UMP Synthase Research Articles

Novel mutation in UMPS gene leads to false positive result of DUMPS (genetic disorder) in buffaloes: need for gene sequencing before confirming results of RFLP in new species

Deficiency of uridine monophosphate synthase (DUMPS) is a lethal genetic disorder associated with early embryonic mortality. Murrah and Mehsana male buffaloes (n = 594) were screened for DUMPS by PCR-RFLP technique. A few Murrah buffalo male calves were found to be carriers of DUMPS in RFLP, which has not been reported earlier. On the Sanger sequencing, a novel A to G substitution mutation was identified in AvaI restriction recognition site of UMPS gene in buffaloes. This mutation hinders digestion of DNA by AvaI which leds to false positive results for DUMPS carrier in RFLP. The results indicated that genome sequencing must be performed before confirming results of RFLP in any new species. All the buffaloes that were tested had only wild-type genotype in exon 5 for DUMPS specific allele.

Simultaneous Genetic Screening of Bovine Leukocyte Adhesion Deficiency, Complex Vertebral Malformation, Deficiency of Uridine Monophosphate Synthase, and Mule foot in Holstein Cows in Taiwan

Simultaneous Genetic Screening of Bovine Leukocyte Adhesion Deficiency, Complex Vertebral Malformation, Deficiency of Uridine Monophosphate Synthase, and Mule foot in Holstein Cows in Taiwan

Występowanie chorób genetycznych (BLAD, DUMPS) w polskiej populacji bydła rasy holsztyńsko-fryzyjskiej

BLAD (bovine leukocyte adhesion deficiency) and DUMPS (deficiency of uridine monophosphate synthase) are hereditary lethal autosomal recessive disorders that may affect Holstein cattle. The aim of the present study was to evaluate the frequency of BLAD and DUMPS genotypes in the Polish population of dairy cows. In the study 5 732 Polish Holstein-Friesian females were genetically tested for BLAD and DUMPS. Results of the present study indicate that the Polish population of dairy cattle is free from DUMPS as there were not carriers nor sick animals. For BLAD the study confirmed the present of carriers in the population to be at a low value (0.21%). Both BLAD and DUMPS may strongly affect the profitability of a dairy farm, therefore, in order to avoid the economic losses caused by the presence of these genetic disorders in the herd it is important to screen the population and detect carriers as soon as possible.

Use of SNPs from Illumina BovineSNP50K BeadChip v3 for imputation of microsatellite alleles for parentage verification and QTL reporting

The present study aimed to test the possibility of avoiding expensive retesting of the whole parental generation for single nucleotide polymorphisms (SNPs), to provide additional analysis of microsatellites in offspring in the transitional period and to analyse the likelihood of imputation of the International Society for Animal Genetics-recommended microsatellite markers from selected SNPs. The imputation and pedigree verification of 9 520 animals (representing 84 dairy bulls, 285 dairy cows, 3 202 beef bulls and 5 949 beef cows) were analysed by the method using 9 410 SNP haplotypes (incorporating an average of 73 SNPs per haplotype). The imputation method was confirmed to allow the parentage verification of up to 87% of the analysed animals without the need for retesting. The most problematic locus was TGLA53, with only 78% successful imputation. Seven loci (BM2113, ETH225, TGLA227, BM1824, SPS115, TGLA122 and TGLA126) had more than 90% imputing accuracy and success of imputation. The success of imputation also depends on the breed and the call rate of the test results. The highest imputation accuracy was found for the Holstein breed; the other six breeds had over 90% successful imputation rates, four breeds had imputation rates between 85.0 and 89.9%, and ten breeds (rarely bred in the Czech Republic) had imputation rates below 85.0%. A call rate of SNP tests lower than 90% indicates problems with haplotype construction and thus deterioration in the success of imputation. The analysis of a possibility of using all possible information from Illumina BovineSNP50K BeadChip v3 showed 109 SNPs encoding 51 quantitative trait loci markers. Haplotypes were designed for interpretation of the most important markers for diseases, exterior and performance. The most important markers for Holstein breeders were chosen as kappa- (variants A, B and E) and beta-casein (variants A1, A2), Holstein haplotypes affecting fertility (HH1, HH3, and HH4) and loci causing genetic defects, bovine leukocyte adhesion deficiency and deficiency of uridine monophosphate synthetase. The results estimated from bovine bead chips corresponded to the expected distribution of the incidence of these traits in the population and were verified by PCR-RFLP tests.

The transcription factors aryl hydrocarbon receptor and MYC cooperate in the regulation of cellular metabolism

The transcription factor AHR (aryl hydrocarbon receptor) drives the expression of genes involved in detoxification pathways in cells exposed to pollutants and other small molecules. Moreover, AHR supports transcriptional programs that promote ribosome biogenesis and protein synthesis in cells stimulated to proliferate by the oncoprotein MYC. Thus, AHR is necessary for the proliferation of MYC-overexpressing cells. To define metabolic pathways in which AHR cooperates with MYC in supporting cell growth, here we used LC-MS-based metabolomics to examine the metabolome of MYC-expressing cells upon AHR knockdown. We found that AHR knockdown reduced lactate, S-lactoylglutathione, N-acetyl-l-alanine, 2-hydroxyglutarate, and UMP levels. Using our previously obtained RNA sequencing data, we found that AHR mediates the expression of the UMP-generating enzymes dihydroorotate dehydrogenase (quinone) (DHODH) and uridine monophosphate synthetase (UMPS), as well as lactate dehydrogenase A (LDHA), establishing a mechanism by which AHR regulates lactate and UMP production in MYC-overexpressing cells. AHR knockdown in glioblastoma cells also reduced the expression of LDHA (and lactate), DHODH, and UMPS but did not affect UMP levels, likely because of compensatory mechanisms in these cells. Our results indicate that AHR contributes to the regulation of metabolic pathways necessary for the proliferation of transformed cells.

Direct Determination of Pseudouridine in RNA by Mass Spectrometry Coupled with Stable Isotope Labeling.

Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.

Metabolic engineering generates a transgene-free safety switch for cell therapy.

Safeguard mechanisms can ameliorate the potential risks associated with cell therapies but currently rely on the introduction of transgenes. This limits their application owing to immunogenicity or transgene silencing. We aimed to create a control mechanism for human cells that is not mediated by a transgene. Using genome editing methods, we disrupt uridine monophosphate synthetase (UMPS) in the pyrimidine de novo synthesis pathway in cell lines, pluripotent cells and primary human T cells. We show that this makes proliferation dependent on external uridine and enables us to control cell growth by modulating the uridine supply, both in vitro and in vivo after transplantation in xenograft models. Additionally, disrupting this pathway creates resistance to 5-fluoroorotic acid, which enables positive selection of UMPS-knockout cells. We envision that this approach will add an additional level of safety to cell therapies and therefore enable the development of approaches with higher risks, especially those that are intended for limited treatment durations.

A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor-α receptor fusion protein etanercept and (b) the heavy and light chains of the anti-Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine- and pyrimidine-free commercial media.

LncRNA SNORD3A specifically sensitizes breast cancer cells to 5-FU by sponging miR-185-5p to enhance UMPS expression

Breast cancer is the most common cancer type in women. Long non-coding RNAs (lncRNAs) have been reported as potential new diagnostic markers, prognostic factors, and therapeutic targets in cancer. However, the specific roles and mechanisms of lncRNAs in breast cancer remain to be elucidated. Here we demonstrated the downregulation of lncRNA SNORD3A in breast cancer cells and tissues and verified its non-protein-coding property. SNORD3A overexpression had no effect on cell proliferation but specifically sensitized breast cancer cells to 5-fluorouracil (5-FU) in vitro and in vivo. Mechanistically, SNORD3A exerts its effect via enhancing uridine monophosphate synthetase (UMPS) protein expression. SNORD3A acts as a competing endogenous RNA for miR-185-5p, leading to UMPS protein upregulation. miR-185-5p overexpression disrupted the effect of SNORD3A on chemosensitization to 5-FU in vitro and in vivo. Moreover, Meis1 overexpression transcriptionally promotes SNORD3A expression, and Meis1 is downregulated in breast cancer cells and tissues. In breast cancer tissues, SNORD3A level positively correlates with Meis1 and UMPS protein levels, whereas miR-185-5p level negatively correlates with UMPS protein level. High SNORD3A transcript and Meis1 and UMPS protein levels predicts a better outcome, but high miR-185-5p level predicts a worse outcome in breast cancer patients receiving 5-FU-based chemotherapy. Our findings indicate that Meis1-regulated SNORD3A specifically sensitizes breast cancer cells to 5-FU via enhancing UMPS expression. The SNORD3A–UMPS axis may serve as a potential biomarker and therapeutic target to improve the efficacy of 5-FU-based chemotherapy for breast cancer patients.

Genetic Screening of Deficiency of Uridine Monophosphate Synthase (Dumps), Bovine Citrullinaemia (BC) and Factor Xi Deficiency (Fxid) in Hardhenu, Sahiwal and Hariana Bulls

Genetic Screening of Deficiency of Uridine Monophosphate Synthase (Dumps), Bovine Citrullinaemia (BC) and Factor Xi Deficiency (Fxid) in Hardhenu, Sahiwal and Hariana Bulls

Genetic analysis of orotic acid predicted with Fourier transform infrared milk spectra

Fourier transform infrared spectral analysis is a cheap and fast method to predict milk composition. A not very well studied milk component is orotic acid. Orotic acid is an intermediate in the biosynthesis pathway of pyrimidine nucleotides and is an indicator for the metabolic cattle disorder deficiency of uridine monophosphate synthase. The function of orotic acid in milk and its effect on calf health, health of humans consuming milk or milk products, manufacturing properties of milk, and its potential as an indicator trait are largely unknown. The aims of this study were to determine if milk orotic acid can be predicted from infrared milk spectra and to perform a large-scale phenotypic and genetic analysis of infrared-predicted milk orotic acid. An infrared prediction model for orotic acid was built using a training population of 292 Danish Holstein and 299 Danish Jersey cows, and a validation population of 381 Danish Holstein cows. Milk orotic acid concentration was determined with nuclear magnetic resonance spectroscopy. For genetic analysis of infrared orotic acid, 3 study populations were used: 3,210 Danish Holstein cows, 3,360 Danish Jersey cows, and 1,349 Dutch Holstein Friesian cows. Using partial least square regression, a prediction model for orotic acid was built with 18 latent variables. The error of the prediction for the infrared model varied from 1.0 to 3.2 mg/L, and the accuracy varied from 0.68 to 0.86. Heritability of infrared orotic acid predicted with the standardized prediction model was 0.18 for Danish Holstein, 0.09 for Danish Jersey, and 0.37 for Dutch Holstein Friesian. We conclude that milk orotic acid can be predicted with moderate to good accuracy based on infrared milk spectra and that infrared-predicted orotic acid is heritable. The availability of a cheap and fast method to predict milk orotic acid opens up possibilities to study the largely unknown functions of milk orotic acid.

Uridine 5’-monophosphate (UMP) metabolising enzymes uracil phosphoribosyltransferase and orotidine-5’-phosphate decarboxylase/ump synthase in camels and Trypanosoma evansi

In this study, the metabolic pathways and the enzymes involved in uridine 5’-monophosphate (UMP) were investigated in camel and the blood parasite Trypanosoma evansi (T. evansi). The pyrimidine pathway of T. evansi was found to be devoid of uridine kinase and uridine 5-nucleotidase. Since this can affect the de novo synthesis using uracil, salvage enzymes as uracil phosphoribosyltransferase (UPRTase) could be important for the parasite life, given that the similarity rate is not more than 32% between the camel and T. evansi UPRTase. The source of UMP in T. evansi could be from uracil by the action of UPRTase. In addition, the bifunctional orotidine 5’-phosphate decarboxylase (OMPdecase)/UMP synthase shares also in UMP homeostasis. Owing to the diverse sources of UMP in T. evansi, the enzymes involved in UMP metabolism are underscored for drug discovery. However, supported by the lack of uridine kinase, further studies are recommended to estimate the impact of UPRTase inhibition on T. evansi growth.

PYROSEQUENCING: ITS POTENTIAL AND LIMITATIONS IN DIAGNOSIS OF INHERITED DISEASES IN CATTLE

A large number of diff erent groups of livestock diseases in which heredity plays a signifi cant role are currently reported. In particular, more than 500 genetically determined morphological and functional disorders have been detected in cattle; for 150 of them, specifi c mutations are known. The sale of bovine genetic materials is associated with the spread of various diseases caused by mutations that occur in the prominent representatives of breeds. The abundance of lethal mutations in the populations requires a broader application of molecular diagnostic methods for detection of monogenic hereditary diseases. DNA pyrosequencing, being the most convenient technique for the rapid diagnosis of single nucleotide mutations in the bovine genome that are located in the regions with known nucleotide sequence, has a potential for meeting this need. Pyrosequencing-based methods for identifi cation of the most common signifi cant mutations in Holstein, Simmental, Brown Swiss and Aberdeen Angus cattle were developed, validated and approved at the FGBU “VGNKI”. Such mutations are associated with leukocyte adhesion defi ciency, complex vertebral malformation, uridine monophosphate synthetase defi ciency, citrullinemia, spinal muscular atrophy, spinal cord demyelination, Brown Swiss haplotype 2, Weaver syndrome, developmental duplications, α-mannosidosis, dwarfi sm, bovine male subfertility, trombocytopathya, arachnomelia syndrome, hypozincemia-like syndrome. These methods are intended to test semen in the breeding centres, scientifi c laboratories working in the fi eld of biotechnology and animal reproduction, livestock reproduction centres. The use of the proposed genetic tests to detect mutant alleles, as well as reduced use of mutation-bearing animals in stock breeding will allow minimizing the occurrence of inherited diseases and thus improving the gene pool of cattle in the country.

BCL-2 family protein BOK is a positive regulator of uridine metabolism in mammals

BCL-2 family proteins regulate the mitochondrial apoptotic pathway. BOK, a multidomain BCL-2 family protein, is generally believed to be an adaptor protein similar to BAK and BAX, regulating the mitochondrial permeability transition during apoptosis. Here we report that BOK is a positive regulator of a key enzyme involved in uridine biosynthesis; namely, uridine monophosphate synthetase (UMPS). Our data suggest that BOK expression enhances UMPS activity, cell proliferation, and chemosensitivity. Genetic deletion of Bok results in chemoresistance to 5-fluorouracil (5-FU) in different cell lines and in mice. Conversely, cancer cells and primary tissues that acquire resistance to 5-FU down-regulate BOK expression. Furthermore, we also provide evidence for a role for BOK in nucleotide metabolism and cell cycle regulation. Our results have implications in developing BOK as a biomarker for 5-FU resistance and have the potential for the development of BOK-mimetics for sensitizing 5-FU-resistant cancers.

Next generation sequencing elucidated a clinically undiagnosed metabolic disorder - An Iranian family with hereditary orotic aciduria

Hereditary orotic aciduria is a rare autosomal recessive metabolic disorder, manifested by retarded growth, developmental delay and elevated levels of orotic acid in the urine. It is the result of defect in the UMPS gene causing a glitch in the pyrimidine synthesis pathway. Orotic aciduria has a broad range of mimicking disorders including diverse metabolic and mitochondrial conditions. Precise diagnosis of the disease is time saving and key to efficient treatment. Based on the patient's clinical diagnosis, age of onset and family history, Whole Exome Sequencing (WES) technique was applied to determine the causative variant. One novel missense mutation was identified in the UMPS gene. Family screening verified heterozygosity of the variant in the parents. This study positively contributes to rapid detection and timely diagnosis of orotic aciduria in Iranian patients. It may also enrich the mutation spectrum of the UMPS gene worldwide.

CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans

Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.

HIGH- RESOLUTION MELTING (HRM) CURVE ANALYSIS: NEW APPROACH USED TO DETECT BLAD AND DUMPS IN URUGUAYAN HOLSTEIN BREED

The widespread use of artificial insemination has allowed the expansion of genetic progress. However, it also brought consequences such as the expansion of lethal hereditary diseases and the increase in inbreeding. The object of this study was to establish a fast and sensitive molecular assay to detect bovine leukocyte adhesion deficiency (BLAD) and deficiency of uridine monophosphate synthase (DUMPS) carriers in Uruguayan Holstein cattle by means of high resolution melting (HRM) curve analysis. By testing previously confirmed carrier and non-carrier animals, we set up a rapid, simple, and inexpensive diagnostic test using PCR followed by HRM curve analysis. The PCR-HRM genotyping method was effective for the discrimination of BLAD and DUMPS homozygous genotypes, and the BLAD heterozygous genotype. We conclude that the PCR-HRM assay is a robust, reliable, and economical tool for the detection of these mutations in the Holstein breed, which may be implemented in genetic selection programs.

Histone deacetylase inhibitors sensitize 5-fluorouracil-resistant MDA-MB-468 breast cancer cells to 5-fluorouracil.

Resistance to 5-fluorouracil (5-FU) is a serious problem in cancer therapy and overcoming it is required in order to improve the efficacy of cancer chemotherapy. Histone deacetylase (HDAC) inhibitors are used in cancer treatments and, recently, it has been reported that HDAC inhibitors can overcome resistance to various anti-cancer drugs in vitro. In the present study, a 5-FU-resistant breast cancer cell line was established, and the effects of HDAC inhibitors in these cells were examined. The 5-FU-resistant cell line MDA-MB-468 (MDA468/FU) was established by continuous exposure of the parental cells to 5-FU. This subline was characterized by high resistance to 5-FU, higher mRNA expression levels of thymidylate synthetase and dihydropyrimidine dehydrogenase (DPD), and lower mRNA expression levels of uridine monophosphate synthetase (UMPS) than the parental cells. Gimeracil, a DPD inhibitor, did not affect the sensitivity of MDA468/FU cells to 5-FU. Oteracil, a UMPS inhibitor, decreased the cytotoxicity of 5-FU in MDA468 cells, but not in MDA468/FU cells. The HDAC inhibitors, valproic acid and suberanilohydroxamic acid sensitized the two cell lines to 5-FU in a concentration-dependent manner. In conclusion, the results of the present study revealed that HDAC inhibitors increase the sensitivity to 5-FU in 5-FU-sensitive and -resistant cells.

Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa

We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene (cUMPS) of Coccomyxa sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura−) of the same alga. Here, we developed a Cre/loxP-based system for the removal of cUMPS flanked by directly repeated loxP sites from the Coccomyxa genome using the intracellular delivery of purified Cre recombinase to generate an Ura− strain that was used as a host for second-round transformation using cUMPS as the selection marker. Employing this marker–gene-recycling system, Coccomyxa strains devoid of foreign DNA except the 34-bp loxP sequence, which overexpressed an acyl-(acyl-carrier-protein) thioesterase gene, and a type-2 diacylglycerol acyltransferase gene, were constructed by the sequential introduction of two expression cassettes for the respective genes. One of the resulting strains showed 1.4-fold higher lipid productivity than the wild-type strain. This method will be applicable to other eukaryotic microalgae to create marker-free transgenic strains.

Screening genetic diseases prevalence in Braunvieh cattle.

Heritable abnormalities can cause a reduction in productive performance, structural defects, or death of the animal. There are reports of hereditary abnormalities in Braunvieh cattle from several countries, but no evidence was found on their existence in Mexico. In this study, 28 genes associated with hereditary diseases were screened with the GGP-LD 30K array (GeneSeek®) in 300 Mexican registered Braunvieh animals. Allelic frequencies of the markers associated with illness were obtained for the following: citrullinaemia, spinal dysmyelination, spinal muscular atrophy, Brows Swiss fertility haplotype 2, congenital muscular dystonia, epidermolysis bullosa, Pompes, maple syrup urine, syndactyly, Weaver syndrome, crooked tail, deficiency of uridine monophosphate synthase, hypotrichosis, Marfan syndrome, and weak calf syndrome. The allelic frequency values were low for all the analysed loci (from 0.0015 to 0.0110), with exception of syndactyly (0.4145). Although homozygous animals for these genetic conditions were detected, no physical or physiological abnormalities associated with the clinical form of the diseases were observed in the sampled animals. Markers associated with a crooked tail, deficiency of uridine monophosphate synthase, hypotrichosis, Marfan syndrome, and weak calf syndrome were absent. The studied Mexican Braunvieh population does not present clinical or subclinical effects for ten diseases in homozygous animals. However, since the assessed animals are considered as breeding stock, the monitoring of carrier animals might be periodically necessary.