The Age-1 and Daf-2 Genes Function in a Common Pathway to Control the Lifespan of Caenorhabditis Elegans

Recessive mutations in two genes, daf-2 and age-1, extend the lifespan of Caenorhabditis elegans significantly. The daf-2 gene also regulates formation of an alternative developmental state called the dauer. Here we asked whether these two genes function in the same or different lifespan pathways. We found that the longevity of both age-1 and daf-2 mutants requires the activities of the same two genes, daf-16 and daf-18. In addition, the daf-2(e1370); age-1(hx546) double mutant did not live significantly longer than the daf-2 single mutant. We also found that, like daf-2 mutations, the age-1(hx546) mutation affects certain aspects of dauer formation. These findings suggest that age-1 and daf-2 mutations do act in the same lifespan pathway and extend lifespan by triggering similar if not identical processes.     

Lethal and Amanitin-Resistance Mutations in the Caenorhabditis Elegans Ama-1 and Ama-2 Genes

Mutants of Caenorhabditis elegans resistant to alpha-amanitin have been isolated at a frequency of about 1.6 x 10(-6) after EMS mutagenesis of the wild-type strain, N2. Four new dominant resistance mutations have been studied genetically. Three are alleles of a previously identified gene, ama-1 IV, encoding the largest subunit of RNA polymerase II. The fourth mutation defines a new gene, ama-2 V. Unlike the ama-1 alleles, the ama-2 mutation exhibits a recessive-lethal phenotype. Growth and reproduction of N2 was inhibited at a concentration of 10 micrograms/ml amanitin, whereas ama-2/+ animals were inhibited at 100 micrograms/ml, and 800 micrograms/ml was required to inhibit growth of ama-1/+ larvae. We have also determined that two reference strains used for genetic mapping, dpy-11(e224)V and sma-1(e30)V, are at least four-fold more sensitive to amanitin that the wild-type strain. Using an amanitin-resistant ama-1(m118) or ama-1(m322) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. The frequency of EMS-induced lethal ama-1 mutations is approximately 1.7 x 10(-3), 1000-fold higher than the frequency of amanitin-resistance alleles. Nine of the lethal alleles are apparent null mutations, and they exhibit L1-lethal phenotypes at both 20 degrees and 25 degrees. Six alleles result in partial loss of RNA polymerase II function as determined by their sterile phenotypes at 20 degrees. All but one of these latter mutations exhibit a more severe phenotype at 25 degrees C. We have also selected seven EMS-induced revertants of three different ama-1 lethals. These revertants restore dominant resistance to amanitin. The selection for revertants also produced eight new dominant amanitin resistance alleles on the balancer chromosome, nT1.     

Genes That Regulate Both Development and Longevity in Caenorhabditis Elegans

The nematode Caenorhabditis elegans responds to conditions of overcrowing and limited food by arresting development as a dauer larva. Genetic analysis of mutations that alter dauer larva formation (daf mutations) is presented along with an updated genetic pathway for dauer vs. nondauer development. Mutations in the daf-2 and daf-23 genes double adult life span, whereas mutations in four other dauer-constitutive genes positioned in a separate branch of this pathway (daf-1, daf-4, daf-7 and daf-8) do not. The increased life spans are suppressed completely by a daf-16 mutation and partially in a daf-2; daf-18 double mutant. A genetic pathway for determination of adult life span is presented based on the same strains and growth conditions used to characterize Daf phenotypes. Both dauer larva formation and adult life span are affected in daf-2; daf-12 double mutants in an allele-specific manner. Mutations in daf-12 do not extend adult life span, but certain combinations of daf-2 and daf-12 mutant alleles nearly quadruple it. This synergistic effect, which does not equivalently extend the fertile period, is the largest genetic extension of life span yet observed in a metazoan.     

Longevity-Determining Genes in Caenorhabditis Elegans: Chromosomal Mapping of Multiple Noninteractive Loci

We have used chromosome mapping with polymorphic markers to define genetic components governing life span in the nematode Caenorhabditis elegans. A complex recombinant-inbred population was derived from an interstrain cross, yielding >1000 genotypes, each a composite of homozygous segments from the two parental strains. Genotypes were analyzed for the last-surviving 1-5% of worms in aging cohorts, and for young controls, by multiplex polymerase chain reaction using polymorphic markers to distinguish the parental alleles. We identified five regions of the genome at which one parental allele was significantly enriched in long-lived subpopulations. At four of five loci, the same alleles were selected in aging cohorts maintained under two different conditions, implying that these genes determine life span in differing environments.     

Genes That Implement the Hermaphrodite Mode of Dosage Compensation in Caenorhabditis Elegans

We report a genetic characterization of several essential components of the dosage compensation process in Caenorhabditis elegans. Mutations in the genes dpy-26, dpy-27, dpy-28, and the newly identified gene dpy-29 disrupt dosage compensation, resulting in elevated X-linked gene expression in XX animals and an incompletely penetrant maternal-effect XX-specific lethality. These dpy mutations appear to cause XX animals to express each set of X-linked genes at a level appropriate for XO animals. XO dpy animals are essentially wild type. Both the viability and the level of X-linked gene expression in XX animals carrying mutations in two or more dpy genes are the same as in animals carrying only a single mutation, consistent with the view that these genes act together in a single process (dosage compensation). To define a potential time of action for the gene dpd-28 we performed reciprocal temperature-shift experiments with a heat sensitive allele. The temperature-sensitive period for lethality begins 5 hr after fertilization at the 300-cell stage and extends to about 9 hr, a point well beyond the end of cell proliferation. This temperature-sensitive period suggests that dosage compensation is functioning in XX animals by mid-embryogenesis, when many zygotically transcribed genes are active. While mutations in the dpy genes have no effect on the sexual phenotype of otherwise wild-type XX or XO animals, they do have a slight feminizing effect on animals whose sex-determination process is already genetically perturbed. The opposite directions of the feminizing effects on sex determination and the masculinizing effects on dosage compensation caused by the dpy mutations are inconsistent with the wild-type dpy genes acting to coordinately control both processes. Instead, the feminizing effects are most likely an indirect consequence of disruptions in dosage compensation caused by the dpy mutations. Based on the cumulative evidence, the likely mechanism of dosage compensation in C. elegans involves reducing X-linked gene expression in XX animals to equal that in XO animals via the action of the dpy genes.     

A Genetic Mosaic Screen of Essential Zygotic Genes in Caenorhabditis Elegans

We have devised a simple genetic mosaic screen, which circumvents the difficulties posed by phenotypic analysis of early lethal mutants, to analyze essential zygotic genes in Caenorhabditis elegans. The screen attempts to distinguish genes involved in cell type and/or lineage specific processes such as determination, differentiation or morphogenesis from genes involved in general processes such as intermediary metabolism by using the pattern of gene function to classify genes: genes required in one or a subset of early blastomeres may have specific functions, whereas genes required in all early blastomeres may have general functions. We found that 12 of 17 genes examined function in specific early blastomeres, suggesting that many zygotic genes contribute to specific early processes. We discuss the advantages and limitations of this screen, which is applicable to other regions of the C. elegans genome.     

秀丽线虫脂肪积累调控的生理与分子机制

脂肪积累是一个复杂的生理过程,模式动物秀丽线虫(以下简称线虫)已经成为目前研究脂肪积累的重要模型.线虫中的脂肪酸代谢通路与其他物种中的代谢是基本一致的,很多关键的代谢调节基因的功能已经得到鉴定.线虫中脂肪积累涉及至少4个核心调控通路,分别为胰岛素和转化生长因子β(TGF-β)信号通路、sbp-1/ mdt-15介导的信号通路、核激素受体nhr-49介导的信号通路与雷帕霉素靶标(TOR)和氨基己糖介导的信号通路.此外,神经递质5-羟色胺、多巴胺和谷氨酸参与了脂肪积累的调控,而tub-1和bbs-1可以介导对脂肪积累的神经调控,暗示了纤毛结构与感觉神经元在脂肪积累中可能的重要功能.线虫中的研究工作对人类肥胖症等代谢疾病的研究具有重要的提示作用.     

Essential Genes in the Hdf6 Region of Chromosome I in Caenorhabditis Elegans

In this paper we describe the analysis of essential genes in the hDf6 region of chromosome I of Caenorhabditis elegans. Nineteen complementation groups have been identified which are required for the growth, survival or fertility of the organism (essential genes). Since ten of these genes were represented by more than one allele, a Poisson calculation predicts a minimum estimate of 25 essential genes in hDf6. The most mutable gene in this region was let-354 with seventeen alleles. An average mutation rate of 5 x 10(-5) mutations/gene/chromosome screened was calculated for an ethyl methanesulfonate dose of 15 mM. Mutations were recovered by screening for lethal mutations using the duplication sDp2 for recovery. Our analysis shows that duplications are very effective for maintenance and mapping of large numbers of lethal mutations. Approximately 600 lethal mutations were mapped in order to identify the 54 that are in the deficiency hDf6. The hDf6 region appears to have a lower proportion of early arresting mutations than other comparably sized regions of the genome.     

Daf-2, Daf-16 and Daf-23: Genetically Interacting Genes Controlling Dauer Formation in Caenorhabditis Elegans

Under conditions of high population density and low food, Caenorhabditis elegans forms an alternative third larval stage, called the dauer stage, which is resistant to desiccation and harsh environments. Genetic analysis of some dauer constitutive (Daf-c) and dauer defective (Daf-d) mutants has revealed a complex pathway that is likely to function in particular neurons and/or responding tissues. Here we analyze the genetic interactions between three genes which comprise a branch of the dauer formation pathway that acts in parallel to or downstream of the other branches of the pathway, the Daf-c genes daf-2 and daf-23 and the Daf-d gene daf-16. Unlike mutations in other Daf-c genes, mutations in both daf-2 and daf-23 cause non-conditional arrest at the dauer stage. Our epistasis analysis suggests that daf-2 and daf-23 are functioning at a similar point in the dauer pathway. First, mutations in daf-2 and daf-23 are epistatic to mutations in the same set of Daf-d genes. Second, daf-2 and daf-23 mutants are suppressed by mutations in daf-16. Mutations in daf-16 do not suppress any of the other Daf-c mutants as efficiently as they suppress daf-2 and daf-23 mutants. Third, double mutants between either daf-2 or daf-23 and several other daf-d mutants exhibit an unusual interaction. Based on these results, we present a model for the function of daf-2, daf-23 and daf-16 in dauer formation.     

Identification and Characterization of Genes That Interact with Lin-12 in Caenorhabditis Elegans

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.     

The Unc-8 and Sup-40 Genes Regulate Ion Channel Function in Caenorhabditis Elegans Motorneurons

Two Caenorhabditis elegans genes, unc-8 and sup-40, have been newly identified, by genetic criteria, as regulating ion channel function in motorneurons. Two dominant unc-8 alleles cause motorneuron swelling similar to that of other neuronal types in dominant mutants of the deg-1 gene family, which is homologous to a mammalian gene family encoding amiloride-sensitive sodium channel subunits. As for previously identified deg-1 family members, unc-8 dominant mutations are recessively suppressed by mutations in the mec-6 gene, which probably encodes a second type of channel component. An unusual dominant mutation, sup-41 (lb125), also co-suppresses unc-8 and deg-1, suggesting the existence of yet another common component of ion channels containing unc-8 or deg-1 subunits. Dominant, transacting, intragenic suppressor mutations have been isolated for both unc-8 and deg-1, consistent with the idea that, like their mammalian homologues, the two gene products function as multimers. The sup-40 (lb130) mutation dominantly suppresses unc-8 motorneuron swelling and produces a novel swelling phenotype in hypodermal nuclei. sup-40 may encode an ion channel component or regulator that can correct the osmotic defect caused by abnormal unc-8 channels.     

The Tc3 Family of Transposable Genetic Elements in Caenorhabditis Elegans

We describe genetic and molecular properties of Tc3, a family of transposable elements in Caenorhabditis elegans. About 15 Tc3 elements are present in the genomes of several different wild-type varieties of C. elegans, but Tc3 transposition and excision are not detected in these strains. Tc3 transposition and excision occur at high frequencies, however, in strain TR679, a mutant identified because of its highly active Tc1 elements. In TR679, Tc3 is responsible for several spontaneous mutations affecting the unc-22 gene. Tc3-induced mutations are unstable, and revertants result from precise or nearly precise excision of Tc3. Although Tc3 is very active in TR679, it is not detectably active in several other mutator mutants, all of which exhibit high levels of Tc1 activity. Tc3 is 2.5 kilobases long, and except for sequences near its inverted repeat termini, it is unrelated to Tc1. The termini of Tc3 are inverted repeats of at least 70 base pairs; the terminal 8 nucleotides of Tc3 are identical to 8 of the terminal 9 nucleotides of Tc1.     

Analysis of Mutations in the Sqt-1 and Rol-6 Collagen Genes of Caenorhabditis Elegans

Different mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans can cause diverse changes in body morphology and display different genetic attributes. We have determined the nucleotide alterations in 15 mutant alleles of these genes. Three mutations in sqt-1 and one in rol-6 that cause dominant right-handed helical twisting (RRol) of animals are arginine to cysteine replacements. These mutations are all within a short conserved sequence, on the amino terminal side of the Gly-X-Y repeats, that is found in all C. elegans cuticle collagens. A recessive RRol mutation of rol-6 is a replacement of one of the same conserved arginines by histidine. In contrast, three sqt-1 mutations that cause recessive left-handed helical twisting (LRol) are replacements of a conserved carboxyterminal cysteine residue with either tyrosine or serine. These results suggest that disulfide bonding is important in collagen organization and that a deficit or surplus of disulfides may cause cuticle alterations of opposite handedness. In contrast to other collagens, glycine replacement mutations in the Gly-X-Y repeats of sqt-1 cause very mild phenotypes. Nonsense mutations of both sqt-1 and rol-6 cause nearly, but not totally, wild-type phenotypes. A nonsense mutation in sqt-1 suppresses the phenotype of rol-6 RRol mutations, suggesting that rol-6 collagen function is dependent on the presence of sqt-1 collagen. Mutations of sqt-1 are not suppressed by a rol-6 nonsense mutation, however, indicating that sqt-1 collagen can function independently of rol-6.     

Par-4, a Gene Required for Cytoplasmic Localization and Determination of Specific Cell Types in Caenorhabditis Elegans Embryogenesis

Specification of some cell fates in the early Caenorhabditis elegans embryo is mediated by cytoplasmic localization under control of the maternal genome. Using nine newly isolated mutations, and two existing mutations, we have analyzed the role of the maternally expressed gene par-4 in cytoplasmic localization. We recovered seven new par-4 alleles in screens for maternal effect lethal mutations that result in failure to differentiate intestinal cells. Two additional par-4 mutations were identified in noncomplementation screens using strains with a high frequency of transposon mobility. All 11 mutations cause defects early in development of embryos produced by homozygous mutant mothers. Analysis with a deficiency in the region indicates that it33 is a strong loss-of-function mutation. par-4(it33) terminal stage embryos contain many cells, but show no morphogenesis, and are lacking intestinal cells. Temperature shifts with the it57ts allele suggest that the critical period for both intestinal differentiation and embryo viability begins during oogenesis, about 1.5 hr before fertilization, and ends before the four-cell stage. We propose that the primary function of the par-4 gene is to act as part of a maternally encoded system for cytoplasmic localization in the first cell cycle, with par-4 playing a particularly important role in the determination of intestine. Analysis of a par-4; par-2 double mutant suggests that par-4 and par-2 gene products interact in this system.     

Properties of a Class of Genes Required for Ray Morphogenesis in Caenorhabditis Elegans

We have identified eight mutations that define at least five terminal differentiation genes (ram genes) whose products are required during the extension of the male-specific ray sensilla in Caenorhabditis elegans. ram gene mutations result in morphological abnormalities in the sensory rays but do not appear to interfere with ray functions. A similar ray morphology phenotype was observed in males harboring mutations in three previously defined genes, dpy-11, dpy-18 and sqt-1, that also affect body shape. One of these genes, sqt-1, is known to encode a collagen. Mutations in different ram genes failed to complement, from which we infer that their gene products functionally interact. For one ram gene, failure to complement was shown to result from haploinsufficiency. Intergenic noncomplementation did not extend to the body morphology genes. The temperature-sensitive periods of both ram and body morphology mutations corresponded to the period of development in which ray extension occurs. We propose that ram gene products act together in a critical interaction between the rays and the cuticle required for wild-type ray morphology.     

Dual Role of Cdc42 in Spindle Orientation Control of Adherent Cells

The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and PtdIns(3,4,5)P3. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. βPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-βPix-actin pathway for this mechanism.Alignment of the mitotic spindles with a predetermined axis, which confines the plane of cell division, occurs in many types of cells and is crucial for morphogenesis and embryogenesis. Cell geometry (30, 32, 47), cell polarity (6, 24, 35), and cell-cell adhesions (20, 22, 48) are proposed to be the determinants for the axis of the spindles. In most cases, spindle alignment along the predetermined axis requires both astral microtubules and the actin cytoskeleton and is believed to involve dynein-dependent microtubule pulling forces functioning at the cell cortex (4, 12, 31).We have previously shown that in nonpolarized adherent cells, such as HeLa cells, integrin-mediated cell-substrate adhesion orients the spindles parallel to the substratum, which ensures that both daughter cells remain attached to the substrate after cell division (42). This mechanism requires the actin cytoskeleton, astral microtubules, the microtubule plus-end-tracking protein EB1, and myosin X. Furthermore, our recent study has shown that the lipid second messenger phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3] is also essential to this mechanism. PtdIns(3,4,5)P3 is accumulated in the midcortex of metaphase cells, which is important for the localized accumulation of dynactin, a dynein-binding partner, at the midcortex. We have proposed that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on the spindle to the midcortex and orients the spindle parallel to the substratum (43). However, the molecular mechanisms that regulate the actin cytoskeleton and PtdIns(3,4,5)P3 in the spindle orientation control remain unknown.The Rho family of GTPases, including Rho, Rac, and Cdc42, plays central roles in the regulation of not only the actin cytoskeleton but also microtubules in the control of various activities of cell motility, including cell adhesion, cell migration, and cell cycle progression (9, 33, 41). Rho family GTPases are also reported to regulate several mitotic events. RhoA plays a crucial role in contractile ring function and localizes to the cleavage furrow along with its effectors, ROCK, citron kinase, and mDia, during cytokinesis (18, 11). Cdc42 and its effector, mDia3, are reported to regulate the alignment of chromosomes during prometaphase and metaphase (49). Interestingly, Cdc42 is also required for proper spindle positioning in polarized cells such as budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans one-cell stage embryos, and mouse oocytes, which undergo asymmetric cell division (1, 23, 13, 28). However, how Cdc42 regulates spindle orientation and whether it has a role in spindle orientation in nonpolarized cells remain unknown.Here, we show that Cdc42 is required for the mechanism that orients the spindle parallel to the substratum in nonpolarized adherent cells. Moreover, our results show that Cdc42 regulates both PtdIns(3,4,5)P3 and the actin cytoskeleton through PI(3)K- and p21-activated kinase 2 (PAK2)/βPix-signaling pathways, respectively. Both pathways are required for the localized accumulation of dynein/dynactin complexes in the midcortex in metaphase cells and, thus, for the proper spindle orientation parallel to the substratum.