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PubMed2001

The oxpA5 mutation of Aspergillus nidulans is an allele of adB, the gene encoding adenylosuccinate synthetase.

The oxpA5 mutation in Aspergillus nidulans results in a pleiotropic phenotype, including resistance to oxypurinol and partial constitutivity of the enzymes of purine catabolism. Here we show that the oxpA5 mutation is an allele of adB, the gene encoding adenylosuccinate synthetase (ASS). Cloning, sequencing and characterisation of the adB gene are reported in this paper. In vivo complementation tests indicate that the oxpA5 mutation is a partial loss-of-function mutation, and altered kinetic parameters of the ASS could account for the pleiotropic phenotype of the oxpA5 mutant. The transcriptional regulation of adB presents some interesting features, including increased gene expression in the presence of ammonium and of AMP, the final product of purine biosynthesis. The adB gene is located adjacent to helA, a newly identified gene coding for a putative RNA helicase.

Adenylosuccinate Synthase↗
PubMed1990

The let-60 locus controls the switch between vulval and nonvulval cell fates in Caenorhabditis elegans.

During induction of the Caenorhabditis elegans hermaphrodite vulva by the anchor cell of the gonad, six multipotent vulval precursor cells (VPCs) have two distinct fates: three VPCs generate the vulva and the other three VPCs generate nonspecialized hypodermis. Genes that control the fates of the VPCs in response to the anchor cell signal are defined by mutations that cause all six VPCs to generate vulval tissue (Multivulva or Muv) or that cause all six VPCs to generate hypodermis (Vulvaless or Vul). Seven dominant Vul mutations were isolated as dominant suppressors of a lin-15 Muv mutation. These mutations are dominant alleles of the gene let-60, previously identified only by recessive lethal mutations. Our genetic studies of these dominant Vul recessive lethal mutations, recessive lethal mutations, intragenic revertants of the dominant Vul mutations, and the closely mapping semi-dominant multivulva lin-34 mutations suggest that: (1) loss-of-function mutations of let-60 are recessive lethal at a larval stage, but they also cause a Vul phenotype if the lethality is rescued maternally by a lin-34 gain-of-function mutation. (2) The dominant Vul alleles of let-60 are dominant negative mutations whose gene products compete with wild-type activity. (3) lin-34 semidominant Muv alleles are either gain-of-function mutations of let-60 or gain-of-function mutations of an intimately related gene that elevates let-60 activity. We propose that let-60 activity controls VPC fates. In a wild-type animal, reception by a VPC of inductive signal activates let-60, and it generates into a vulval cell type; in absence of inductive signal, let-60 activity is low and the VPC generates hypodermal cells. Our genetic interaction studies suggest that let-60 acts downstream of let-23 and lin-15 and upstream of lin-1 and lin-12 in the genetic pathway specifying the switch between vulval and nonvulval cell types.

Alleles↗
PubMed1999

Role of SpoVG in asymmetric septation in Bacillus subtilis.

Deletion of the citC gene, coding for isocitrate dehydrogenase, arrests sporulation of Bacillus subtilis at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum. A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the spoVG gene. The suppressing mutation and other spoVG loss-of-function mutations enabled citC mutant cells to form asymmetric septa and to activate the forespore-specific sigma factor sigmaF. However, little induction of mother cell-specific, sigmaE-dependent sporulation genes was observed in a citC spoVG double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the citC mutant. These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl2. Overexpression of the spoVG gene in wild-type cells significantly delayed sigmaF activation. Increased expression and stability of SpoVG in citC mutant cells may contribute to the citC mutant phenotype. Inactivation of the spoVG gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal. Unlike the case for inactivation of the cell division inhibitor gene minD, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active. These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation.

Bacillus subtilis↗
PubMed2005

Analysis of pyrimidine catabolism in Drosophila melanogaster using epistatic interactions with mutations of pyrimidine biosynthesis and beta-alanine metabolism.

The biochemical pathway for pyrimidine catabolism links the pathways for pyrimidine biosynthesis and salvage with beta-alanine metabolism, providing an array of epistatic interactions with which to analyze mutations of these pathways. Loss-of-function mutations have been identified and characterized for each of the enzymes for pyrimidine catabolism: dihydropyrimidine dehydrogenase (DPD), su(r) mutants; dihydropyrimidinase (DHP), CRMP mutants; beta-alanine synthase (betaAS), pyd3 mutants. For all three genes, mutants are viable and fertile and manifest no obvious phenotypes, aside from a variety of epistatic interactions. Mutations of all three genes disrupt suppression by the rudimentary gain-of-function mutation (r(Su(b))) of the dark cuticle phenotype of black mutants in which beta-alanine pools are diminished; these results confirm that pyrimidines are the major source of beta-alanine in cuticle pigmentation. The truncated wing phenotype of rudimentary mutants is suppressed completely by su(r) mutations and partially by CRMP mutations; however, no suppression is exhibited by pyd3 mutations. Similarly, su(r) mutants are hypersensitive to dietary 5-fluorouracil, CRMP mutants are less sensitive, and pyd3 mutants exhibit wild-type sensitivity. These results are discussed in the context of similar consequences of 5-fluoropyrimidine toxicity and pyrimidine catabolism mutations in humans.

Amidohydrolases↗
PubMed2001

[From gene to disease; thyroid stimulating hormone receptor, hyperthyroidism and hypothyroidism].

The thyroid-stimulating hormone receptor (TSH-R) gene appears to be very sensitive to mutagenesis, in view of the vast number of reported mutations. Loss-of-function germline mutations occur preferentially in the hormone-binding extracellular domain of the TSH-R, resulting in familial TSH resistance. Gain-of-function germline mutations occur preferentially in the transmembrane domain of the TSH-R, resulting in familial non-autoimmune hyperthyroidism. Familial gestational hyperthyroidism is due to a mutant TSH-R which is hypersensitive to chorionic gonadotropin. Somatic gain-of-function mutations are a major cause of toxic thyroid adenomas.

Adult↗
PubMed1997

Mutations in the alpha1 subunit of an L-type voltage-activated Ca2+ channel cause myotonia in Caenorhabditis elegans.

The control of excitable cell action potentials is central to animal behavior. We show that the egl-19 gene plays a pivotal role in regulating muscle excitation and contraction in the nematode Caenorhabditis elegans and encodes the alphal subunit of a homologue of vertebrate L-type voltage-activated Ca2+ channels. Semi-dominant, gain-of-function mutations in egl-19 cause myotonia: mutant muscle action potentials are prolonged and the relaxation delayed. Partial loss-of-function mutations cause slow muscle depolarization and feeble contraction. The most severe loss-of-function mutants lack muscle contraction and die as embryos. We localized two myotonic mutations in the sixth membrane-spanning domain of the first repeat (IS6) region, which has been shown to be responsible for voltage-dependent inactivation. A third myotonic mutation implicates IIIS4, a region involved in sensing plasma-membrane voltage change, in the inactivation process.

Action Potentials↗
PubMed2004

The Caenorhabditis elegans MAPK phosphatase VHP-1 mediates a novel JNK-like signaling pathway in stress response.

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and to a wide variety of environmental stresses. MAPK cascades can be inactivated at the MAPK activation step by members of the MAPK phosphatase (MKP) family. However, the components that act in MKP-regulated pathways have not been well characterized in the context of whole organisms. Here we characterize the Caenorhabditis elegans vhp-1 gene, encoding an MKP that acts preferentially on the c-Jun N-terminal kinase (JNK) and p38 MAPKs. We found that animals defective in vhp-1 are arrested during larval development. This vhp-1 defect is suppressed by loss-of-function mutations in the kgb-1, mek-1, and mlk-1 genes encoding a JNK-like MAPK, an MKK7-type MAPKK, and an MLK-type MAPKKK, respectively. The genetic and biochemical data presented here demonstrate a critical role for VHP-1 in the KGB-1 pathway. Loss-of-function mutations in each component in the KGB-1 pathway result in hypersensitivity to heavy metals. These results suggest that VHP-1 plays a pivotal role in the integration and fine-tuning of the stress response regulated by the KGB-1 MAPK pathway.

Amino Acid Motifs↗
PubMed2001

Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay.

Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that 'hot-spot' p53 mutants (which comprise approximately 30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants. We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

Base Sequence↗
PubMed2006

A homozygous R262Q mutation in the gonadotropin-releasing hormone receptor presenting as constitutional delay of growth and puberty with subsequent borderline oligospermia.

CONTEXT: The GnRH receptor plays a central role in regulating gonadotropin synthesis and release, and several mutations in the GNRHR gene have been reported in patients with idiopathic or familial forms of isolated hypogonadotropic hypogonadism (IHH). OBJECTIVE: The objective of the study was to investigate whether partial loss-of-function mutations in the GnRH receptor might be responsible for delayed puberty phenotypes. PATIENTS: Patients included sibling pairs with delayed puberty (n = 8) or those in whom one brother had delayed puberty and another had hypogonadotropic hypogonadism (n = 3). METHODS: Methods included mutational analysis of the GNRHR gene. RESULTS: A homozygous R262Q mutation in the GnRH receptor was identified in two brothers from one family. In this kindred, the proband presented at 15 yr of age with delayed puberty. After a short course of testosterone, he seemed to be progressing through puberty appropriately and was discharged from follow-up. His younger brother was also referred with delayed puberty but showed little progress after treatment. Frequent sampling revealed detectable but apulsatile LH and FSH release. His clinical progress was consistent with IHH, and he requires ongoing testosterone replacement. CONCLUSIONS: Homozygous partial loss-of-function mutations in the GnRH receptor, such as R262Q, can present with variable phenotypes including apparent delayed puberty. Ongoing clinical vigilance might be required when patients are discharged from follow-up, especially when there is a family history of delayed puberty or IHH because oligospermia and reduced bone mineralization can occur with time.

Adolescent↗
PubMed2002

Defective de novo methylation of viral and cellular DNA sequences in ICF syndrome cells.

ICF syndrome (immunodeficiency, centromere instability and facial anomalies) is a recessive human genetic disorder resulting from mutations in the DNA methyltransferase 3B (DNMT3B) gene. Patients with this disease exhibit numerous chromosomal abnormalities, including anomalous decondensation, pairing, separation and breakage, primarily involving the pericentromeric regions of chromosomes 1 and 16. Global levels of DNA methylation in ICF cells are only slightly reduced; however, certain repetitive sequences and genes on the inactive X chromosome of female ICF patients are significantly hypomethylated. In the present report, we analyze the molecular defect of de novo methylation in ICF cells in greater detail by making use of a model Epstein-Barr virus (EBV)-based system and three members of the unique cellular cancer-testis (C-T) gene family. Results with the EBV-based system indicate that de novo methylation of newly introduced viral sequences is defective in ICF syndrome. Limited de novo methylation capacity is retained in ICF cells, indicating that the mutations in DNMT3B are not complete loss-of-function mutations or that other DNMTs cooperate with DNMT3B. Analysis of three C-T genes (two on the X chromosome and one autosomal) revealed that loss of methylation from cellular gene sequences is heterogeneous, with both autosomal and X chromosome-based genes demonstrating sensitivity to mutations in DNMT3B. Aberrant hypomethylation at a number of loci examined correlated with altered gene expression levels. Lastly, no consistent changes in the protein levels of the DNA methyltransferases were noted when normal and ICF cell lines were compared.

Abnormalities, Multiple↗
PubMed2006

A gain-of-function screen identifying genes required for vein formation in the Drosophila melanogaster wing.

The formation of the Drosophila wing involves developmental processes such as cell proliferation, pattern formation, and cell differentiation that are common to all multicellular organisms. The genes controlling these cellular behaviors are conserved throughout the animal kingdom, and the genetic analysis of wing development has been instrumental in their identification and functional characterization. The wing is a postembryonic structure, and most loss-of-function mutations are lethal in homozygous flies before metamorphosis. In this manner, loss-of-function genetic screens aiming to identify genes affecting wing formation have not been systematically utilized. As an alternative, a number of genetic searches have utilized the phenotypic consequences of gene gain-of-expression, as a method more efficient to search for genes required during imaginal development. Here we present the results of a gain-of-function screen designed to identify genes involved in the formation of the wing veins. We generated 13,000 P-GS insertions of a P element containing UAS sequences (P-GS) and combined them with a Gal4 driver expressed mainly in the developing pupal veins. We selected 500 P-GSs that, in combination with the Gal4 driver, result in modifications of the veins, changes in the morphology of the wing, or defects in the differentiation of the trichomes. The P-element insertion sites were mapped to the genomic sequence, identifying 373 gene candidates to participate in wing morphogenesis and vein formation.

Animals↗
PubMed2001

Functional heterogeneity of ROMK mutations linked to hyperprostaglandin E syndrome.

BACKGROUND: The renal K(+) channel ROMK (Kir1.1) controls salt reabsorption in the kidney. Loss-of-function mutations in this channel cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), which is characterized by severe renal salt and fluid wasting. METHODS: We investigated 10 HPS/aBS patients for mutations in the ROMK gene by single-strand conformation polymorphism analysis (SSCA) and direct sequencing. To assess the functional consequences, Ba(2+)-sensitive K(+) currents were measured in five mutants of the core region as well as one mutant with truncated C-terminus, using the two-electrode voltage-clamp technique after an injection of mutant cRNA into Xenopus oocytes. RESULTS: Three novel ROMK mutations were identified together with six mutations described previously. The mutations were categorized into three groups: (1) amino acid exchanges in the core region (M1-H5-M2), (2) truncation at the cytosolic C-terminus, and (3) deletions of putative promoter elements. While the core mutations W99C, N124K, and I142T led to significantly reduced macroscopic K(+) currents (1 to 8% of wild-type currents), the A103V and P110L variants retained substantial K(+) conductivity (23 and 35% of wild-type currents, respectively). Coexpression of A103V and P110L, resembling the compound heterozygous state of the affected individual, further reduced macroscopic currents to 9% of the wild-type currents. All mutants in the core region exerted a dominant-negative effect on wild-type ROMK1. The C-terminal frameshift (fs) mutation (H354fs) did not change current amplitudes compared with ROMK1 wild type, suggesting that a mechanism other than alteration of the electrophysiological properties may responsible for loss of channel activity. CONCLUSIONS: Analysis of ROMK mutants linked to HPS/aBS revealed a spectrum of mechanisms accounting for loss of channel function. Further characterization of the molecular defects might be helpful for the development of new therapeutic approaches.

Animals↗
PubMed2003

Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1.

Fragile X syndrome is caused by loss-of-function mutations in the fragile X mental retardation 1 gene. How these mutations affect neuronal development and function remains largely elusive. We generated specific point mutations or small deletions in the Drosophila fragile X-related (Fmr1) gene and examined the roles of Fmr1 in dendritic development of dendritic arborization (DA) neurons in Drosophila larvae. We found that Fmr1 could be detected in the cell bodies and proximal dendrites of DA neurons and that Fmr1 loss-of-function mutations increased the number of higher-order dendritic branches. Conversely, overexpression of Fmr1 in DA neurons dramatically decreased dendritic branching. In dissecting the mechanisms underlying Fmr1 function in dendrite development, we found that the mRNA encoding small GTPase Rac1 was present in the Fmr1-messenger ribonucleoprotein complexes in vivo. Mosaic analysis with a repressor cell marker (MARCM) and overexpression studies revealed that Rac1 has a cell-autonomous function in promoting dendritic branching of DA neurons. Furthermore, Fmr1 and Rac1 genetically interact with each other in controlling the formation of fine dendritic branches. These findings demonstrate that Fmr1 affects dendritic development and that Rac1 is partially responsible for mediating this effect.

Animals↗
PubMed2004

Screening for presenilin inhibitors using the free-living nematode, Caenorhabditis elegans.

Caenorhabditis elegans contains 3 homologs of presenilin genes that are associated with Alzheimer s disease. Loss-of-function mutations in C. elegans genes cause a defect in egg laying. In humans, loss of presenilin-1 (PS1) function reduces amyloid-beta peptide processing from the amyloid protein precursor. Worms were screened for compounds that block egg laying, phenocopying presenilin loss of function. To accommodate even relatively high throughput screening, a semi-automated method to quantify egg laying was devised by measuring the chitinase released into the culture medium. Chitinase is released by hatching eggs, but little is shed into the medium from the body cavity of a hermaphrodite with an egg laying deficient (egl) phenotype. Assay validation involved measuring chitinase release from wild-type C. elegans (N2 strain), sel-12 presenilin loss-of-function mutants, and 2 strains of C. elegans with mutations in the egl-36 K(+) channel gene. Failure to find specific presenilin inhibitors in this collection likely reflects the small number of compounds tested, rather than a flaw in screening strategy. Absent defined biochemical pathways for presenilin, this screening method, which takes advantage of the genetic system available in C. elegans and its historical use for anthelminthic screening, permits an entry into mechanism-based discovery of drugs for Alzheimer's disease.

Animals↗
PubMed2003

Epilepsy with auditory features: a LGI1 gene mutation suggests a loss-of-function mechanism.

Autosomal dominant partial epilepsy with auditory features (ADPEAF) is a genetically heterogeneous disorder. Some patients exhibit mutations in the leucine-rich glioma inactivated (LGI1) gene. In an ADPEAF family, a novel mutation in the Lgi1 signal peptide is predicted to interfere with the protein cell sorting, resulting in altered processing. This finding suggests a loss-of-function mechanism for LGI1 gene mutations causing ADPEAF even if other mechanisms cannot be ruled out.

Acoustic Stimulation↗
PubMed2005

The fluG-BrlA pathway contributes to the initialisation of autolysis in submerged Aspergillus nidulans cultures.

The fluG gene proved to be essential in the initialisation of autolysis in Aspergillus nidulans (teleomorph Emericella nidulans) cultures, while a loss-of-function mutation in only one out of the flbB-E genes had only minor effects on autolysis. In contrast to its important role in sporulation, brlA regulated only some, but not all, elements of the autolytic process. The tightly coupled autolytic events (chitinase and proteinase production, hyphal fragmentation, disorganisation of pellets, autolytic loss of biomass) observable in ageing cultures of A. nidulans were disconnected by loss-of-function mutations in some genes of the FluG-BrlA regulatory network. The tight correlation between pellet morphology and size and hydrolase production was also erased by these mutations. On the other hand, the mutations studied did not affect the glutathione metabolism of the fungus.

Aspergillus nidulans↗
PubMed2000

Lesions in the sterol delta reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis.

The brassinosteroid (BR) biosynthetic pathway, and the sterol pathway which is prerequisite to the BR pathway, are rapidly being characterized because of the availability of a large number of characteristic dwarf mutants in Arabidopsis. Here we show that the Arabidopsis dwarf5 mutants are disrupted in a sterol Delta7 reduction step. dwf5 plants display the characteristic dwarf phenotype typical of other BR mutants. This phenotype includes small, round, dark-green leaves, and short stems, pedicels, and petioles. Metabolite tracing with 13C-labeled precursors in dwf5 verified a deficiency in a sterol Delta7 reductase activity. All six independent alleles contain loss-of-function mutations in the sterol Delta7 reductase gene. These include a putative mRNA instability mutation in dwf5-1, 3' and 5' splice-site mutations in dwf5-2 and dwf5-6, respectively, premature stop codons in dwf5-3 (R400Z) and dwf5-5 (R409Z), and a mis-sense mutation in dwf5-4 (D257N). The dwf5 plant could be restored to wild type by ectopic overexpression of the wild-type copy of the gene. Both the Arabidopsis dwf5 phenotype and the human Smith-Lemli-Opitz syndrome are caused by loss-of-function mutations in a sterol Delta7 reductase gene, indicating that it is required for the proper growth and development of these two organisms.

Alleles↗
PubMed2006

The Caenorhabditis elegans rhy-1 gene inhibits HIF-1 hypoxia-inducible factor activity in a negative feedback loop that does not include vhl-1.

Hypoxia-inducible factor (HIF) transcription factors implement essential changes in gene expression that enable animals to adapt to low oxygen (hypoxia). The stability of the C. elegans HIF-1 protein is controlled by the evolutionarily conserved EGL-9/VHL-1 pathway for oxygen-dependent degradation. Here, we describe vhl-1-independent pathways that attenuate HIF-1 transcriptional activity in C. elegans. First, the expression of HIF-1 target genes is markedly higher in egl-9 mutants than in vhl-1 mutants. We show that HIF-1 protein levels are similar in animals carrying strong loss-of-function mutations in either egl-9 or vhl-1. We conclude that EGL-9 inhibits HIF-1 activity, as well as HIF-1 stability. Second, we identify the rhy-1 gene and show that it acts in a novel negative feedback loop to inhibit expression of HIF-1 target genes. rhy-1 encodes a multi-pass transmembrane protein. Although loss-of-function mutations in rhy-1 cause relatively modest increases in hif-1 mRNA and HIF-1 protein expression, some HIF-1 target genes are expressed at higher levels in rhy-1 mutants than in vhl-1 mutants. Animals lacking both vhl-1 and rhy-1 function have a more severe phenotype than either single mutant. Collectively, these data support models in which RHY-1 and EGL-9 function in VHL-1-independent pathway(s) to repress HIF-1 transcriptional activity.

Alleles↗