Genetic Screen For Mutants Research Articles

Role of thioredoxin reductase in the Yap1p-dependent response to oxidative stress in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Yap1p transcription factor is required for the H2O2-dependent activation of many antioxidant genes including the TRX2 gene encoding thioredoxin 2. To identify factors that regulate Yap1p activity, we carried out a genetic screen for mutants that show elevated expression of a TRX2-HIS3 fusion in the absence of H2O2. Two independent mutants isolated in this screen carried mutations in the TRR1 gene encoding thioredoxin reductase. Northern blot and whole-genome expression analysis revealed that the basal expression of most Yap1p targets and many other H2O2-inducible genes is elevated in Deltatrr1 mutants in the absence of external stress. In Deltatrr1 mutants treated with H2O2, the Yap1p targets, as well as genes comprising a general environmental stress response and genes encoding protein-folding chaperones, are hyperinduced. However, despite the elevated expression of genes encoding antioxidant enzymes, Deltatrr1 mutants are extremely sensitive to H2O2. The results suggest that cells lacking thioredoxin reductase have diminished capacity to detoxify oxidants and/or to repair oxidative stress-induced damage and that the thioredoxin system is involved in the redox regulation of Yap1p transcriptional activity.

New ways for old genes

We already know from classical physiological studies that plant signals interact with each other to generate downstream developmental responses. Arabidopsis genetics is now about to identify the molecular links between signaling pathways in plants. Recently, five groups have identified mutations in genes that had been previously implicated in other pathways, revealing interactions between abscisic acid (ABA) and ethylene signal transduction as well as between ABA and sugar signaling.Recently, Majid Ghassemian et al. 1xRegulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis. Ghassemian, M. et al. Plant Cell. 2000; 12: 1117–1126PubMedSee all References1 and Nathalie Beaudoin et al. 2xInteractions between abscisic acid and ethylene signaling cascades. Beaudoin, N. et al. Plant Cell. 2000; 12: 1103–1115PubMedSee all References2 reported that they recovered alleles of ein2 in genetic screens for mutants with enhanced responses to ABA at seed germination. The EIN2 locus had previously been shown to determine sensitivity to another plant hormone, ethylene. In parallel, Francisco Arenas-Huertero et al. 3xAnalysis of Arabidopsis glucose insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar. Arenas-Huertero, F. et al. Genes Dev. 2000; 14: 2085–2096PubMedSee all References3, Ron Laby et al. 4xThe Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response. Laby, R.J. et al. Plant J. 2000; 23: 587–596Crossref | PubMedSee all References4 and Casper Huijser et al. 5xThe Arabidopsis SUCROSE UNCOUPLED-6 gene is identical to ABSCISIC ACID INSENSITIVE-4: involvement of abscisic acid in sugar responses. Huijser, C. et al. Plant J. 2000; 23: 577–585Crossref | PubMedSee all References5 reported that they isolated alleles of abi4 in mutant screens for sugar insensitivity during seedling development. A mutation in ABI4 was originally shown to confer resistance to ABA at seed germination. Altogether, these findings show that ABA and ethylene, along with sugar and ABA transduction pathways, share common genetic determinants. However, the connections between these two pairs of signaling cascades are not restricted to one genetically defined step in the cascade. Mutations in other elements of the ethylene transduction pathway, such as ctr1, which confers constitutive response to ethylene, or etr1, which is a member of the ethylene receptor family, also modulate seed sensitivity to ABA during germination. In fact, Beaudoin et al. also recovered ctr1 in a screen for decreased sensitivity to ABA. This suggests that the ethylene transduction pathway impinges on the ABA signaling cascade to regulate seed germination.In the case of the interplay between sugar sensing and ABA signaling, ABI4 plays a prominent role whereas mutations in other ABI genes have little or no influence on the sugar sensitivity of seedling development. However, mutants impaired in ABA biosynthesis are also insensitive to sugar, suggesting that ABA production is required for sugar sensing. These interesting findings point to an extensive cross talk between ABA and another stress signal molecule, ethylene, and between ABA and the sugar nutrient-sensing pathways.However, these are probably just flashes of light on parts of the transduction networks that allow plants to integrate diverse signals such as environmental signals, nutrient status and developmental signals to produce adaptive responses. Further studies show that ethylene and ABA signaling pathways also interact to regulate root development but, in this organ, mutations that confer ethylene insensitivity also decrease ABA-mediated inhibition of root growth1,2. Hopefully, new tools such as DNA microarrays, which enable us to monitor changes in gene expression triggered by a signal or a mutation on a genome wide scale, should help us to uncover the molecular basis for signal cross talk in plants in more detail.

Protruding vulva mutants identify novel loci and Wnt signaling factors that function during Caenorhabditis elegans vulva development.

The Caenorhabditis elegans vulva develops from the progeny of three vulval precursor cells (VPCs) induced to divide and differentiate by a signal from the somatic gonad. Evolutionarily conserved Ras and Notch extracellular signaling pathways are known to function during this process. To identify novel loci acting in vulval development, we carried out a genetic screen for mutants having a protruding-vulva (Pvl) mutant phenotype. Here we report the initial genetic characterization of several novel loci: bar-1, pvl-4, pvl-5, and pvl-6. In addition, on the basis of their Pvl phenotypes, we show that the previously identified genes lin-26, mom-3/mig-14, egl-18, and sem-4 also function during vulval development. Our characterization indicates that (1) pvl-4 and pvl-5 are required for generation/survival of the VPCs; (2) bar-1, mom-3/mig-14, egl-18, and sem-4 play a role in VPC fate specification; (3) lin-26 is required for proper VPC fate execution; and (4) pvl-6 acts during vulval morphogenesis. In addition, two of these genes, bar-1 and mom-3/mig-14, are known to function in processes regulated by Wnt signaling, suggesting that a Wnt signaling pathway is acting during vulval development.

Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology.

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.

Coordinating Mitosis and Morphogenesis

Two groups have identified the gene for tribbles in Drosophila , whose product acts to inhibit the activity of the Drosophila homolog of the CDC25 phosphatase, String. String activity allows the phase transition from G2 to M in the cell cycle. Großhans and Wieschaus identified tribbles in a genetic screen for mutants in which the embryonic cells in the ventral region have accelerated entry into mitosis and, thus, do not properly form the ventral furrow. Mata et al . identified tribbles in a genetic screen for mutants that have increased number of cells (cystocytes) in the ovary. Mata et al . demonstrated that Tribbles promotes the proteasome-mediated degradation of String, thereby delaying mitosis. Both groups show that overexpression of tribble inhibits mitosis. Tribbles shows homology to serine-threonine kinases; however, conserved residues in the catalytic domain and ATP-binding domain are missing. Furthermore, Großhans and Wieschaus mutated a critical lysine in the predicted catalytic center of Tribbles and found this mutant of Tribbles was able to delay the cell cycle when injected into embryos, suggesting that Tribbles is not functioning as a kinase. During embryogenesis, it is essential for cells to coordinate between mitotic programs and morphogenic programs. Tribbles is one component in the pathway controlling the switch between these two programs. Großhans, J., and Wieschaus, E. (2000) A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila . Cell 101 : 523-531. [Online Journal] Mata, J., Curado, S., Ephrussi, A., and Rørth, P. (2000) Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating String/CDC25 proteolysis. Cell 101 : 511-522. [Online Journal]

Glc7p protein phosphatase inhibits expression of glutamine-fructose-6-phosphate transaminase from GFA1.

Inhibitor-1 (I-1) is a specific inhibitor of protein phosphatase-1 (PP1). We assayed the ability of I-1 to inhibit Saccharomyces cerevisiae PP1, Glc7p, in vivo. Glc7p like other PP1 catalytic subunits associates with a variety of noncatalytic subunits, and Glc7p holoenzymes perform distinct physiological roles. Our results show that I-1 inhibits Glc7p holoenzymes that regulate transcription and mitosis, but holoenzymes responsible for meiosis and glycogen metabolism were unaffected. Additionally, we exploited a genetic screen for mutants that were dependent on I-1 to grow. This scheme can identify processes that are negatively regulated by Glc7p-catalyzed dephosphorylation. In this paper I-1-dependent gfa1 mutations were analyzed in detail. GFA1 encodes glutamine-fructose-6-phosphate transaminase. One or more phosphorylated proteins activate GFA1 transcription because the pheromone response and Pkc1p/mitogen-activated protein kinase pathways positively regulate GFA1 transcription. Our findings show that an I-1-sensitive Glc7p holoenzyme reduces GFA1 transcription. Therefore, GFA1 is a member of a growing list of genes that are negatively regulated by Glc7p dephosphorylation.

A Cytoskeletal Localizing Domain in the Cyclase-associated Protein, CAP/Srv2p, Regulates Access to a Distant SH3-binding Site

In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2). CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase. CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2). In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling.

The Caenorhabditis elegans gene ncc-1 encodes a cdc2-related kinase required for M phase in meiotic and mitotic cell divisions, but not for S phase.

We have identified six protein kinases that belong to the family of cdc2-related kinases in Caenorhabditis elegans. Results from RNA interference experiments indicate that at least one of these kinases is required for cell-cycle progression during meiosis and mitosis. This kinase, encoded by the ncc-1 gene, is closely related to human Cdk1/Cdc2, Cdk2 and Cdk3 and yeast CDC28/cdc2(+). We addressed whether ncc-1 acts to promote passage through a single transition or multiple transitions in the cell cycle, analogous to Cdks in vertebrates or yeasts, respectively. We isolated five recessive ncc-1 mutations in a genetic screen for mutants that resemble larval arrested ncc-1(RNAi) animals. Our results indicate that maternal ncc-1 product is sufficient for embryogenesis, and that zygotic expression is required for cell divisions during larval development. Cells that form the postembryonic lineages in wild-type animals do not enter mitosis in ncc-1 mutants, as indicated by lack of chromosome condensation and nuclear envelope breakdown. However, progression through G1 and S phase appears unaffected, as revealed by expression of ribonucleotide reductase, incorporation of BrdU and DNA quantitation. Our results indicate that C. elegans uses multiple Cdks to regulate cell-cycle transitions and that ncc-1 is the C. elegans ortholog of Cdk1/Cdc2 in other metazoans, required for M phase in meiotic as well as mitotic cell cycles.

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.

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes.

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'-->3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

Pop3p is essential for the activity of the RNase MRP and RNase P ribonucleoproteins in vivo.

RNase MRP is a ribonucleoprotein (RNP) particle which is involved in the processing of pre-rRNA at site A3 in internal transcribed spacer 1. Although RNase MRP has been analysed functionally, the structure and composition of the particle are not well characterized. A genetic screen for mutants which are synthetically lethal (sl) with a temperature-sensitive (ts) mutation in the RNA component of RNase MRP (rrp2-1) identified an essential gene, POP3, which encodes a basic protein of 22.6 kDa predicted molecular weight. Over-expression of Pop3p fully suppresses the ts growth phenotype of the rrp2-1 allele at 34 degrees C and gives partial suppression at 37 degrees C. Depletion of Pop3p in vivo results in a phenotype characteristic of the loss of RNase MRP activity; A3 cleavage is inhibited, leading to under-accumulation of the short form of the 5.8S rRNA (5.8S(S)) and formation of an aberrant 5.8S rRNA precursor which is 5'-extended to site A2. Pop3p depletion also inhibits pre-tRNA processing; tRNA primary transcripts accumulate, as well as spliced but 5'- and 3'-unprocessed pre-tRNAs. The Pop3p depletion phenotype resembles those previously described for mutations in components of RNase MRP and RNase P (rrp2-1, rpr1-1 and pop1-1). Immunoprecipitation of epitope-tagged Pop3p co-precipitates the RNA components of both RNase MRP and RNase P. Pop3p is, therefore, a common component of both RNPs and is required for their enzymatic functions in vivo. The ubiquitous RNase P RNP, which has a single protein component in Bacteria and Archaea, requires at least two protein subunits for its function in eukaryotic cells.

1. Phototransduction and Circadian Clock Pathways Regulating Gene Transcription in Higher Plants

Publisher Summary Plants serve as one of the best model systems for the investigation of the integration of the phototransduction and circadian clock pathways regulating gene transcription. The use of combined biochemical, molecular, and genetic approaches provide information on each of the three components of the circadian system involved in the regulation of plant gene transcription. The three basic components are: (1) input signal transduction pathways from the environmental cues (primarily light-dark signals), which entrain the activity of the circadian oscillator and determine the phase of the free-running rhythm; (2) a circadian oscillator that generates the rhythm; and (3) output signal transduction pathways for the temporal regulation of specific biological processes. This chapter discusses the identity and function of plant photoreceptors, the input signal transduction pathways from the photoreceptors, the analysis of the circadian oscillator by genetic screens for mutants with altered regulation of CAB expression, and the characterization of the output signal transduction pathways starting from the identification of circadian clock- and light-responsive cis-acting CAB promoter elements. Expression of the plant CAB gene family, encoding the chlorophyll a and b binding proteins of the light-harvesting complex of photosystem II, is one of the most extensively investigated plant gene families whose expression is circadian clock-regulated at the level of transcription. Analysis of CAB gene expression has revealed that a network of regulatory pathways controls CAB transcription. The potential for the combinatorial interaction of a multiplicity of inputs to CAB regulation provides a means of generating complex expression patterns.

Yeast pip3/mec3 Mutants Fail To Delay Entry into S Phase and To Slow DNA Replication in Response to DNA Damage, and They Define a Functional Link between Mec3 and DNA Primase

The catalytic DNA primase subunit of the DNA polymerase alpha-primase complex is encoded by the essential PRI1 gene in Saccharomyces cerevisiae. To identify factors that functionally interact with yeast DNA primase in living cells, we developed a genetic screen for mutants that are lethal at the permissive temperature in a cold-sensitive pril-2 genetic background. Twenty-four recessive mutations belonging to seven complementation groups were identified. Some mutants showed additional phenotypes, such as increased sensitivity to UV irradiation, methyl methanesulfonate, and hydroxyurea, that were suggestive of defects in DNA repair and/or checkpoint mechanisms. We have cloned and characterized the gene of one complementation group, PIP3, whose product is necessary both for delaying entry into S phase or mitosis when cells are UV irradiated in G1 or G2 phase and for lowering the rate of ongoing DNA synthesis in the presence of methyl methanesulfonate. PIP3 turned out to be the MEC3 gene, previously identified as a component of the G2 DNA damage checkpoint. The finding that Mec3 is also required for the G1- and S-phase DNA damage checkpoints, together with the analysis of genetic interactions between a mec3 null allele and several conditional DNA replication mutations at the permissive temperature, suggests that Mec3 could be part of a mechanism coupling DNA replication with repair of DNA damage, and DNA primase might be involved in this process.

Isolation and analysis of the yeast TEA1 gene, which encodes a zinc cluster Ty enhancer-binding protein.

A genetic screen for mutants that affect the activity of internal regulatory sequences of Ty retrotransposons led to the identification of a new gene encoding a DNA-binding protein that interacts with the downstream enhancer-like region of Ty1 elements. The TEA1 (Ty enhancer activator) gene sequence predicts a protein of 86.9 kDa whose N terminus contains a zinc cluster and dimerization motif typical of the Gal4-type family of DNA-binding proteins. The C terminus encodes an acidic domain with a net negative charge of -10 and the ability to mediate transcriptional activation. Like other zinc cluster proteins, purified Tea1 was found to bind to a partially palindromic CGGNxCCG repeat motif located in the Ty1 enhancer region. The Ty1 Tea1 binding site has a spacing of 10 and is located near binding sites for the DNA-binding proteins Rap1 and Mcm1. Analysis of the phenotype of tea1 deletion mutants confirmed that the TEA1 gene is required for activation from the internal Ty1 enhancer characterized in this study and makes a modest contribution to normal Ty1 levels in the cell. Hence, Tea1, like Rap1, is a member of a small family of downstream activators in Saccharomyces cerevisiae. Further analysis of the Tea1 protein and its interactions may provide insight into the mechanism of downstream activation in yeast cells.

Suppression of Mutations in Two Saccharomyces cerevisiae Genes by the Adenovirus E1A Protein

The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.

Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation.

A genetic screen for mutants of Arabidopsis that are hypersensitive to UV light was developed and used to isolate a new mutant designated uvh1. UV hypersensitivity in uvh1 was due to a single recessive trait that is probably located on chromosome 3. Although isolated as hypersensitive to an acute exposure to UV-C light, uvh1 was also hypersensitive to UV-B wavelengths, which are present in sunlight that reaches the earth's surface. UV-B damage to both wild-type and uvh1 plants could be significantly reduced by subsequent exposure of UV-irradiated plants to photoreactivating light, showing that photoreactivation of UV-B damage is important for plant viability and that uvh1 plants are not defective in photoreactivation. A new assay for DNA damage, the Dral assay, was developed and used to show that exposure of wild-type and uvh1 plants to a given dose of UV light induces the same amount of damage in chloroplast and nuclear DNA. Thus, uvh1 is not defective in a UV protective mechanism. uvh1 plants were also found to be hypersensitive to ionizing radiation. These results suggest that uvh1 is defective in a repair or tolerance mechanism that normally provides plants with resistance to several types of DNA damage.

Sequence of the PAS8 gene, the product of which is essential for biogenesis of peroxisomes in Saccharomyces cerevisiae

In a genetic screen for mutants disturbed in peroxisomal functions we found that the laboratory ‘wild type’ strain YP102 behaved like a typical peroxisome assembly mutant. Here, we report the sequence of the complementing gene ( PAS8), coding for a protein of 1030 amino acids that appears to be a novel member of the AAA-protein family which also includes NSFp and PAS1p.

A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1.

NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth. To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1. Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups. The genes of two complementation groups, NSP116 and NSP49, were cloned. Like the previously described nucleoporins, these genes encode proteins with many repeat sequences. NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins. NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope. These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.

Nucleotide sequence and characterization of temperature-sensitive pol1 mutants of Saccharomyces cerevisiae

We have analyzed the effects of temeperature-sensitivity (ts)-conferring mutations in the Saccharomyces cerevisiae DNA polymerase I-encoding gene on cell growth, in vivo DNA synthesis, intrachromosomal gene conversion and pop-out recombination. Also, we have identified the molecular defect responsible for the ts phenotype. Two mutant alleles ( cdc17-1, cdc17-2) were originally identified as cell-cycle mutations, while a third mutation ( hpr3) was found during a genetic screening for mutants with a hyper-recombination phenotype. Both cdc17-2 and hpr3 cells complete one round of cell division and DNA replication after shift to nonpermissive temperature, before being arrestedas a dumbbell-shaped cells. Conversely, the cdc17-1 mutation immediately blocks growth and DNA synthesis at 37°C. No substantial difference was observed in the frequency of intrachromosomal gene conversion and pop-out recombination events, when hpr3 and cdc17-1 were compared to the previously characterized pol1-1 mutant. These two frequencies were ten- to 30-fold above wild-type level at semipermissive temperature. In each mutant, a single bp substitution, causing the replacement of Gly residues by either Asp ( cdc17-1, cdc17-2) or Glu ( hpr3) in yeast DNA polymerase I is responsible for the ts phenotype.

THE MEDICAL TREATMENT OF SCHOOL CHILDREN

The MAPKKK dual leucine zipper-containing kinase (DLK, Wallenda in <i>Drosophila</i>) is an evolutionarily conserved component of the axonal injury response pathway. After nerve injury, DLK promotes degeneration of distal axons and regeneration of proximal axons. This dual role in coordinating degeneration and regeneration suggests that DLK may be a sensor of axon injury, and so understanding how DLK is activated is important. Two mechanisms are known to activate DLK. First, increasing the levels of DLK via overexpression or loss of the PHR ubiquitin ligases that target DLK activate DLK signaling. Second, in <i>Caenorhabditis elegans</i>, a calcium-dependent mechanism, can activate DLK. Here we describe a new mechanism that activates DLK in <i>Drosophila</i>: loss of the spectraplakin <i>short stop</i> (<i>shot</i>). In a genetic screen for mutants with defective neuromuscular junction development, we identify a hypomorphic allele of <i>shot</i> that displays synaptic terminal overgrowth and a precocious regenerative response to nerve injury. We demonstrate that both phenotypes are the result of overactivation of the DLK signaling pathway. We further show that, unlike mutations in the PHR ligase Highwire, loss of function of <i>shot</i> activates DLK without a concomitant increase in the levels of DLK. As a spectraplakin, Shot binds to both actin and microtubules and promotes cytoskeletal stability. The DLK pathway is also activated by downregulation of the TCP1 chaperonin complex, whose normal function is to promote cytoskeletal stability. These findings support the model that DLK is activated by cytoskeletal instability, which is a shared feature of both spectraplakin mutants and injured axons.