Increased activity of a model heterologous protein in Saccharomyces cerevisiae strains with reduced vacuolar proteinases

Strains of Saccharomyces cerevisiae with reduced activity of the four major vacuolar proteinases were constructed and used as an expression system for a model heterologous gene product (-galactosidase from Escherichia coli). The vacuolar proteinases were inactivated by mutation within the structural genes encoding proteinase A (PRA1), proteinase B (PRB1), carboxypeptidase Y (PRC1) and carboxypeptidase S (CPS1). Strains were constructed with mutations in one or more of these structural genes. Having constructed the strains, the E. coli -galactosidase (lacZ) gene was introduced by transformation. Batch cultures of each strain were grown and the activity of -galactosidase measured. An assessment of the effect of the loss of specific proteinases on the heterologous gene product was then made. The results indicated that strains with reduced vacuolar proteinase activity showed as much as 173% higher -galactosidase activity than a strain with wild-type proteinase activity carrying the lacZ gene. The most productive strains of all were those with reduced carboxypeptidase activity and/or reduced proteinase A activity. At first sight the inclusion of a pra1 mutation and/or the pra1 and cps1 mutations would appear wortwhile for significantly enhanced expression of a heterologous gene product in yeast. However this conclusion is too simplistic: each heterologous protein will require a host specifically tailored to ensure optimum expression. Correspondence to: J. R. Dickinson     

Evidence for chemically-induced structural gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells

L5178Y mouse lymphoma cells normally appear to possess two functional thymidine kinase alleles (TK^+/+). TK-deficient (TK^?/?) clonal lines can be derived from these cells by treatment with EMS or other mutagens. Mezger-Freed [12] has argued that such stable phenotypic variants do not arise as the result of gene mutations but instead represent epigenetic events such as normally occur during differentiation without any permanent gene alteration. If this be so, then rare TK^+/? revertants arising in TK^?/? cultures should possess TK enzyme identical with one of those present in the original TK^+/+ cells, since only depression of the TK gene is involved. Our studies show that this is not the case.Among the mutant TK enzymes analyzed in vitro (those from parental TK^+/? lines, each derived in turn from separate TK^?/? lines) differences were found in (1) solubility in saline; (2) solubility in3 M LiCl; (3) K_m′s; and (4) ATP-Mg²⁺ requirements. These findings were incompatible with a non-mutational model for the production of these stable variants and, in conjunction with reversion-rate data, they tended to favor either direct structural gene modifications or mutations affecting the expression of adult and fetal enzymes.     

Proteolytic inactivation of the NADP-dependent glutamate dehydrogenase in proteinase-deficient mutants of Saccharomyces cerevisiae

The proteolytic inactivation of NADP-dependent glutamate dehydrogenase (l-glutamate: NADP⁺ oxidoreductase, EC 1.4.1.4) during carbon starvation was studied using several proteinase-deficient mutants of Saccharomyces cerevisiae. Strains bearing mutations in the structural genes for proteinase B, proteinase C (carboxypeptidase Y), or in both genes catalyzed the inactivation and initial proteolytic cleavage of NADP-glutamate dehydrogenase at a rate indistinguishable from that of the wild-type parent strain. In addition, the pleiotropic mutation, pep4-3, which results in a deficiency for proteinases A, B, and C, did not affect the inactivation or initial proteolytic cleavage of NADP-glutamate dehydrogenase.     

Involvement of the PS03 gene of Saccharomyces cerevisiae in intrachromosomal mitotic recombination and gene amplification

Using a genetic system of haploid strains of Saccharomyces cerevisiae carrying a duplication of the his4 region on chromosome III, the pso3-1 mutation was shown to decrease the rate of spontaneous mitotic intrachromosomal recombination 2- to 13-fold. As previously found for the rad52-1 mutant, the pso3-1 mutant is specifically affected in mitotic gene conversion. Moreover, both mutations reduce the frequency of spontaneous recombination. However, the two mutations differ in the extent to which they affect recombination between either proximally or distally located markers on the two his4 heteroalleles. In addition, amplifications of the his4 region were detected in the pso3-1 mutant. We suggest that the appearance of these amplifications is a consequence of the inability of the pso3-1 mutant to perform mitotic gene conversion.     

Hereditary hemochromatosis: An update vision of the laboratory diagnosis

Haemochromatosis (HC) is an inherited disorder of iron metabolism. The 85–90% of Hereditary hemochromatosis cases are caused by mutations in HFE gene (HC type 1). The remaining 10–15% of HC cases are caused by mutations in other non-HFE genes (HJV, HAMP, TRF2, SLC40A1, BMP6). The study of patients for the diagnosis of HC has an important laboratory approached: analysis of biochemical parameters and genetic studies. To confirm a case, it is necessary to carry out a genetic study of the C282Y and H63D mutations. The presence of C282Y mutation in homozygosis is compatible with the diagnosis of HC type 1. Due to the incomplete penetrance of this mutation and the variable phenotypic expression, the severe forms of the disease are relatively rare. The study of variants in non-HFE genes allows more detailed study of both non-classic HC cases and those with more severe clinical expression. The genotype characterization of a patient not always justified the phenotype expression of the symptoms in this disease. All laboratory clinicians must consider recommendation provide by the experts in the Materia.     

Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high α-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum–derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in α-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.     

Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment

Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced.     

PEP4 Gene Function Is Required for Expression of Several Vacuolar Hydrolases in SACCHAROMYCES CEREVISIAE

The pep4-3 mutation results in a 90–95% reduction in the levels of five vacuolar hydrolases in yeast, including proteinases A and B, carboxypeptidase Y, RNase(s) and the repressible alkaline phosphatase. The mutation is without effect on two secreted glycoproteins, on an enzyme of the vacuolar membrane, and on a proteinase located outside of the vacuole. Mutations at the PEP4 locus exhibit a dosage effect on the levels of some, but not all, of the enzymes whose expression requires the function of the gene.     

Positive Regulation in a Eukaryote, a Study of the uaY Gene of ASPERGILLUS NIDULANS: I. Characterization of Alleles, Dominance and Complementation Studies, and a Fine Structure Map of the uaY -oxpA Cluster

In this paper we characterize genetically a positive eukaryotic regulatory gene: the uaY gene of the ascomycete Aspergillus nidulans. Several steps in the uptake and degradation of purines are under the control of the uaY gene (summarized in Scazzocchio and Gorton 1977). In the present paper 12 uaY^- mutations are characterized with respect to their inducibility for adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase and by the absence of the uric acid-xanthine permease scored in vivo by resistance to 2-thiouric acid. While 10 mutations are uniformly unleaky, two others are almost wild type for the induction of urate oxidase. A fine structure map of the uaY gene shows that the two "leaky" mutations are not clustered. The fine structure mapping unambiguously positions six uaY alleles and provides preliminary but interesting trends regarding the pattern of gene conversion in the uaY gene. The enzyme levels in all uaY^-/uaY⁺ heterozygous diploids are intermediate between the corresponding uaY^-/uaY^- and uaY⁺/uaY⁺ homozygous diploids, suggesting that one functional copy of the uaY gene is able to mediate the complete induction of only one set of structural genes. No complementation was found between any two uaY^- alleles. This establishes that the mutations showing either of the phenotypes are alleles in the same gene; it fails to provide evidence for intracistronic complementation. A mutation, oxpA5, causes resistance to the xanthine analogue oxypurinol (4, 6-dihydroxypyrazolo-(3, 4-d)-pyrimidine) and partial constitutivity of adenine deaminase, xanthine dehydrogenase (purine hydroxylase I) and urate oxidase. The constitutive phenotype is suppressed by mutations blocking the synthesis of intracellular inducers. The mutation is recessive and complements fully with the 11 uaY^- mutations tested. It maps to the left of all 12 uaY mutations to which it has been crossed. The data indicate that both the resistance and constitutivity arise from one mutational event in a gene, oxpA, different from uaY and possibly adjacent to it. We propose that the oxpA gene codes for a protein involved in limiting the flow of inducers into the cell nucleus. Thus oxpA and uaY constitute a regulatory gene cluster, indicating that uaY is the regulatory gene.     

Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae

The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature alpha-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30 degrees C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.     

Dystrophic mdx mouse myoblasts exhibit elevated ATP/UTP-evoked metabotropic purinergic responses and alterations in calcium signalling

Pathophysiology of Duchenne Muscular Dystrophy (DMD) is still elusive. Although progressive wasting of muscle fibres is a cause of muscle deterioration, there is a growing body of evidence that the triggering effects of DMD mutation are present at the earlier stage of muscle development and affect myogenic cells. Among these abnormalities, elevated activity of P2X7 receptors and increased store-operated calcium entry myoblasts have been identified in mdx mouse. Here, the metabotropic extracellular ATP/UTP-evoked response has been investigated. Sensitivity to antagonist, effect of gene silencing and cellular localization studies linked these elevated purinergic responses to the increased expression of P2Y2 but not P2Y4 receptors. These alterations have physiological implications as shown by reduced motility of mdx myoblasts upon treatment with P2Y2 agonist. However, the ultimate increase in intracellular calcium in dystrophic cells reflected complex alterations of calcium homeostasis identified in the RNA seq data and with significant modulation confirmed at the protein level, including a decrease of Gq11 subunit α, plasma membrane calcium ATP-ase, inositol-2,4,5-trisphosphate-receptor proteins and elevation of phospholipase Cβ, sarco-endoplamatic reticulum calcium ATP-ase and sodium‑calcium exchanger. In conclusion, whereas specificity of dystrophic myoblast excitation by extracellular nucleotides is determined by particular receptor overexpression, the intensity of such altered response depends on relative activities of downstream calcium regulators that are also affected by Dmd mutations. Furthermore, these phenotypic effects of DMD emerge as early as in undifferentiated muscle. Therefore, the pathogenesis of DMD and the relevance of current therapeutic approaches may need re-evaluation.     

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. II. Genetic Properties of Group II Suppressors

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. One of these groups (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) appears to include a set of informational suppressors in which the vehicle of suppression is glycyl-tRNA. Some of the genetic properties of Group II suppressors are described in this communication.——Corevertants of the Group II frameshift mutations his4–519 and leu2–3 have been characterized to determine the spectrum of reversion events induced by the frameshift mutagen ICR-170. Seventythree ICR-induced corevertants were analyzed. With the exception of one corevertant, which carried an allele of SUF1, all carried alleles of SUF3 or SUF5. SUF1, SUF3, SUF4 and SUF6 were represented among spontaneous and UV-induced corevertants. In the course of these experiments one of the suppressors was mapped. SUF5, the probable structural gene for tRNA^GLY1, is located between ade2 and ade9 on chromosome XV.——SUF1, SUF4 and SUF6 have novel properties and comprise a distinct subset of suppressors. Although these suppressors show no genetic linkage to each other, they share several common features including lethality in haploid pairwise combinations, reduced tRNA^GLY3 isoacceptor activity and increased efficiency of suppression in strains carrying the cytoplasmically inherited [PSI] element. In addition, strains carrying SUF1, SUF4 or SUF6 are phenotypically unstable and give rise to mitotic Suf⁺ segregants at high frequency. These segregants invariably contain a linked, second-site mutation that maps in or adjacent to the suppressor gene itself. Strains carrying any of these suppressors also give rise to mitotic segregants that exhibit enhanced efficiency of suppression; mutations responsible for this phenotype map at two loci, upf1 and upf2. These genes show no genetic linkage to any of the Group II suppressors.——Methods that permit positive selection for mutants with decreased or enhanced efficiency of suppression have been devised in order to examine large numbers of variants. The importance of these interacting mutants is underscored by their potential utility in studying suppressor function at the molecular level.     

The HIV-1 Integrase Mutations Y143C/R Are an Alternative Pathway for Resistance to Raltegravir and Impact the Enzyme Functions

Resistance to HIV-1 integrase (IN) inhibitor raltegravir (RAL), is encoded by mutations in the IN region of the pol gene. The emergence of the N155H mutation was replaced by a pattern including the Y143R/C/H mutations in three patients with anti-HIV treatment failure. Cloning analysis of the IN gene showed an independent selection of the mutations at loci 155 and 143. Characterization of the phenotypic evolution showed that the switch from N155H to Y143C/R was linked to an increase in resistance to RAL. Wild-type (WT) IN and IN with mutations Y143C or Y143R were assayed in vitro in 3′end-processing, strand transfer and concerted integration assays. Activities of mutants were moderately impaired for 3′end-processing and severely affected for strand transfer. Concerted integration assay demonstrated a decrease in mutant activities using an uncleaved substrate. With 3′end-processing assay, IC₅₀ were 0.4 µM, 0.9 µM (FC = 2.25) and 1.2 µM (FC = 3) for WT, IN Y143C and IN Y143R, respectively. An FC of 2 was observed only for IN Y143R in the strand transfer assay. In concerted integration, integrases were less sensitive to RAL than in ST or 3′P but mutants were more resistant to RAL than WT.     

The C-Terminal Domain of Sin1 Interacts with the SWI-SNF Complex in Yeast

In the yeast Saccharomyces cerevisiae, the SWI-SNF complex has been proposed to antagonize the repressive effects of chromatin by disrupting nucleosomes. The SIN genes were identified as suppressors of defects in the SWI-SNF complex, and the SIN1 gene encodes an HMG1-like protein that has been proposed to be a component of chromatin. Specific mutations (sin mutations) in both histone H3 and H4 genes produce the same phenotypic effects as do mutations in the SIN1 gene. In this study, we demonstrate that Sin1 and the H3 and H4 histones interact genetically and that the C terminus of Sin1 physically associates with components of the SWI-SNF complex. In addition, we demonstrate that this interaction is blocked in the full-length Sin1 protein by the N-terminal half of the protein. Based on these and additional results, we propose that Sin1 acts as a regulatable bridge between the SWI-SNF complex and the nucleosome.     

Cell Fate Decisions in Malignant Hematopoiesis: Leukemia Phenotype Is Determined by Distinct Functional Domains of the MN1 Oncogene

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.     

Structure-function defects of the TWINKLE linker region in progressive external ophthalmoplegia

TWINKLE is the helicase at the mitochondrial DNA (mtDNA) replication fork in mammalian cells. Mutations in the PEO1 gene, which encodes TWINKLE, cause autosomal dominant progressive external ophthalmoplegia (AdPEO), a disorder associated with deletions in mtDNA. Here, we characterized seven different AdPEO-causing mutations in the linker region of TWINKLE and we identified distinct molecular phenotypes. For some mutations, protein hexamerization and DNA helicase activity are completely abolished whereas others display more subtle effects. To better understand these distinct phenotypes, we constructed a molecular model of TWINKLE based on the three-dimensional structure of the bacteriophage T7 gene 4 protein. The structural model explains the molecular phenotypes and also predicts the functional consequences of other AdPEO-causing mutations. Our findings provide a molecular platform for further studies in cell- and animal-based model systems and demonstrate that knowledge of the bacteriophage T7 DNA replication machinery may be key to understanding the molecular and phenotypic consequences of mutations in the mtDNA replication apparatus.