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PubMed2003

Leucine-rich repeat-mediated intramolecular interactions in nematode recognition and cell death signaling by the tomato resistance protein Mi.

The root-knot nematode resistance gene Mi from tomato encodes a nucleotide-binding/leucine-rich repeat (NB/LRR) protein with a novel amino-terminal domain compared to related disease-resistance genes. The closely linked paralog Mi-1.1, which does not confer nematode resistance, encodes a protein 91% identical to the functional copy, Mi-1.2. The chimeric construct Mi-DS3, which encodes the 161 amino-terminal residues from Mi-1.1 fused to the remainder of Mi-1.2, induces localized necrosis when transiently expressed in Nicotiana benthamiana leaves. We produced mutant constructs that exchanged sequences encoding each of the 40 amino acid differences from the Mi-1.1 LRR region into Mi-DS3 and into Mi-1.2. For 23 of the substitutions, necrosis was lost upon transient expression of the mutated Mi-DS3 in N. benthamiana, and nematode resistance was lost when the altered Mi-1.2 was expressed in the tomato roots. One substitution, R961D, failed to give Mi-DS3-induced necrosis, but produced a dominant lethal phenotype when introduced into Mi-1.2. This gain-of-function phenotype was suppressed by co-expression with the amino-terminal region of Mi-1.1, suggesting that residue 961 is critical for negative regulation by the corresponding N-terminal region. Substitutions of Mi-1.1 residues 984-986 retained the ability to cause necrosis in Mi-DS3, but resulted in loss-of-nematode resistance in Mi-1.2, suggesting that these residues are essential for nematode recognition. None of the loss-of-function mutations in Mi-1.2 had a dominant negative phenotype. These results indicate that the Mi-1.2 LRR is involved in regulation of the transmission of the resistance response as well as in recognition of the nematode.

Amino Acid Motifs↗
PubMed1994

Heterogeneous AVPR2 gene mutations in congenital nephrogenic diabetes insipidus.

Mutations in the AVPR2 gene encoding the receptor for arginine vasopressin in the kidney (V2 ADHR) have been reported in patients with congenital nephrogenic diabetes insipidus, a predominantly X-linked disorder of water homeostasis. We have used restriction-enzyme analysis and direct DNA sequencing of genomic PCR product to evaluate the AVPR2 gene in 11 unrelated affected males. Each patient has a different DNA sequence variation, and only one matches a previously reported mutation. Cosegregation of the variations with nephrogenic diabetes insipidus was demonstrated for two families, and a de novo mutation was documented in two additional cases. Carrier detection was accomplished in one family. All the variations predict frameshifts, truncations, or nonconservative amino acid substitutions in evolutionarily conserved positions in the V2 ADHR and related receptors. Of interest, a 28-bp deletion is found in one patient, while another, unrelated patient has a tandem duplication of the same 28-bp segment, suggesting that both resulted from the same unusual unequal crossing-over mechanism facilitated by 9-mer direct sequence repeats. Since the V2 ADHR is a member of the seven-transmembrane-domain, G-protein-coupled receptor superfamily, the loss-of-function mutations from this study and others provide important clues to the structure-function relationship of this and related receptors.

Arginine Vasopressin↗
PubMed1998

Diabetes insipidus, diabetes mellitus, optic atrophy and deafness (DIDMOAD) caused by mutations in a novel gene (wolframin) coding for a predicted transmembrane protein.

Wolfram syndrome is an autosomal recessive disorder characterized by juvenile diabetes mellitus, diabetes insipidus, optic atrophy and a number of neurological symptoms including deafness, ataxia and peripheral neuropathy. Mitochondrial DNA deletions have been described in a few patients and a locus has been mapped to 4p16 by linkage analysis. Susceptibility to psychiatric illness is reported to be high in affected individuals and increased in heterozygous carriers in Wolfram syndrome families. We screened four candidate genes in a refined critical linkage interval covered by an unfinished genomic sequence of 600 kb. One of these genes, subsequently named wolframin, codes for a predicted transmembrane protein which was expressed in various tissues, including brain and pancreas, and carried loss-of-function mutations in both alleles in Wolfram syndrome patients.

Adolescent↗
PubMed2003

Attenuation of FLOWERING LOCUS C activity as a mechanism for the evolution of summer-annual flowering behavior in Arabidopsis.

Plant species have evolved a wide variety of flowering habits, each adapted to maximize reproductive success in their local environment. Even within a species, accessions from different environments can exhibit markedly different flowering behavior. In Arabidopsis, some accessions are rapid-cycling summer annuals, whereas others accessions are late flowering and vernalization responsive and thus behave as winter annuals. Two genes, FLOWERING LOCUS C (FLC) and FRIGIDA (FRI), interact synergistically to confer the winter-annual habit. Previous work has shown that many summer-annual accessions contain null mutations in the FRI gene; thus it appears that these summer-annual accessions have arisen from winter-annual ancestors by losing FRI function. In this work we demonstrate that naturally occurring allelic variation in FLC has provided another route to the evolution of summer-annual flowering behavior in Arabidopsis. We have identified two summer-annual accessions, Da (1)-12 and Shakhdara, that contain functional alleles of FRI, but are early flowering because of weak alleles of FLC. We have also determined that the weak allele of FLC found in Landsberg erecta is naturally occurring. Unlike accessions that have arisen because of loss-of-function mutations in FRI, the FLC alleles from Da (1)-12, Shakhdara, and Landsberg erecta are not nulls; however, they exhibit lower steady-state mRNA levels than strong alleles of FLC. Sequence analysis indicates that these weak alleles of FLC have arisen independently at least twice during the course of evolution.

Alleles↗
PubMed2003

Mutations in PRKCSH cause isolated autosomal dominant polycystic liver disease.

Autosomal dominant polycystic liver disease (ADPLD) is a distinct clinical and genetic entity that can occur independently from autosomal dominant polycystic kidney disease (ADPKD). We previously studied two large kindreds and reported localization of a gene for ADPLD to an approximately 8-Mb region, flanked by markers D19S586/D19S583 and D19S593/D19S579, on chromosome 19p13.2-13.1. Expansion of these kindreds and identification of an additional family allowed us to define flanking markers CA267 and CA048 in an approximately 3-Mb region containing >70 candidate genes. We used a combination of denaturing high-performance liquid chromatography (DHPLC) heteroduplex analysis and direct sequencing to screen a panel of 15 unrelated affected individuals for mutations in genes from this interval. We found sequence variations in a known gene, PRKCSH, that were not observed in control individuals, that segregated with the disease haplotype, and that were predicted to be chain-terminating mutations. In contrast to PKD1, PKD2, and PKHD1, PRKCSH encodes a previously described human protein termed "protein kinase C substrate 80K-H" or "noncatalytic beta-subunit of glucosidase II." This protein is highly conserved, is expressed in all tissues tested, and contains a leader sequence, an LDLa domain, two EF-hand domains, and a conserved C-terminal HDEL sequence. Its function may be dependent on calcium binding, and its putative actions include the regulation of N-glycosylation of proteins and signal transduction via fibroblast growth-factor receptor. In light of the focal nature of liver cysts in ADPLD, the apparent loss-of-function mutations in PRKCSH, and the two-hit mechanism operational in dominant polycystic kidney disease, ADPLD may also occur by a two-hit mechanism.

Alternative Splicing↗
PubMed2000

Defects of intergenomic communication: where do we stand?

An expanding number of autosomal diseases has been associated with mitochondrial DNA (mtDNA) depletion and multiple deletions. These disorders have been classified as defects of intergenomic communication because mutations of the nuclear DNA are thought to disrupt the normal cross-talk that regulates the integrity and quantity of mtDNA. In 1989, autosomal dominant progressive external ophthalmoplegia with multiple deletions of mitochondrial DNA was the first of these disorders to be identified. Two years later, mtDNA depletion syndrome was initially reported in infants with severe hepatopathy or myopathy. The causes of these diseases are still unclear, but genetic linkage studies have identified three chromosomal loci for AD-PEO. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder associated with both mtDNA depletion and multiple deletions, is now known to be due to loss-of-function mutations in the gene encoding thymidine phosphorylase. Increased plasma thymidine levels in MNGIE patients suggest that imbalanced nucleoside and nucleotide pools in mitochondria may lead to impaired replication of mtDNA. Future research will certainly lead to the identification of additional genetic causes of intergenomic communication defects and will likely provide insight into the normal "dialogue" between the two genomes.

Cell Nucleus↗
PubMed2006

The Arabidopsis zinc finger-homeodomain genes encode proteins with unique biochemical properties that are coordinately expressed during floral development.

Arabidopsis (Arabidopsis thaliana) contains approximately 100 homeobox genes, many of which have been shown to play critical roles in various developmental processes. Here we characterize the zinc finger-homeodomain (ZF-HD) subfamily of homeobox genes, consisting of 14 members in Arabidopsis. We demonstrate that the HDs of the ZF-HD proteins share some similarities with other known HDs in Arabidopsis, but they contain distinct features that cluster them as a unique class of plant HD-containing proteins. We have carried out mutational analyses to show that the noncanonical residues present in the HDs of this family of proteins are important for function. Yeast (Saccharomyces cerevisiae) two-hybrid matrix analyses of the ZF-HD proteins reveal that these proteins both homo- and heterodimerize, which may contribute to greater selectivity in DNA binding. These assays also show that most of these proteins do not contain an intrinsic activation domain, suggesting that interactions with other factors are required for transcriptional activation. We also show that the family members are all expressed predominantly or exclusively in floral tissue, indicating a likely regulatory role during floral development. Furthermore, we have identified loss-of-function mutations for six of these genes that individually show no obvious phenotype, supporting the idea that the encoded proteins have common roles in floral development. Based on these results, we propose the ZF-HD gene family encodes a group of transcriptional regulators with unique biochemical activities that play overlapping regulatory roles in Arabidopsis floral development.

Arabidopsis↗
PubMed1995

The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential.

We report the cloning and primary structure of the Drosophila insulin receptor gene (inr), functional expression of the predicted polypeptide, and the isolation of mutations in the inr locus. Our data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The INR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin-binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The INR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension which contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage which leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90 and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature INR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation of both types of beta subunits, which in turn allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of INR have different signaling potentials. Finally, we show that loss of function mutations in the inr gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of inr appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are affected by inr mutations.

Amino Acid Sequence↗
PubMed2004

Activation of the hedgehog pathway in advanced prostate cancer.

BACKGROUND: The hedgehog pathway plays a critical role in the development of prostate. However, the role of the hedgehog pathway in prostate cancer is not clear. Prostate cancer is the second most prevalent cause of cancer death in American men. Therefore, identification of novel therapeutic targets for prostate cancer has significant clinical implications. RESULTS: Here we report that activation of the hedgehog pathway occurs frequently in advanced human prostate cancer. We find that high levels of hedgehog target genes, PTCH1 and hedgehog-interacting protein (HIP), are detected in over 70% of prostate tumors with Gleason scores 8-10, but in only 22% of tumors with Gleason scores 3-6. Furthermore, four available metastatic tumors all have high expression of PTCH1 and HIP. To identify the mechanism of the hedgehog signaling activation, we examine expression of Su(Fu) protein, a negative regulator of the hedgehog pathway. We find that Su(Fu) protein is undetectable in 11 of 27 PTCH1 positive tumors, two of them contain somatic loss-of-function mutations of Su(Fu). Furthermore, expression of sonic hedgehog protein is detected in majority of PTCH1 positive tumors (24 out of 27). High levels of hedgehog target genes are also detected in four prostate cancer cell lines (TSU, DU145, LN-Cap and PC3). We demonstrate that inhibition of hedgehog signaling by smoothened antagonist, cyclopamine, suppresses hedgehog signaling, down-regulates cell invasiveness and induces apoptosis. In addition, cancer cells expressing Gli1 under the CMV promoter are resistant to cyclopamine-mediated apoptosis. All these data suggest a significant role of the hedgehog pathway for cellular functions of prostate cancer cells. CONCLUSION: Our data indicate that activation of the hedgehog pathway, through loss of Su(Fu) or overexpression of sonic hedgehog, may involve tumor progression and metastases of prostate cancer. Thus, targeted inhibition of hedgehog signaling may have significant implications of prostate cancer therapeutics.

Gene Expression↗
PubMed1990

Heterozygote effects in dreher mice.

The dreher mutation (gene symbol: dr) is an autosomal recessive mutation located on chromosome 1 of the mouse. Homozygous dreher mice (dr/dr) are ataxic, have a white belly spot, a short-tail, inner ear and skeletal malformations, and a variety of CNS abnormalities. Recently in our dreher colony (the drsst-J allele on a B6C3Fe background), we noticed mice with one or more white belly spots typical of drsst-J/drsst-J mice but which were non-ataxic and had a normal tail length; wild-type mice (+/+) of the same genetic background do not have simialr belly spots. Results of three breeding experiments indicate that a new mutation had not occurred, but rather that the spotted, non-ataxic mice are heterozygous dreher mice (drsst-J/+). Histological examination showed that drsst-J/+ mice have abnormalities in the hippocampal formation that are qualitatively similar to those found in drsst-J/drsst-J mice. Most frequently there is an increase in the number of pyramidal cells in CA3 and a marked thickening of the pyramidal cell layer. In contrast to dreher homozygotes the cerebellum appears to have a normal foliation pattern and no discernible laminar abnormalities. Thus, both breeding experiments and histological examination indicate that drsst-J is semidominant. We speculate that drsst-J is a "loss of function" mutation, but, in any event, the presence of phenotypic abnormalities in drsst-J/+ mice may be useful in identifying the primary developmental defect in dreher mice.

Animals↗
PubMed1996

Minimal region of loss at 13q14 in B-cell chronic lymphocytic leukemia.

Allelic loss at nonrandom chromosomal sites is thought to mark the position of tumor suppressor genes involved in the pathogenesis and progression of human malignancies. Solid tumors in particular have been found to harbor multiple genetic changes resulting in loss of function mutations. Tumor suppressor genes have also been found to be involved in the progression of lymphoid tumors. Previous reports have suggested the involvement of a tumor suppressor gene located on the long arm of chromosome 13, between the retinoblastoma (RB) and D13S25 loci, in the pathogenesis and or progression of more than 40% of B-cell chronic lymphocytic leukemia (B-CLL), a common lymphoid malignancy whose molecular etiology remains largely unknown. In the present study, we report the construction and characterization of a YAC contig spanning a region of approximately 3 cM between the RB gene and the D13S31 locus. We also screened 60 paired normal/tumor B-CLL samples for allelic loss on chromosome 13 with nine microsatellite markers located between RB and D13S25. This analysis has allowed us to narrow the smallest region of loss to a segment of 550 kb located between the 206XF12 and D13S25 markers.

Alleles↗
PubMed1999

Molecular basis of autosomal dominant polycystic kidney disease.

Recent studies have identified the genes mutated in the two major forms of autosomal dominant polycystic kidney disease, PKD1 and PKD2. The PKD1 gene product is likely to be a very large membrane-associated glycoprotein that functions as a receptor for cell-cell or cell-matrix interactions. PKD2 has significant homology to the family of voltage-activated calcium channels. Both proteins are expressed in the developing kidney and appear to have an overlapping pattern of expression. Several studies suggest that the gene products are interacting partners of a signaling pathway. Studies of human tissue suggest a two-hit genetic mechanism is responsible for both forms of the disease. Consistent with this hypothesis, murine models engineered with loss-of-function mutations of Pkd1 or Pkd2 develop cystic disease in the homozygous state. In these animals, renal development proceeds normally through day 15, at approximately which time renal cysts begin to form. The studies suggest an essential role for the PKD proteins in regulating later stages of tubular maturation. The animal models will be useful resources for defining the pathogenesis of autosomal dominant polycystic kidney disease and testing various therapeutic interventions. The two-hit model has potentially important clinical implications.

Animals↗
PubMed1994

Tumor suppressor genes and signal transduction in Drosophila.

Several studies in Drosophila have revealed the existence of loss-of-function mutations that lead to overproliferation of epithelial tissues. The molecular analysis of these tumor suppressor genes has provided important clues in the search for signalling mechanisms controlling growth. We discuss here two genes that act to control epithelial cell proliferation. Mutations in the fat locus cause hyperplastic imaginal disc overgrowth, in which the imaginal disc cells retain their epithelial structure, junctional complexes, and ability to differentiate. However, mutations in fat also cause defective cell-cell adhesion, as indicated by the separation of epithelial vesicles from the epithelial sheet in the mutant tissue. The fat gene encodes a giant relative of cadherins, providing the first evidence that this class of junction-associated molecules can act to regulate growth. Loss of the discs large (dlg) gene product results in neoplastic overgrowth of imaginal discs, causes the epithelial cells to lose apical/basal polarity, and prevents them from differentiating. The gene encodes a protein made up of a series of peptide motifs, including an SH3 and a potential enzymatic domain, a region homologous to guanylate kinases. The presence of these domains suggests a role for Dlg in guanine nucleotide-mediated signal transduction. The Dlg protein is restricted to a specific apical junctional complex in Drosophila epithelial cells, the septate junction. It is the first identified member of a new family of junction-associated proteins that includes ZO-1 and ZO-2, major components of vertebrate tight junctions. These results suggest that junctional contacts could play previously unrecognized roles in regulating the growth of epithelial tissues and that ZO-1 and/or ZO-2 may act as tumor suppressor proteins in humans.

Amino Acid Sequence↗
PubMed1990

The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger.

The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans has been sequenced and its transcript mapped and orientated. A single ORF can encode a protein of 719 amino acids. A 52 amino acid region including a putative 'zinc finger' strongly resembles putative DNA binding regions of the major regulatory protein of erythroid cells. The derived protein sequence also contains a highly acidic region possibly involved in gene activation and 22 copies of the motif S(T)PXX, abundant in DNA binding proteins. Analysis of chromosomal rearrangements and transformation with deletion clones identified 342 N-terminal and 124 C-terminal residues as inessential and localized a C-terminal region required for nitrogen metabolite repressibility. A -1 frameshift eliminating the inessential 122 C-terminal amino acids is a surprising loss-of-function mutation. Extraordinary basicity of the replacement C terminus might explain its phenotype. Mutant sequencing also identified a polypeptide chain termination and several missense mutations, but most interesting are sequence changes associated with specificity mutations. A mutation elevating expression of some structural genes under areA control whilst reducing or not affecting expression of others is a leucine to valine change in the zinc finger loop. It reverts to a partly reciprocal phenotype by replacing the mutant valine by methionine.

Amino Acid Sequence↗
PubMed1999

An essential role for the Drosophila Pax2 homolog in the differentiation of adult sensory organs.

The adult peripheral nervous system of Drosophila includes a complex array of mechanosensory organs (bristles) that cover much of the body surface of the fly. The four cells (shaft, socket, sheath, and neuron) which compose each of these organs adopt distinct fates as a result of cell-cell signaling via the Notch (N) pathway. However, the specific mechanisms by which these cells execute their conferred fates are not well understood. Here we show that D-Pax2, the Drosophila homolog of the vertebrate Pax2 gene, has an essential role in the differentiation of the shaft cell. In flies bearing strong loss-of-function mutations in the shaven function of D-Pax2, shaft structures specifically fail to develop. Consistent with this, we find that D-Pax2 protein is expressed in all cells of the bristle lineage during the mitotic (cell fate specification) phase of bristle development, but becomes sharply restricted to the shaft and sheath cells in the post-mitotic (differentiative) phase. Two lines of evidence described here indicate that D-Pax2 expression and function is at least in part downstream of cell fate specification mechanisms such as N signaling. First, we find that the lack of late D-Pax2 expression in the socket cell (the sister of the shaft cell) is controlled by N pathway activity; second, we find that loss of D-Pax2 function is epistatic to the socket-to-shaft cell fate transformation caused by reduced N signaling. Finally, we show that misexpression of D-Pax2 is sufficient to induce the production of ectopic shaft structures. From these results, we propose that D-Pax2 is a high-level transcriptional regulator of the shaft cell differentiation program, and acts downstream of the N signaling pathway as a specific link between cell fate determination and cell differentiation in the bristle lineage.

Alleles↗
PubMed2004

Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability.

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant loss-of-function mutations in SERCA indicates novel intra- and intermolecular interactions for SERCA in vivo, overlooked by current structural models.

Animals↗
PubMed1992

Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells.

Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.

Animals↗
PubMed2001

The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae.

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4alpha-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4alpha-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719-2727). Studies presented here have shown that this sphingolipid-dependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.

Carboxy-Lyases↗