GULO Gene Research Articles

Potentiation of Tumor Hallmarks by the Loss of GULO, a Vitamin C Biosynthesis Gene in Humans.

Vitamin C plays a significant role in various physiological functions. Humans depend on external sources of vitamin C due to the loss of the L-gulono-γ-lactone oxidase (GULO) gene that contributes to the synthesis of vitamin C. During the evolutionary loss of the GULO gene, physical, chemical, and biological factors were different from the present environmental settings. Besides the evolutionary genetic loss of the GULO gene, there is a gap in the insightful discussion on the potential implications of the non-functional GULO gene towards the predisposition of humans to cancer that faces hostile and carcinogenic environments. Various methods by which vitamin C modulates cellular processes related to cancer, including DNA repair, epigenetic changes, and redox balance, are discussed. Furthermore, we present experimental and clinical evidence indicating that vitamin C deficiency promotes tumor growth, metastasis, and therapy resistance, emphasizing its potential as a cancer phenotypic modulator. Therapeutic implications of restoring vitamin C levels in cancer treatment range from improving the efficacy of conventional medicines to exploiting metabolic vulnerabilities in tumors. The relevance of assessing vitamin C status in cancer patients and the basis for additional research into vitamin C supplementation as an adjuvant therapy is emphasized. This paper presents a comprehensive overview of the implications associated with the functional deficiency of the GULO gene in human subjects exhibiting diverse tumor hallmarks, encompassing ECM remodeling, hypoxia, epigenetic reprogramming, oxidative stress, and drug responsiveness.

L-gulono-γ-lactone Oxidase, the Key Enzyme for L-Ascorbic Acid Biosynthesis.

L-ascorbic acid (AsA, vitamin C) plays a vital role in preventing various diseases, particularly scurvy. AsA is known for its antioxidant properties, which help protect against reactive oxygen species generated from metabolic activities; however, at high doses, it may exhibit pro-oxidative effects. The final step in AsA biosynthesis is catalyzed by L-gulono-γ-lactone oxidase (GULO). This enzyme is present in many organisms, but some animals, including humans, guinea pigs, bats, and other primates, are unable to synthesize AsA due to the absence of a functional GULO gene. The GULO enzyme belongs to the family of aldonolactone oxidoreductases (AlORs) and contains two conserved domains, an N-terminal FAD-binding region and a C-terminal HWXK motif capable of binding the flavin cofactor. In this review, we explore AsA production, the biosynthetic pathways of AsA, and the localization of GULO-like enzymes in both animal and plant cells. Additionally, we compare the amino acid sequences of AlORs across different species and summarize the findings related to their enzymatic activity. Interestingly, a recombinant C-terminal rat GULO (the cytoplasmic domain of the rat GULO expressed in Escherichia coli) demonstrated enzymatic activity. This suggests that the binding of the flavin cofactor to the HWXK motif at the C-terminus is sufficient for the formation of the enzyme's active site. Another enzyme, GULLO7 from Arabidopsis thaliana, also lacks the N-terminal FAD-binding domain and is strongly expressed in mature pollen, although its activity has not been specifically measured.

The golden bell flower genome provides insights into its evolutionary history and reveals the potential genomic bases of its ecological divergence from weeping forsythia

The golden bell flower (GBF), a beloved flower that blooms in early spring, faces a significant challenge in genetic research and breeding due to the lack of available genomic data. Here, a genome of GBF with a genome size of 735.31 Mb and tiny gaps of 3,782 N were assembled. Our results showed GBF underwent two round of whole genome doubling (WGD) events. The more recent WGD events, along with subsequent structural variations in GBF and weeping forsythia, might lead to the divergence of GBF and weeping forsythia. Analysis of enriched KEGG pathways and Gene Ontology categories of the expanded and contracted genes indicated an increase in the GBF abilities of faster growth and stronger cold and drought tolerance since the separation of the two species. Our results showed that the effective population size (Ne) of the GBF species has been decreasing, declining from 0.352 Mya to 0.018 Mya. However, the stronger cold tolerance of GBF likely helped to alleviate the reduction of Ne during the Pleistocene glacial periods. The expansion of peroxidase (POD) and l-gulonolactone oxidase (GULO) genes and more POD and GULO genes with anaerobic and ABA-induced cis elements in GBF genome might be responsible for the strong waterlogging tolerance of GBF. Our study characterized the evolutionary history of GBF and the genomic bases for the ecological divergenece between GBF and weeping forsythia. Our genome provides genetic resources for future genetic and breeding studies on GBF and its related species.

Fatal Epileptic Seizures in Mice Having Compromised Glutathione and Ascorbic Acid Biosynthesis.

Reduced glutathione (GSH) and ascorbic acid (AA) are the two most abundant low-molecular-weight antioxidants in mammalian tissues. GclmKO knockout mice lack the gene encoding the modifier subunit of the rate-limiting enzyme in GSH biosynthesis; GclmKO mice exhibit 10-40% of normal tissue GSH levels and show no overt phenotype. GuloKO knockout mice, lacking a functional Gulo gene encoding L-gulono-γ-lactone oxidase, cannot synthesize AA and depend on dietary ascorbic acid for survival. To elucidate functional crosstalk between GSH and AA in vivo, we generated the GclmKO/GuloKO double-knockout (DKO) mouse. DKO mice exhibited spontaneous epileptic seizures, proceeding to death between postnatal day (PND)14 and PND23. Histologically, DKO mice displayed neuronal loss and glial proliferation in the neocortex and hippocampus. Epileptic seizures and brain pathology in young DKO mice could be prevented with AA supplementation in drinking water (1 g/L). Remarkably, in AA-rescued adult DKO mice, the removal of AA supplementation for 2-3 weeks resulted in similar, but more severe, neocortex and hippocampal pathology and seizures, with death occurring between 12 and 21 days later. These results provide direct evidence for an indispensable, yet underappreciated, role for the interplay between GSH and AA in normal brain function and neuronal health. We speculate that the functional crosstalk between GSH and AA plays an important role in regulating glutamatergic neurotransmission and in protecting against excitotoxicity-induced brain damage.

The evolution of vitamin C biosynthesis and transport in animals

BackgroundVitamin C (VC) is an indispensable antioxidant and co-factor for optimal function and development of eukaryotic cells. In animals, VC can be synthesized by the organism, acquired through the diet, or both. In the single VC synthesis pathway described in animals, the penultimate step is catalysed by Regucalcin, and the last step by l-gulonolactone oxidase (GULO). The GULO gene has been implicated in VC synthesis only, while Regucalcin has been shown to have multiple functions in mammals.ResultsBoth GULO and Regucalcin can be found in non-bilaterian, protostome and deuterostome species. Regucalcin, as here shown, is involved in multiple functions such as VC synthesis, calcium homeostasis, and the oxidative stress response in both Deuterostomes and Protostomes, and in insects in receptor-mediated uptake of hexamerin storage proteins from haemolymph. In Insecta and Nematoda, however, there is no GULO gene, and in the latter no Regucalcin gene, but species from these lineages are still able to synthesize VC, implying at least one novel synthesis pathway. In vertebrates, SVCT1, a gene that belongs to a family with up to five members, as here shown, is the only gene involved in the uptake of VC in the gut. This specificity is likely the result of a subfunctionalization event that happened at the base of the Craniata subphylum. SVCT-like genes present in non-Vertebrate animals are likely involved in both VC and nucleobase transport. It is also shown that in lineages where GULO has been lost, SVCT1 is now an essential gene, while in lineages where SVCT1 gene has been lost, GULO is now an essential gene.ConclusionsThe simultaneous study, for the first time, of GULO, Regucalcin and SVCTs evolution provides a clear picture of VC synthesis/acquisition and reveals very different selective pressures in different animal taxonomic groups.

Growth and Antioxidant-Related Effects of the Reestablished Ascorbic Acid Pathway in Zebrafish (Danio rerio) by Genomic Integration of L-Gulonolactone Oxidase From Cloudy Catshark (Scyliorhinus torazame).

Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.

Decreased ascorbic acid biosynthesis in response to PMSG in the pre-pubertal female rat ovary

Ascorbic acid (AA) is known to be an important antioxidant serving as a cofactor in collagen synthesis, and thus facilitates follicular growth in the ovary. Many studies have shown that AA is synthesized in the liver and transported to other organs including ovary, however, there is no direct evidence of ascorbic acid synthesis in the ovary. Hence, we examined the expression pattern of different proteins (SMP30/GNL and GULO) involved in the AA synthesis in pre-pubertal rat, which showed significant expression of these proteins, suggesting the synthesis of AA in the ovary. Accumulation of AA in the ovary during follicular growth has been well demonstrated. However, the effect of Pregnant Mare Serum Gonadotropin (PMSG) on the AA synthesis in the ovary has not been studied in detail. Hence to decipher the effect, different doses of PMSG were injected subcutaneously into the pre-pubertal female rats, and ovarian AA level was measured after 48 h. A significant increase in AA content was observed in PMSG treated animal groups. Further, to understand the mechanism underlying ovarian AA accumulation, the expression levels of SMP30/GNL and GULO genes were measured. Expression of both the genes was significantly suppressed, which suggested a lowered AA synthesis in the PMSG treated rat ovary. For further understanding, mRNA expression of AA transporters SVCT1 and SVCT2 encoded by SLC23A1 and SLC23A2 genes respectively were measured, which showed increased level of SVCT1 expression. These observations suggested that the increased AA content might not be due to increased synthesis of AA within the ovary but possibly due to increased uptake from blood during the stimulation of follicular growth.

Multiple independent L-gulonolactone oxidase (GULO) gene losses and vitamin C synthesis reacquisition events in non-Deuterostomian animal species

BackgroundL-ascorbate (Vitamin C) is an important antioxidant and co-factor in eukaryotic cells, and in mammals it is indispensable for brain development and cognitive function. Vertebrates usually become L-ascorbate auxothrophs when the last enzyme of the synthetic pathway, an L-gulonolactone oxidase (GULO), is lost. Since Protostomes were until recently thought not to have a GULO gene, they were considered to be auxothrophs for Vitamin C.ResultsBy performing phylogenetic analyses with tens of non-Bilateria and Protostomian genomes, it is shown, that a GULO gene is present in the non-Bilateria Placozoa, Myxozoa (here reported for the first time) and Anthozoa groups, and in Protostomians, in the Araneae family, the Gastropoda class, the Acari subclass (here reported for the first time), and the Priapulida, Annelida (here reported for the first time) and Brachiopoda phyla lineages. GULO is an old gene that predates the separation of Animals and Fungi, although it could be much older. We also show that within Protostomes, GULO has been lost multiple times in large taxonomic groups, namely the Pancrustacea, Nematoda, Platyhelminthes and Bivalvia groups, a pattern similar to that reported for Vertebrate species. Nevertheless, we show that Drosophila melanogaster seems to be capable of synthesizing L-ascorbate, likely through an alternative pathway, as recently reported for Caenorhabditis elegans.ConclusionsNon-Bilaterian and Protostomians seem to be able to synthesize Vitamin C either through the conventional animal pathway or an alternative pathway, but in this animal group, not being able to synthesize L-ascorbate seems to be the exception rather than the rule.

Bioinformatics Protocols for Quickly Obtaining Large-Scale Data Sets for Phylogenetic Inferences.

Useful insight into the evolution of genes and gene families can be provided by the analysis of all available genome datasets rather than just a few, which are usually those of model species. Handling and transforming such datasets into the desired format for downstream analyses is, however, often a difficult and time-consuming task for researchers without a background in informatics. Therefore, we present two simple and fast protocols for data preparation, using an easy-to-install, open-source, cross-platform software application with user-friendly, rich graphical user interface (SEDA; http://www.sing-group.org/seda/index.html ). The first protocol is a substantial improvement over one recently published (López-Fernández et al. Practical applications of computational biology and bioinformatics, 12th International conference. Springer, Cham, pp 88-96 (2019)[1]), which was used to study the evolution of GULO, a gene that encodes the enzyme responsible for the last step of vitamin C synthesis. In this paper, we show how the sequence data file used for the phylogenetic analyses can now be obtained much faster by changing the way coding sequence isoforms are removed, using the newly implemented SEDA operation "Remove isoforms". This protocol can be used to easily show that putative functional GULO genes are present in several Prostotomian groups such as Molluscs, Priapulida and Arachnida. Such findings could have been easily missed if only a few Protostomian model species had been used. The second protocol allowed us to identify positively selected amino acid sites in a set of 19 primate HLA immunity genes. Interestingly, the proteins encoded by MHC class II genes can show just as many positively selected amino acid sites as those encoded by classical MHC class I genes. Although a significant percentage of codons, which can be as high as 14.8%, are evolving under positive selection, the main mode of evolution of HLA immunity genes is purifying selection. Using a large number of primate species, the probability of missing the identification of positively selected amino acid sites is lower. Both projects were performed in less than one week, and most of the time was spent running the analyses rather than preparing the files. Such protocols can be easily adapted to answer many other questions using a phylogenetic approach.

Metadichol®, Vitamin C and GULO Gene Expression in Mouse Adipocytes

Metadichol, a novel nano emulsion of lipid alcohols, up regulates the expression of GULO gene in mouse at picogram levels. 3T3-L1 pre adipocyte cells were differentiated using differentiating media. Post differentiation, the cells were treated with different concentrations of Metadichol for 24 hours and untreated cells served as control. Whole RNA was isolated after the treatment period and semi-quantitative reverse transcription PCR was run with GULO gene specific primer to obtain cDNA. Relative gene expression of GULO was determined by analysis of GULO gene amplicons using image J software. The GULO gene expression in the treated cells was up regulated fourfold relative to basal level of untreated cells.. Relative gene expression at concentrations 1 μg/ml, 100 ng/ml, 1 ng/ml, 100 pg/ml, 1 pg/ml was found to be 2.11, 2.64, 2.96, 3.96, 3.25 fold compared to control.

Effect of Fluorosis on Liver Cells of VC Deficient and Wild Type Mice

For decades, mouse and other rodents have been used for the study of oxidative or related studies such as the effect of fluoride. It is known that rodents normally synthesize their own vitamin C (VC) due to the presence of a key enzyme in ascorbic acid synthesis, l-gulono-lactone-γ-oxidase (Gulo), while humans do not have the capacity of VC synthesis due to the deletion of most parts of the GULO gene. The spontaneous fracture (sfx) mouse recently emerged as a model for study of VC deficiency. We investigated the effect of fluoride on liver cells from wild type Balb/c and sfx mice. We found that activities of SOD, GPx, and CAT were reduced in both wild type and sfx mice; however, the amount of reduction in the sfx cells is more than that in Balb/c cells. In addition, while both cells increased MDA, the increase in the sfx cells is greater than that in Balb/c cells. Gene networks of Sod, Gpx, and Cat in the liver of humans and mice are also different. Our study suggests that reaction to fluoride in vitamin C deficient mice might be different from that of wild type mice.

Conserved or Lost: Molecular Evolution of the Key Gene GULO in Vertebrate Vitamin C Biosynthesis

L-gulono-gamma-lactone oxidase (GULO) catalyzes the final step in vertebrate vitamin C biosynthesis. Vitamin C-incapable vertebrates lack the GULO gene. Gene structure and phylogenetic analyses showed that vertebrate GULO genes are 64-95% identical at the amino acid level and consist of 11 conserved exons. GULO pseudogenes have multiple indel mutations and premature stop codons in higher primates, guinea pigs, and some bats. No GULO-like sequences were identified in teleost fishes. During animal GULO evolution, exon F was subdivided into F1 and F2. Additional GULO retropseudogenes were identified in dogs, cats, and giant pandas. GULO-flanking genome regions acquired frequent segment translocations and inversions during vertebrate evolution. Purifying selection was detected across vertebrate GULO genes (d(N)/d(S) = 0.069), except for some positively selected sites identified in sharks and frogs. These positive sites demonstrated little functional significance when mapped onto the three-dimensional GULO protein structure. Vertebrate GULO genes are conserved except for those that are lost.

A “Forward Genomics” Approach Links Genotype to Phenotype using Independent Phenotypic Losses among Related Species

Genotype-phenotype mapping is hampered by countless genomic changes between species. We introduce a computational "forward genomics" strategy that-given only an independently lost phenotype and whole genomes-matches genomic and phenotypic loss patterns to associate specific genomic regions with this phenotype. We conducted genome-wide screens for two metabolic phenotypes. First, our approach correctly matches the inactivated Gulo gene exactly with the species that lost the ability to synthesize vitamin C. Second, we attribute naturally low biliary phospholipid levels in guinea pigs and horses to the inactivated phospholipid transporter Abcb4. Human ABCB4 mutations also result in low phospholipid levels but lead tosevere liver disease, suggesting compensatory mechanisms in guinea pig and horse. Our simulation studies, counts of independent changes in existing phenotype surveys, and the forthcoming availability of many new genomes all suggest that forward genomics can be applied to many phenotypes, including those relevant for human evolution and disease.

Evaluation of gene expression in muscle in mouse model lacking of vitamin C synthesis

Background Human species and guinea pigs have to obtain vitamin C (VC) from food because they are unable to synthesize ascorbic acid due to the absence of the gene that encodes L-gulonolactone oxidase (Gulo). The spontaneous bone fracture (sfx) mouse is a mouse model which is deficient in the synthesis of VC because of the deletion in Gulo gene. Because muscle forces are a strong determinant of bone structure, particularly during the process of growth and development, we examined the gene expression of muscle in sfx mice.

Effect of vitamin C deficiency during postnatal development on adult behavior: functional phenotype of Gulo(−/−) knockout mice

Organisms using oxygen for aerobic respiration require antioxidants to balance the production of reactive oxygen species during metabolic processes. Various species--including humans and other primates--suffer mutations in the GULO gene encoding L-gulono-γ-lactone oxidase; GULO is the rate-limiting enzyme in the biosynthesis of ascorbate, an important cellular antioxidant. Animals lacking the ability to synthesize vitamin C develop scurvy without dietary supplementation. The Gulo-/- knockout (KO) mouse requires oral supplemental vitamin C; without this supplementation the animal dies with a scorbutic condition within several weeks. Vitamin C is known to be most abundant in the brain, where it is believed to play important roles in neuroprotection, neurotransmission and neuromodulation. We therefore hypothesized that ascorbate deficiency in Gulo-/- KO mice might lead to an abnormal behavioral phenotype. We established the amount of ascorbate in the drinking water (220 ppm) necessary for generating a chronic low-ascorbate status in the brain, yet clinically the mice appeared healthy throughout 100 days postpartum at which time all behavioral-phenotyping tests were completed. Compared with Gulo+/+ wild-type littermates, ascorbate-deficient Gulo-/- mice were found to be less active in moving in their environment; when in water, these mice swam more slowly in some tests, consistent with a mild motor deficit. We found no evidence of cognitive, anxiety or sensorimotor-gating problems. Despite being less active, Gulo-/- mice exhibited exaggerated hyperactivity to the dopaminergic agonist methamphetamine. The subnormal movement, combined with hypersensitivity to a dopamine agonist, point to developmental ascorbate deficiency causing long-term striatal dysfunction.

Effect of vitamin C deficiency during postnatal development on adult behavior: functional phenotype of Gulo(-/-) knockout mice

Organisms using oxygen for aerobic respiration require antioxidants to balance the production of reactive oxygen species during metabolic processes. Various species – including humans and other primates – suffer mutations in the GULO gene encoding L-gulono-γ-lactone oxidase; GULO is the rate-limiting enzyme in the biosynthesis of ascorbate, an important cellular antioxidant. Animals lacking the ability to synthesize vitamin C develop scurvy without dietary supplementation. The Gulo(−/−) knockout (KO) mouse requires oral supplemental vitamin C; without this supplementation the animal dies with a scorbutic condition within several weeks. Vitamin C is known to be most abundant in the brain, where it is believed to play important roles in neuroprotection, neurotransmission and neuromodulation. We therefore hypothesized that ascorbate deficiency in Gulo(−/−) KO mice might lead to an abnormal behavioral phenotype. We established the amount of ascorbate in the drinking water (220 ppm) necessary for generating a chronic low-ascorbate status in the brain, yet clinically the mice appeared healthy throughout 100 days postpartum at which time all behavioral-phenotyping tests were completed. Compared with Gulo(+/+) wild-type littermates, ascorbate-deficient Gulo(−/−) mice were found to be less active in moving in their environment; when in water, these mice swam more slowly in some tests, consistent with a mild motor deficit. We found no evidence of cognitive, anxiety or sensorimotor-gating problems. Despite being less active, Gulo(−/−) mice exhibited exaggerated hyperactivity to the dopaminergic agonist methamphetamine. The subnormal movement, combined with hypersensitivity to a dopamine agonist, point to developmental ascorbate deficiency causing long-term striatal dysfunction.

Recent Loss of Vitamin C Biosynthesis Ability in Bats

The traditional assumption that bats cannot synthesize vitamin C (Vc) has been challenged recently. We have previously shown that two Old World bat species (Rousettus leschenaultii and Hipposideros armiger) have functional L-gulonolactone oxidase (GULO), an enzyme that catalyzes the last step of Vc biosynthesis de novo. Given the uncertainties surrounding when and how bats lost GULO function, exploration of gene evolutionary patterns is needed. We therefore sequenced GULO genes from 16 bat species in 5 families, aiming to establish their evolutionary histories. In five cases we identified pseudogenes for the first time, including two cases in the genus Pteropus (P. pumilus and P. conspicillatus) and three in family Hipposideridae (Coelops frithi, Hipposideros speoris, and H. bicolor). Evolutionary analysis shows that the Pteropus clade has the highest ω ratio and has been subjected to relaxed selection for less than 3 million years. Purifying selection acting on the pseudogenized GULO genes of roundleaf bats (family Hipposideridae) suggests they have lost the ability to synthesize Vc recently. Limited mutations in the reconstructed GULO sequence of the ancestor of all bats contrasts with the many mutations in the ancestral sequence of recently emerged Pteropus bats. We identified at least five mutational steps that were then related to clade origination times. Together, our results suggest that bats lost the ability to biosynthesize vitamin C recently by exhibiting stepwise mutation patterns during GULO evolution that can ultimately lead to pseudogenization.

The transcription of l-gulono-gamma-lactone oxidase, a key enzyme for biosynthesis of ascorbate, during development of Persian sturgeon Acipenser persicus

l-Gulono-gamma-lactone oxidase (GULO) is a key enzyme for the biosynthesis of ascorbate, which is essential for several cellular functions. In the present study, mRNA expression of GULO gene was evaluated during the early development of Persian sturgeon. First, because there are no comparative studies that have established suitable quantitative real-time PCR reference genes in sturgeons for any physiological conditions, we evaluated six candidate reference genes (ACTB, RPL13, UBQ, RPL6, GAPDH and EF1A) during the early development of Persian sturgeon. The most stable mRNA expression was obtained with RPL6 and ACTB, whereas the least stable was RPL13. After normalization using RPL6, ACTB and RPL6/ACTB combination, the mRNA expression of GULO was highest at the embryonic stage (2days before hatching; P<0.05) and started to decline from hatching of larvae to the rest of the developmental time-points. This suggests that the vitamin C requirements are highest during early life stages, and it is likely that the changes in GULO mRNA expression are associated with changes in GULO enzyme activity.

Differential gene expression between wild-type and Gulo-deficient mice supplied with vitamin C

The aim of this study was to test the hypothesis that hepatic vitamin C (VC) levels in VC deficient mice rescued with high doses of VC supplements still do not reach the optimal levels present in wild-type mice. For this, we used a mouse scurvy model (sfx) in which the L-gulonolactone oxidase gene (Gulo) is deleted. Six age- (6 weeks old) and gender- (female) matched wild-type (WT) and sfx mice (rescued by administering 500 mg of VC/L) were used as the control (WT) and treatment (MT) groups (n = 3 for each group), respectively. Total hepatic RNA was used in triplicate microarray assays for each group. EDGE software was used to identify differentially expressed genes and transcriptomic analysis was used to assess the potential genetic regulation of Gulo gene expression. Hepatic VC concentrations in MT mice were significantly lower than in WT mice, even though there were no morphological differences between the two groups. In MT mice, 269 differentially expressed transcripts were detected (≥ twice the difference between MT and WT mice), including 107 up-regulated and 162 down-regulated genes. These differentially expressed genes included stress-related and exclusively/predominantly hepatocyte genes. Transcriptomic analysis identified a major locus on chromosome 18 that regulates Gulo expression. Since three relevant oxidative genes are located within the critical region of this locus we suspect that they are involved in the down-regulation of oxidative activity in sfx mice.

Progressive Pseudogenization: Vitamin C Synthesis and Its Loss in Bats

For the past 50 years, it was believed that all bats, like humans and guinea pigs, did not synthesize vitamin C (Vc) because they lacked activity of L-gulonolactone oxidase (GULO) in their livers. Humans and guinea pigs lack the activity due to pseudogenization of GULO in their genomes, but there is no genetic evidence to show whether such loss in bats is caused by pseudogenization. Unexpectedly, our successful molecular cloning in one frugivorous bat (Rousettus leschenaultii) and one insectivorous bat (Hipposideros armiger) ascertains that no pseudogenization occurs in these species. Furthermore, we find normal GULO protein expression using bat-specific anti-GULO polyclonal antibodies in bats, evaluated by Western blotting. Most surprisingly, GULO activity assays reveal that these two bat species have retained the ability to synthesize Vc, but at low levels compared with the mouse. It is known that bats in the genus Pteropus have lost GULO activity. We then found that functional constraints acting on the GULO of Pteropus vampyrus (which lost its function) are relaxed. These results imply that the ability to synthesize Vc in bats has not been lost completely in species as previously thought. We also suggest that the evolution of bat GULO genes can be a good model to study genetic processes associated with loss-of-function.