcloning and sequencing of the COX17 gene revealed that it codes for a cysteine-rich protein with a molecular weight of 8,057. Unlike other previously described accessory factors involved in cytochrome oxidase assembly, all of which are components of mitochondria, Cox17p is a cytoplasmic protein. The cytoplasmic location of Cox17p suggested that it might have a function in delivery of a prosthetic group to the holoenzyme. A requirement of Cox17p in providing the copper prosthetic group of cytochrome oxidase is supported by the finding that a cox17 null mutant is rescued by the addition of copper to the growth medium. Evidence is presented indicating that Cox17p is not involved in general copper metabolism in yeast but rather has a more specific function in the delivery of copper to mitochondria.