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Genetic interactions at the FLA10 locus: suppressors and synthetic phenotypes that affect the cell cycle and flagellar function in Chlamydomonas reinhardtii.

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.

Cell Cycle↗

Genetic interaction of Gsc and Dkk1 in head morphogenesis of the mouse.

Mouse embryos lacking Gsc and Dkk1 function display severe deficiencies in craniofacial structures which are not found in either Dkk1 homozygous null or Gsc homozygous null mutant embryos. Loss of Gsc has a dosage-related effect on the severity of head truncation phenotype in Dkk1 heterozygous embryos. The synergistic effect of these mutations in enhancing head truncation provides direct evidence of a genetic interaction between Gsc and Dkk1, which display overlapping expression in the prechordal mesoderm. In the absence of Gsc activity, the expression of Dkk1, WNT genes and a transgenic reporter for WNT signalling are altered. Our results show that Gsc and Dkk1 functions are non-redundant in the anterior mesendoderm for normal anterior development and Gsc may influence Wnt signalling as a negative regulator.

Journal Article↗

Two domains of the tolloid protein contribute to its unusual genetic interaction with decapentaplegic.

The zygotic dorsal-ventral patterning gene tolloid (tld) exhibits a complex structure consisting of an N-terminal domain with sequence similarity to the astacin family of metalloproteases and a C-terminal domain composed of two EGF-like repeats and five copies of the CUB repeat which was first found in human complement proteins C1r and C1s. The overall structure of TLD is similar to the human bone morphogenetic protein, BMP-1. Previous genetic studies (Ferguson and Anderson, 1992) have shown that certain tld alleles behave antagonistically toward decapentaplegic (dpp), a second zygotic dorsal-ventral patterning gene whose product is most closely related to the TGF-beta superfamily members BMP-2 and BMP-4. The common phenotype and genetic interaction data may suggest a direct physical association between these two proteins. In this study, we use an allelic series of haplo-viable dpp mutations to order the strength of the tld antagonistic interaction. In addition, we have identified the lesions associated with 20 tld mutations by DNA sequencing. These mutations consist of six alleles that behave in an antimorphic manner with respect to dpp, five alleles that revert the antagonistic interaction, and 9 loss-of-function alleles with a range of phenotypes from weak to strong. We find that antimorphic mutations and second-site revertants cluster to the protease domain and to a second site located at the junction of the first two CUB repeats. In contrast, loss-of-function mutations are dispersed throughout the length of the protein. Those mutations that map within the CUB repeats invariably affect consensus residues, thereby establishing the functional significance of certain residues in the CUB repeat consensus sequence. Possible molecular models for TLD action are discussed.

Alleles↗

Hlx is induced by and genetically interacts with T-bet to promote heritable T(H)1 gene induction.

Type 1 helper T (T(H)1) cells are essential for cellular immunity, but their ontogeny, maturation and durability remain poorly understood. By constructing a dominant-negative form of T-bet, we were able to determine the role played by this lineage-inducing trans-activator in the establishment and maintenance of heritable T(H)1 gene expression. Optimal induction of interferon-gamma (IFN-gamma) expression required genetic interaction between T-bet and its target, the homeoprotein Hlx. In fully mature T(H)1 cells, reiteration of IFN-gamma expression and stable chromatin remodeling became relatively independent of T-bet activity and coincided with demethylation of DNA. In contrast, some lineage attributes, such as expression of IL-12R beta 2 (interleukin 12 receptor beta 2), required ongoing T-bet activity in mature T(H)1 cells and their progeny. These findings suggest that heritable states of gene expression might be maintained by continued expression of the inducing factor or by a mechanism that confers a stable imprint of the induced state.

Animals↗

The mouse rib-vertebrae mutation disrupts anterior-posterior somite patterning and genetically interacts with a Delta1 null allele.

Rib-vertebrae (rv) is an autosomal recessive mutation in mouse that affects the morphogenesis of the vertebral column. Axial skeleton defects vary along the anterior-posterior body axis, and include split vertebrae and neural arches, and fusions of adjacent segments. Here, we show that defective somite patterning underlies the vertebral malformations and altered Notch signaling may contribute to the phenotype. Somites in affected regions are irregular in size and shape, epithelial morphology is disrupted, and anterior-posterior somite patterning is abnormal, reminiscent of somite defects obtained in loss-of-function alleles of Notch signaling pathway components. Expression of Dll1, Dll3, Lfng and Notch1 is altered in rv mutant embryos, and rv and Dll1(lacZ), a null allele of the Notch ligand Delta1, genetically interact. Mice double heterozygous for rv and Dll1(lacZ), show vertebral defects, and one copy of Dll1(lacZ) on the homozygous rv background enhances the mutant phenotype and is lethal in the majority of cases. However, fine genetic mapping places rv into an interval on chromosome seven that does not contain a gene encoding a known component of the Notch signaling pathway.

Animals↗

The Caenorhabditis elegans unc-64 locus encodes a syntaxin that interacts genetically with synaptobrevin.

We describe the molecular cloning and characterization of the unc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63-64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those of Drosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.

Amino Acid Sequence↗

Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae.

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.

Alleles↗

Genetic interactions between atm and p53 influence cellular proliferation and irradiation-induced cell cycle checkpoints.

Ataxia-telangiectasia and Li-Fraumeni syndrome, pleiotropic disorders caused by mutations in the genes atm and p53, share a marked increase in cancer rates. A number of studies have argued for an interaction between these two genes (for comprehensive reviews, see M. S. Meyn, Cancer Res., 55: 5991-6001, 1995, and M. F. Lavin and Y. Shiloh, Annu. Rev., Immunol., 15: 177-202, 1996). Specifically, atm is placed upstream of p53 in mediating G1-S cell cycle checkpoint control, and both atm and p53 are believed to influence cellular proliferation. To analyze the genetic interactions of atm and p53, mouse embryonic fibroblasts (MEFs) homozygously deficient for both atm and p53 were used to assess cell cycle and growth control. These double-null fibroblasts proliferate rapidly and fail to exhibit the premature growth arrest seen with atm-null MEFs. MEFs null for both atm and p53 do not express any p21(cipl/wafl), showing that p53 is required for p21(cipl/wafl) expression in an atm-null background. By contrast, homozygous loss of either atm, p53, or both results in similar abnormalities of the irradiation-induced G1-S cell cycle checkpoint. Our results suggest two separate pathways of interaction between atm and p53, one linear, involving G1-S cell cycle control, and another more complex, involving aspects of growth regulation.

Animals↗

The budding yeast cohesin gene SCC1/MCD1/RHC21 genetically interacts with PKA, CDK and APC.

Cohesin is a protein that plays a key role in the cohesion and separation of sister chromatids. During the duplication of chromatids, cohesin holds sister chromatids together until the onset of anaphase, and thereby prevents the premature separation of sister chromatids which would otherwise jeopardize the faithful segregation of chromosomes. To investigate the molecular mechanisms of sister chromatid cohesion, we have isolated multicopy suppressors of a temperature-sensitive (ts) mutation in the SCC1/MCD1/RHC21 gene which encodes a component of the cohesin complex in budding yeast. Isolation of multicopy suppressors of rhc21-sk16 and further genetic analyses revealed that several distinct biological pathways are involved in the regulation of SCC1/MCD1/RHC21 function. Firstly, PDE2 and BCY1, each of which inhibits the activity of protein kinase A (PKA), suppressed the temperature sensitivity of the rhc21-sk16 mutant. Secondly, PDE2 suppressed the temperature sensitivity of the cdc16-1 mutant. These results suggest that SCC1/MCD1/RHC21 is negatively regulated by the PKA pathway via the anaphase promoting complex (APC). Thirdly, ZDS1, a multicopy suppressor of cdc28-1N, and its homologue ZDS2 were isolated as multicopy suppressors of rhc21-sk16. Furthermore, the rhc21-sk16 mutant did not grow in the presence of the cdc28-1N mutation. Hence, SCC1/MCD1/RHC21 is positively regulated by the mitotic CDK, CDC28. Finally, SCC1/MCD1/RHC21 was found to interact genetically with CDC20, an activator of APC. Overexpression of CDC20 suppressed the temperature sensitivity of rhc21-sk16, and rhc21-sk16 was shown to be synthetically lethal with cdc20-1. In addition, the growth of the rhc21-sk16 mutant was inhibited by overproduction of the anaphase inhibitor Pds1p, whose degradation is mediated by Cdc20p in APC-dependent proteolysis. The functional relationships between SCC1/MCD1/RHC21 and PKA, CDK or APC are discussed.

Cdc20 Proteins↗

Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish.

Recent evidence indicates a specific role for vascular endothelial growth factor a (Vegfa) during artery development in both zebrafish and mouse embryos, whereas less is known about signals that govern vein formation. In zebrafish, loss of vegfa blocks segmental artery formation and reduces artery-specific gene expression, whereas veins are largely unaffected. Here, we describe a mutation in the zebrafish vegf receptor-2 homolog, kdra, which eliminates its kinase activity and leads to specific defects in artery development. We further find that Flt4, a receptor for Vegfc, cooperates with Kdr during artery morphogenesis, but not differentiation. We also identify an additional zebrafish vegfr-2 ortholog, referred to as kdrb, which can partially compensate for loss of kdra but is dispensable for vascular development in wild-type embryos. Interestingly, we find that these Vegf receptors are also required for formation of veins but in distinct genetic interactions that differ from those required for artery development. Taken together, our results indicate that formation of arteries and veins in the embryo is governed in part by different Vegf receptor combinations and suggest a genetic mechanism for generating blood vessel diversity during vertebrate development.

Animals↗

Severe epilepsy resulting from genetic interaction between Scn2a and Kcnq2.

A mutation in the voltage-gated sodium-channel Scn2a results in moderate epilepsy in transgenic Scn2a(Q54) mice maintained on a C57BL/6J strain background. The onset of progressive epilepsy begins in adults with short-duration partial seizures that originate in the hippocampus. The underlying abnormality is an increase in persistent sodium current in hippocampal neurons. The voltage-gated potassium channel Kcnq2 is responsible for generating M current (I(KM)) that is thought to control excitability and limit repetitive firing of hippocampal neurons. To determine whether impaired M current would exacerbate the seizure phenotype of Scn2a(Q54) mice, we carried out genetic crosses with two mutant alleles of Kcnq2. Szt1 mice carry a spontaneous deletion that removes the C-terminal domain of Kcnq2. A novel Kcnq2 missense mutation V182M was identified by screening the offspring of ENU-treated males for reduced threshold to electrically evoked minimal clonic seizures. Double mutant mice carrying the Scn2a(Q54) transgene together with either of the Kcnq2 mutations exhibited severe epilepsy with early onset, generalized tonic-clonic seizures and juvenile lethality by 3 weeks of age. This dramatic exacerbation of the sodium-channel mutant phenotype indicates that M current plays a critical role in preventing seizure initiation and spreading in this animal model. The genetic interaction between Scn2a and Kcnq2 demonstrates that combinations of mild alleles of monogenic epilepsy genes can result in severe disease and provides a model for complex inheritance of human epilepsy. The data suggest that interaction between these genes might contribute to the variable expressivity observed in human families with sodium-channel mutations. In a screen of 23 SMEI patients with missense mutations of SCN1A, no second-site mutations in KCNQ2 were identified.

Amino Acid Sequence↗

Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae.

Evidence obtained from recent studies supports the existence of an error-free postreplication repair (PRR) and a mutagenesis pathway within the Saccharomyces cerevisiae RAD6 DNA repair group. The MMS2 gene is the only known yeast gene involved in error-free PRR that, when mutated, significantly increases the spontaneous mutation rate. In this study, the mutational spectrum of the mms2 mutator was determined and compared to the wild type strain. In addition, mutagenenic effects and genetic interactions of the mms2 mutator and rev3 anti-mutator were examined with respect to forward mutations, frameshift reversions as well as amber and ochre suppressions. It was concluded from these results that the mms2 mutator phenotype is largely dependent on the functional REV3 gene. The synergistic effects of mms2 and rev3 mutations towards killing by a variety of DNA-damaging agents ruled out the possibility that MMS2 simply acts to suppress REV3 activity and favored the hypothesis that MMS2 and REV3 form two alternative subpathways within the RAD6 DNA repair pathway. Taken together, we propose that two pathways represented by MMS2 and REV3 deal with a similar range of endogenous and environmental DNA damage but with different biological consequences, namely, error-free repair and mutagenesis, respectively.

Base Sequence↗

Genetic interactions between Drosophila melanogaster menin and Jun/Fos.

Menin is a tumor suppressor required to prevent multiple endocrine neoplasia in humans. Mammalian menin protein is associated with chromatin modifying complexes and has been shown to bind a number of nuclear proteins, including the transcription factor JunD. Menin shows bidirectional effects acting positively on c-Jun and negatively on JunD. We have produced protein null alleles of Drosophila menin (mnn1) and have over expressed the Mnn1 protein. Flies homozygous for protein-null mnn1 alleles are viable and fertile. Localized over-expression of Mnn1 causes defects in thoracic closure, a phenotype that sometimes results from insufficient Jun activity. We observed complex genetic interactions between mnn1 and jun in different developmental settings. Our data support the idea that one function of menin is to modulate Jun activity in a manner dependent on the cellular context.

Alleles↗

Studies on the association of Fc gamma receptor IIA, IIB, IIIA and IIIB polymorphisms with rheumatoid arthritis in the Japanese: evidence for a genetic interaction between HLA-DRB1 and FCGR3A.

We recently detected a new single nucleotide polymorphism of FcgammaRIIB gene, which alters an amino acid within the transmembrane domain from Ile to Thr (I232T), and its association with SLE in the Japanese. This study was performed to examine whether FCGR2B-I232T was associated with susceptibility to rheumatoid arthritis in the Japanese. At the same time, FCGR2A, 3A and 3B polymorphisms were also examined. Genotyping of FCGR2B-I232T, FCGR2A-H131R, FCGR3A-F176V and FCGR3B-NA1/2 polymorphisms were performed using genomic DNA. Association with RA was analyzed in 382 Japanese patients with RA and 303 healthy individuals using a case-control approach. In addition, the same groups of patients and controls were genotyped for HLA-DRB1 to examine possible interaction with FCGR genes. Significantly different distribution of genotype, allele carrier and allele frequencies was not observed between patients with RA and healthy individuals in any of the four polymorphisms. When the subjects were stratified according to the carriage of HLA-DRB1 shared epitope (SE), significant increase of FCGR3A-176F/F genotype was observed in SE positive patients compared with SE positive healthy individuals (P=0.009, P(corr)=0.07). In conclusion, FCGR3A-176F/F genotype was considered to confer risk through genetic interaction with HLA-DRB1 SE.

Adult↗

Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations.

We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.

Genes↗

RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae.

The RGD1 gene was identified during systematic genome sequencing of Saccharomyces cerevisiae. To further understand Rgd1p function, we set up a synthetic lethal screen for genes interacting with RGD1. Study of one lethal mutant made it possible to identify the SLG1 and MID2 genes. The gene SLG1/HCS77/WSC1 was mutated in the original synthetic lethal strain, whereas MID2/SMS1 acted as a monocopy suppressor. The SLG1 gene has been described to be an upstream component in the yeast PKC pathway and encodes a putative cell surface sensor for the activation of cell integrity signalling. First identified by viability loss of shmoos after pheromone exposure, and since found in different genetic screens, MID2 was recently reported as also encoding an upstream activator of the PKC pathway. The RGD1 gene showed genetic interactions with both sensors of cell integrity pathway. The rgd1 slg1 synthetic lethality was rescued by osmotic stabilization, as expected for mutants altered in cell wall integrity. The slight viability defect of rgd1 in minimal medium, which was exacerbated by mid2, was not osmoremediated. As for mutants altered in PKC pathway, the accumulation of small-budded dead cells in slg1, rgd1 and mid2 after heat shock was prevented by 1 M sorbitol. In addition, the rgd1 strain also displayed dead shmoos after pheromone treatment, like mid2. Taken together, the present results indicate close functional links between RGD1, MID2 and SLG1 and suggest that RGD1 and MID2 interact in a cell integrity signalling functionally linked to the PKC pathway.

Calcium-Binding Proteins↗

A novel delta 0 mutation in cis with Hb Knossos: a study of different genetic interactions in three Egyptian families.

We have defined the molecular basis of normal HbA2 beta-thalassaemia associated with Hb Knossos. DNA sequence analysis of the delta globin gene in cis with beta Knossos showed deletion of a single A in codon 59 leading to a premature termination at codon 60. This delta 0/beta Knossos allele has been observed in three unrelated Egyptian families and associated with a single beta haplotype (+----++). One individual who was homozygous for the delta 0/beta Knossos allele as well as heterozygous for a non-deletional alpha thalassaemia, was completely clinically asymptomatic, while others have coinherited the delta 0/beta Knossos allele with different beta and alpha thalassaemia determinants. A study of the different genetic interactions giving rise to a spectrum of clinical phenotypes is reported.

Adult↗

Genetic interaction between the AS1-AS2 and RDR6-SGS3-AGO7 pathways for leaf morphogenesis.

In higher plants, class I KNOTTED1-like homeobox (KNOX) gene suppression and leaf polarity establishment are two processes crucial for leaf morphogenesis. The Arabidopsis genes, ASYMMETRIC LEAVES1 and 2 (AS1 and AS2), are required for repressing the class I KNOX genes and promoting leaf adaxial cell fates. In addition, the RNA-DEPENDENT RNA POLYMERASE6 (RDR6) gene acts synergistically with AS1 and AS2 to specify the adaxial polarity and repress the KNOX genes in leaves. It is known that RDR6 is one of the key components in plant post-transcriptional gene silencing (PTGS), and is likely to function with other silencing components in a genetic pathway in regulating leaf patterning. Here we report phenotypic analyses of double mutants combining as1 or as2 with other mutations relating to different RNA silencing pathways. We show that plants carrying rdr6, suppressor of gene silencing3 (sgs3) or zippy (zip, also called ago7) in combination with as1 or as2 demonstrate severe morphological defects, and the double mutant plants are generally similar to one another. Detailed phenotypic and molecular analyses reveal that leaves of rdr6 as2(1), sgs3 as2(1) and zip as2(1) all show an abnormal adaxial identity, and contain high levels of microRNA165/166 and FILAMENTOUS FLOWER (FIL) transcripts. These results suggest that RDR6, SGS3 and AGO7 act in the same pathway, which genetically interacts with the AS1-AS2 pathway for leaf development. The RDR6-SGS3-AGO7 pathway was previously identified as regulating the plant vegetative phase change. Our results reveal a new function of the pathway, which is also required for normal leaf morphogenesis.

Arabidopsis↗