Heterozygous Deletion Mutants Research Articles

Chemical-Genetic Approaches for Exploring Mode of Action of Antifungal Compounds in the Fungal Pathogen Candida albicans.

Candida albicans is a prevalent fungal pathogen of humans that can cause both superficial and life-threatening disease, primarily in immunocompromised populations. Currently, antifungal drug classes available to treat fungal infections remain limited and the emergence of drug-resistant strains threatens antifungal efficacy, necessitating the discovery and development of additional therapeutics. The construction of the C. albicans double-barcoded heterozygous deletion collection (DBC) enables the rapid and systematic assessment of haploinsufficiency phenotypes in a pooled format. Specifically, this functional genomics resource can be used to identify heterozygous deletion mutants that are hypersensitive to compounds in order to define putative cellular targets and/or other modifiers of compound activity. Here, we describe protocols to characterize the mode of action of small molecules using the C. albicans DBC, including how to prepare compound-treated cultures, isolate genomic DNA, amplify strain-specific barcodes, and prepare DNA libraries for high-throughput sequencing. This technique provides a powerful approach to elucidate the compound mechanism of action in order to bolster the antifungal pipeline.

S3.4a The repurposing approach identifies pitavastatin (calcium) making fluconazole fungicidal by inhibiting ergosterol synthesis

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMObjectivesMaking fluconazole (FLC) fungicidal in combination with adjuvants is a promising strategy to avoid the emergence of FLC resistance and eliminate the persistence and recurrence of fungal infections. To address this question, we combined in vitro screening of a library of FDA-approved drugs to identify compounds for making FLC fungicidal.MethodsWe performed a high-throughput screen of an FDA-approved compound library (HY-L022, MCE®), which contains 2372 drugs, to identify potentially novel FLC synergistic lethal adjuvants using broth microdilution and dose-matrix titration assays. The abilities of candidate drugs to turn FLC from fungistatic to fungicidal were further investigated by FLC disk diffusion assays carried out by four tested strains with different FLC tolerance levels (SC5314, SN152, cmp1-aid∆/cmp1-aid∆, and ADH1p-UPC2 strains). We determined the median lethal dose (LD50) of Candidate compounds by the Up-and-Down procedure (UDP) (OECD 425, 2008) via the intraperitoneal route in adult mice and used cyclosporine A and geldanamycin as control drugs to screen FLC synergistic lethal adjuvants with lower toxicity. Finally, we constructed heterozygous deletion mutants for ergosterol synthesis-related genes to identify the mechanism of action of the synergistic lethality of pitavastatin (calcium) (PIT) and FLC (Fig. 1a).ResultsWe found that 200 compounds (≤100 μm) could make FLC (4 μg/ml) fungicidal and further confirmed that 30 compounds turned FLC (4 μg/ml) from fungistatic to fungicidal at a concentration lower than 12.5 μm by broth microdilution assays (Fig. 1b). We further identified that 12/30 compounds (≤3.125 μm) can make FLC fungicidal (≤4 μg/ml) using dose-matrix titration assays. Among these compounds, PIT can make FLC fungicidal at as low as 0.78 μm (Fig. 1c). In the FLC disk diffusion assay, we identified 8 compounds (5 μm) that were superior to or equivalent to the abilities of the control drugs to eliminate the FLC tolerance of four tested strains. It was worth noting that PIT could make FLC fungicidal against all four tested strains (Fig. 1d). The LD50 value of PIT is 103.6 mg/kg and the highest of the tested compounds. Spot assay results showed feeding exogenous 100 μm ergosterol counteracted the antifungal activity of PIT (16 μm) (Fig. 2a), but did not restore the growth defect of Tet-HMG1/hmg1∆ mutant, in which the HMG1 gene expression would be inhibited by tetracycline (Fig. 2b). This paradoxical phenotype suggests that Hmg1 is unlikely to be a target of PIT. We found four heterozygous deletion mutants increased susceptibility to PIT (Fig. 2c) and the growth defect of ERG8/erg8∆ and IDI1/idi1∆ mutants exposed to PIT can be restored by ergosterol (Fig. 2d), suggesting that Erg8 and Idi1 are potential targets of PIT.ConclusionsOur study demonstrates that PIT has a better synergistic lethal effect with FLC by inhibiting ergosterol synthesis and higher safety and suggests that PIT may be repurposed as a promising adjuvant to make FLC fungicidal and enhance the efficacy of FLC in treating invasive fungal infections caused by pathogenic fungi with high FLC tolerance.

Construction of Double Heterozygous Deletion Strains for Complex Haploinsufficiency-Based Genetic Analysis in Candida albicans.

Complex haploinsufficiency refers to the genetic interaction that occurs in strains with heterozygous mutations at two different loci (a double heterozygous deletion mutant). Double heterozygous deletion mutants can be used to identify gene partners that act within the same pathway or to determine expression-dependent genetic interactions that result in phenotypic changes outside of what would be expected based on the phenotypes of the single heterozygous deletion mutants. The approach outlined here uses a lithium acetate transformation method on a parental "query" strain to introduce a transcription factor deletion DNA construct that is derived from the Homann et al. Candida albicans transcription factor deletion library (Homann et al. PLoS Genet 5(12):e1000783, 2009). We also outline the steps to confirming the genotype of the resulting transformants as well as an example of the use of double heterozygous deletion mutants for complex haploinsufficiency analysis of biofilm formation.

An FYVE-Domain-Containing Protein, PsFP1, Is Involved in Vegetative Growth, Oxidative Stress Response and Virulence of Phytophthora sojae.

Proteins that contain the FYVE zinc-finger domain are recruited to PtdIns3P-containing membranes, participating in numerous biological processes such as membrane trafficking, cytoskeletal regulation, and receptor signaling. However, the genome-wide distribution, evolution, and biological functions of FYVE-containing proteins are rarely reported for oomycetes. By genome mining of Phytophthora sojae, two proteins (PsFP1 and PsFP2) with a combination of the FYVE domain and the PX domain (a major phosphoinositide binding module) were found. To clarify the functions of PsFP1 and PsFP2, the CRISPR/Cas9-mediated gene replacement system was used to knock out the two genes respectively. Only heterozygous deletion mutants of PsFP1 were recovered, and the expression level of PsFP1 in the heterozygous knockout transformants was significantly down-regulated. These PsFP1 mutants showed a decrease in mycelial growth and pathogenicity and were more sensitive to hydrogen peroxide. These phenotypes were recovered to the level of wild-type by overexpression PsFP1 gene in the PsFP1 heterozygous knockout transformant. In contrast, deletion of PsFP2 had no significant effect on vegetative growth, asexual and sexual reproduction, pathogenicity, or oxidative stress sensitivity. PsFP1 was primarily localized in vesicle-like structures and both the FYVE and PX domains are important for its localization. Overall, our results indicate that PsFP1 plays an important role in the vegetative growth and virulence of P. sojae.

RUNX1 Mutations Identify an Entity of Blast Phase Chronic Myeloid Leukemia (BP-CML) Patients with Distinct Phenotype, Transcriptional Profile and Drug Vulnerabilities

Introduction: Genetic alterations of RUNX1 gene (mutations and fusion genes) are common in hematological malignancies including AML, ALL, MDS, and MPN. Mutations of RUNX1 occur in 10% of newly diagnosed AML patients and associate with inferior prognosis. Similarly, point mutations in RUNX1 are frequent in BP-CML, but little is known about their role in BP patients.Methods: Bone marrow samples from four BP patients with RUNX1 mutations were collected from Helsinki University Hospital, and samples from four other BP patients without RUNX1 mutations were used as controls. Samples were analyzed by exome and RNA sequencing. For drug sensitivity and resistance testing (DSRT), a high-throughput platform comprising 195 approved and investigational oncology drugs was used. To further study the role of RUNX1 mutations in BP-CML, Baf3/BCR-ABL1+ RUNX1-/- knock-out and RUNX1-/mut heterozygous deletion mutant models were created using CRISPR-CAS9 technology. The effects of the mutations in the cell line models were characterized by flow cytometry, RNA sequencing, and DSRT analyses.Results: The median age of RUNX1-mutated BP patients was 47 years (range 36-56 years) and included 3 patients with myeloid-BP and 1 with lymphoid-BP phenotype. RUNX1-mutated patients tended to have higher mutational load and co-occurrence with mutations to the BCOR, EZH2 and PHF6 genes in agreement with the mutational profile of RUNX1-mutated AML. Signature analysis of RUNX1-mutated BP patients showed dominance of signature 1 (age-related), signature 6, and 15 (DNA mismatch repair) correlating with the control group signature profiles. In addition, the RUNX1 mutated group showed specific enrichment of signature 9, related to somatic hypermutation and AID/RAG axis activity, that was not observed in the control group. RNA sequencing revealed that 388 genes were significantly differentially expressed between RUNX1-mutated and control BP patients (139 upregulated, 249 downregulated). Interestingly, the antigen processing and presentation pathway was among the top upregulated pathways in RUNX1 mutated patients with overexpression of CIITA and HLA-DR genes. Stem cell differentiation, complement, and hemostasis pathways were enriched in the downregulated gene set. Lymphoid-specific transcription factors (DNTT, PAX5 and VPERB1) in addition to CD19 and CD25 were significantly upregulated in the RUNX1-mutated group. DSRT data showed greater sensitivity to glucocorticoids, MEK-, mTOR- and VEGFR-inhibitors in RUNX1-mutated patients compared to the control group. Data from RUNX1-/- and RUNX1-/mut cell line models confirmed the induced expression of CD19 surface marker with RUNX1 mutation. DSRT results showed increased activity of MEK inhibitor and mTOR inhibitors in RUNX1-/- knock-out and RUNX1-/mut cell lines.Conclusions:RUNX1 mutations in BP-CML patients associate with distinct phenotypic and transcriptional features. DSRT results together with transcriptional data support the potential benefits of glucocorticoids and MEK and mTOR inhibitors as well as immunotherapy targeting CD19 and CD25 in this group of BP-CML patients. DisclosuresKankainen:Medix Biochemica: Consultancy. Heckman:Celgene: Research Funding; Orion Pharma: Research Funding; Novartis: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria.

Altered synaptic transmission and maturation of hippocampal CA1 neurons in a mouse model of human chr16p11.2 microdeletion.

The pathophysiology of neurodevelopmental disorders is often observed early in infancy and toddlerhood. Mouse models of syndromic disorders have provided insight regarding mechanisms of action, but most studies have focused on characterization in juveniles and adults. Insight into developmental trajectories, particularly those related to circuit and synaptic function, will likely yield important information regarding disorder pathogenesis that leads to symptom progression. Chromosome 16p11.2 microdeletion is one of the most common copy number variations associated with a spectrum of neurodevelopmental disorders. Yet, how haploinsufficiency of chr16p11.2 affects early synaptic maturation and function is unknown. To address this knowledge gap, the present study focused on three key components of circuit formation and function, basal synaptic transmission, local circuit function, and maturation of glutamatergic synapses, in developing hippocampal CA1 neurons in a chr16p11.2 microdeletion mouse model. The data demonstrate increased excitability, imbalance in excitation and inhibition, and accelerated maturation of glutamatergic synapses in heterozygous deletion mutant CA1 neurons. Given the critical role of early synaptic development in shaping neuronal connectivity and circuitry formation, these newly identified synaptic abnormalities in chr16p11.2 microdeletion mice may contribute to altered developmental trajectory and function of the developing brain. NEW & NOTEWORTHY The synaptic pathophysiology underlying neurodevelopmental disorders often emerges during infancy and toddlerhood. Therefore, identifying initial changes in synaptic function is crucial for gaining a mechanistic understanding of the pathophysiology, which ultimately will facilitate the design of early interventions. Here, we investigated synaptic and local circuit properties of hippocampal CA1 neurons in a human chr16p11.2 microdeletion mouse model during early postnatal development (preweaning). The data demonstrate increased neuronal excitability, excitatory/inhibitory imbalance, and accelerated maturation of glutamatergic synapses. These perturbations in early hippocampal circuit function may underlie the early pathogenesis of the heterozygous chr16p11.2 microdeletion, which is often associated with epilepsy and intellectual disability.

Abstract 242: Functional Characterization of a Cis-eQTL Locus for Plasma Cholesterol Using CRISPR/Cas Genome Editing in Human Pluripotent Stem Cells

Genome-wide association studies (GWASs) have discovered many novel genetic loci linked to serum lipid levels, yet pinpointing the causal variants remains a major challenge. Expression quantitative trait locus (eQTL) analysis of liver and adipose indicates that many lipid-associated variants influence gene expression in a cis-regulatory manner. To identify causal variants at 57 lipid-associated eQTL loci, we performed a massively parallel reporter assay (MPRA) in which the genomic region surrounding each candidate SNP was coupled to a reporter gene with a unique barcode identifier. This construct pool was transfected into 3T3-L1 adipocytes, and the copy number of each expressed barcode determined by RNAseq. Variants were prioritized according to allele-specific regulatory activity. The top-ranked variant, rs2277862, is associated with total cholesterol and is flanked by the ERGIC3, CPNE1, and CEP250 genes, none of which have previous connections to lipid metabolism. We hypothesized that rs2277862 is causal for the eQTL at the 20q11 locus, and that it lies within a transcriptional regulatory site to influence local gene expression. We identified human pluripotent stem cell (hPSC) lines with different genotypes at rs2277862 and, using CRISPR/Cas genome-editing technology, either deleted the putative regulatory site encompassing rs2277862 or knocked in the alternate SNP allele. Analysis of undifferentiated rs2277862 homozygous major deletion mutants revealed diminished expression of ERGIC3, CPNE1, and CEP250; conversely, heterozygous deletion mutants had increased expression of the same three genes. Our results suggest that in hPSCs the major and minor alleles of rs2277862 have opposing effects on gene expression. In ongoing work, we are validating these findings in differentiated adipocytes and seek to identify the factor(s) that binds the putative regulatory site to mediate allele-specific gene expression. We anticipate that this work will offer fresh insight into the mechanisms by which GWAS-implicated SNPs influence expression of causal genes for lipid metabolism and, more broadly, establish a novel methodology for finding causal variants for any phenotype of interest.

Comprehensive analysis of genes involved in the oxidative stress tolerance using yeast heterozygous deletion collection

In Saccharomyces cerevisiae, oxidative stress plays an inhibitory role during industrial fermentation. Although previous reports have identified genes required for oxidative stress tolerance, employing the yeast genome-wide screening, these screenings used a homozygous mutant collection which did not include the essential genes whose deletions result in lethality. Here, we report a truly genome-wide screening for the genes required for oxidative stress tolerance, using a heterozygous mutant collection which includes both essential and nonessential genes. Approximately 6300 heterozygous deletion mutants were grown in the presence or absence of H2 O2 . The screening identified a total of 331 genes whose heterozygotes conferred hypersensitivity to H2 O2 , indicating that these genes are required for oxidative stress tolerance. Notably, among these genes, 71 were essential genes. We classified these 71 essential genes based on localization, indicating that the localization of gene products from these essential genes was enriched in the nucleus and nucleolus. Classification of these essential genes based on functional categorizations showed that rRNA synthesis and tRNA synthesis were over-represented, suggesting that nuclear function, such as RNA synthesis, is important in the response to oxidative stress. These results provide a helpful resource for the understanding of the molecular basis of oxidative stress-tolerant mechanisms.

The Natural Anticancer Agent Plumbagin Induces Potent Cytotoxicity in MCF-7 Human Breast Cancer Cells by Inhibiting a PI-5 Kinase for ROS Generation

Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast Schizosaccharomyces pombe, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI50; 10 µM) and induced cell elongation and septum formation in wild-type S. pombe. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in S. pombe. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in S. pombe deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.

P101. Cleft palate in compound heterozygote of sonic hedgehog and MFCS4

Although cleft palate is the most common birth defect of human, detailed molecular mechanism in it is not fully elucidated. Palate development is achieved through descending, reorientation and fusion of palatal shelves in association with neighboring organs including mandible and tongue. Sonic hedgehog (Shh) plays essential roles in midline formation and development of many organs such as central nerve system, limb and secondary palate. During palate development, expression of Shh is in the epithelia of tongue, pharynx and rugae. MFCS4 is one of the cis-regulatory sequences for Shh and it is exclusively activated in the epithelium of pharynx from E10.5 to E15.5. Although heterozygous deletion mutants of Shh (Shh+/−) or MFCS4 (MFCS4+/−) do not show cleft palate, compound heterozygotes that yield both Shh+/− and MFCS4+/− (Shh+/− MFCS4−/+) exhibit cleft palate with full penetrance. By analyzing this compound heterozygote, we aimed to clarify the function of Shh in palate development. Arrested development of palatal shelves of Shh+/− MFCS4−/+ in reorientation phase was found by histological observation. The failure of palatal shelves reorientation was rescued by rotary organ culture of Shh+/− MFCS4−/+ maxillae, suggesting the development of the tongue is disrupted. In situ hybridization for myogenic differentiation 1 (MyoD1) showed that the development of intrinsic and extrinsic lingual muscles was hypoplastic and orientation of genioglossus muscle was abnormal. These alterations of the tongue muscle development corresponded to expanded expression of stromal cell-derived factor 1 (Sdf-1) that recruits CXC chemokine receptor 4 (Cxcr4)-positive migrating myoblasts. These findings suggest that because myoblasts fail in proliferation, condensation and subsequent differentiation due to expanded and disrupted signaling between Sdf-1 and corresponding receptor Cxcr4, movement of lingual muscles is insufficient, which blocks palatal shelf reorientation. In human MFCS4, we have identified single nucleotide polymorphisms that are specific for the cleft palate population, further suggesting the involvement of Shh regulated by MFCS4 in palate development.

An integrated platform of genomic assays reveals small-molecule bioactivities

Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.

Genome-Wide Fitness Test and Mechanism-of-Action Studies of Inhibitory Compounds in Candida albicans

Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections. Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis. Here, we exploit the diploid status of C. albicans by constructing a library of 2,868 heterozygous deletion mutants and screening this collection using 35 known or novel compounds to survey chemically induced haploinsufficiency in the pathogen. In this reverse genetic assay termed the fitness test, genes related to the mechanism of action of the probe compounds are clearly identified, supporting their functional roles and genetic interactions. In this report, chemical–genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms. We also demonstrate how chemically induced haploinsufficiency profiles can be used to identify the mechanism of action of novel antifungal agents, thereby illustrating the potential utility of this approach to antifungal drug discovery.

P2-030: Elucidating function and proteolytic properties of families of aspartic intramembrane proteases

Regulated intramembrane proteolysis (RIP) is a key mechanism in the pathogenesis of Alzheimer's Disease (AD). Presenilins are putative aspartic proteases required for the RIP cleavage of Amyloid Precursor Protein and other type I proteins including Notch receptors that are critical in early development. We and others previously found a novel family of genes related to presenilins and termed IMPAS (or IMP/SPP/SPPL/PSH). The aim of this study was to elucidate functional properties of IMPAS proteins. We applied mammalian cell culture proteolytic assays and soil nematode C.elegans model. Three IMPAS genes in C.elegans (Ce–imp–1, Ce–imp–2, Ce–imp–3) and five paralogous human genes genes (IMP1–IMP5) were cloned and studied. Based on original alignment comparisons we predicted that human hIMP1/SPP and Ce–imp–2 are structural and functional orthologs. Inhibition of Ce–imp–2 gene by dsRNAi or in knock–out mutant strains resulted in slow growth, uncoordinated movement, reduced brood size and larval death due to the incomplete shedding of cuticle (ICS) in the animals. The progeny of heterozygous deletion mutant Ce–imp–2 was analyzed by single worm PCR. The data demonstrated that homozygous mutants showed development pathology of late embryo death and ISC defects that is unrelated to Notch defects described for presenilin (sel–12, hop–1) mutants. This specific phenotype mimics features observed in worms with low function of megalin gene or on cholesterol diet. Inhibition of other IMPAS homologues in C.elegans (Ce–imp–1, Ce–imp–3) did not result in any obvious phenotype. We revealed that in co–expressing experiments with PS1 both human IMP1 and Ce–imp–2 were capable to cleave C–terminal part of PS1 in transmembrane domain. The data indicate that Ce–imp–2 is likely orthologous gene to hIMP1/SPP with similar proteolytic functions. The IMP1/Ce–imp–2–induced and PS1–induced proteolytic cleavage assays were used to delineate essential domains and amino–acid signatures important for enzymatic or conformation properties critical for proteolytic functions of two types of intramembrane proteases (IMPAS and presenilin families).

Disruption of the Candida albicans TPS2 gene encoding trehalose-6-phosphate phosphatase decreases infectivity without affecting hypha formation.

Deletion of trehalose-6-phosphate phosphatase, encoded by TPS2, in Saccharomyces cerevisiae results in accumulation of trehalose-6-phosphate (Tre6P) instead of trehalose under stress conditions. Since trehalose is an important stress protectant and Tre6P accumulation is toxic, we have investigated whether Tre6P phosphatase could be a useful target for antifungals in Candida albicans. We have cloned the C. albicans TPS2 (CaTPS2) gene and constructed heterozygous and homozygous deletion strains. As in S. cerevisiae, complete inactivation of Tre6P phosphatase in C. albicans results in 50-fold hyperaccumulation of Tre6P, thermosensitivity, and rapid death of the cells after a few hours at 44 degrees C. As opposed to inactivation of Tre6P synthase by deletion of CaTPS1, deletion of CaTPS2 does not affect hypha formation on a solid glucose-containing medium. In spite of this, virulence of the homozygous deletion mutant is strongly reduced in a mouse model of systemic infection. The pathogenicity of the heterozygous deletion mutant is similar to that of the wild-type strain. CaTPS2 is a new example of a gene not required for growth under standard conditions but required for pathogenicity in a host. Our results suggest that Tre6P phosphatase may serve as a potential target for antifungal drugs. Neither Tre6P phosphatase nor its substrate is present in mammals, and assay of the enzymes is simple and easily automated for high-throughput screening.

Functional analysis of six ORFs from Saccharomyces cerevisiae chromosome IV: two-spored asci produced by disruptant of YDR027c and strain-dependent DNA heterogeneity around YDR036c.

Six S. cerevisiae FY1679 heterozygous deletion mutants were made by replacing six open reading frames (ORFs) of the chromosome IV right arm with kanMX4 selection marker. Haploid and homozygous diploid deletion mutants were obtained from sporulation, dissection and mating experiments. No essential genes were found. The basic phenotypic analysis showed that the haploid and homozygous deletants for the ORF YDR027c (LUV1, VSP54 or RKI1) grew slowly. The diploid homozygous deletants for this ORF had a low frequency of sporulation. They produced asci with no more than one or two haploid spores and the majority of these spores formed were not viable. The deletion of the other ORFs, YDR022c (CIS1), YDR030c (RAD28), YDR032c (PST2), YDR033w (MRH1) and YDR036c, did not change the phenotypes tested in strain FY1679 or the first four ORFs in strain CEN.PK2. This work showed some differences in the DNA sequences between FY1679 and CEN.PK2: the regions immediately 1 kb upstream from YDR036c in these two strains are too different to hybridize properly, preventing deletion of YDR036c in the CEN.PK2 background by recombination with a disruption cassette designed for FY1679. In addition, there are different sets of transposable elements on the other side of the ORF, the differences starting at about 3.5 kb downstream from YDR036c.

Dissection of the D-region of the human major histocompatibility complex by means of induced mutations in a lymphoblastoid cell line

This paper describes part of a mutagenic dissection of the human D-region. Twenty-six human lymphoblastoid cell mutants that had lost expressions of HLA-DR were created with a two-step procedure: (i) A mutant from which one entire haplotype had been physically deleted by γ-rays was isolated by means of immunoselection against cells expressing a specific HLA-B antigen (ii) This heterozygous deletion mutant was irradiated with γ-rays or treated with ICR 191. A frameshift mutagen, and mutants that no longer expressed the remaining DR1 antigen were selected with a monoclonal antibody directed against a monomorphic DR determinant. Monoclonal antibody GENOX 3.53 was used to show that four of the γ-ray induced DR-null mutants did not express the cis-linked MB1/MT1 locus. Since MB1/MT1 was still expressed in the other 16 γ-ray induced and 6 ICR 191-induced DR-null mutants, the separate loss of expression of MB1/MT1 and DR1 is strong evidence that the DR1 and MB1/MT1 alloantigens are under separate genetic control in the cells we used. Since DR-null mutants bound SB2-specific monoclonal antibody ILR1, whether or not they expressed MB1/MT1, the results mean that γ-rays resolved the genetic determinants for DR1, MB1/MT1, and SB2. Additional complexity of determinants encoded by D-region genes is indicated by the following results. The amount of MB1/MT1 antigen that was detected with ELISA tests for binding of GENOX 3.53 antibody to cells varied inversely with the number of expressed copies of DR or of a locus near DR. This could result from an increased amount of MB1/MT1 antigen or from increased binding accessibility of GENOZ 3.53-reactive antigen in DR-null mutants. Monoclonal antibodies CC 11.23 and CC 6.4 displayed patterns of binding to parental and diverse mutant cells that differed from that of GENOX 3.53, suggesting the existence of at least one additional D-region antigen that is neither SB. DR, nor MB/MT.