Polyubiquitin Gene Research Articles

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.

Ubiquitin levels: the next target against gynecological cancers?

Ubiquitylation is a tightly regulated process that is essential for appropriate cell survival and function, and the ubiquitin pathway has shown promise as a therapeutic target for several forms of cancer. In this issue of the JCI, Kedves and colleagues report the identification of a subset of gynecological cancers with repressed expression of the polyubiquitin gene UBB, which renders these cancer cells sensitive to further decreases in ubiquitin production by inhibition of the polyubiquitin gene UBC. Moreover, inducible depletion of UBC in mice harboring tumors with low UBB levels dramatically decreased tumor burden and prolonged survival. Together, the results of this study indicate that there is a synthetic lethal relationship between UBB and UBC that has potential to be exploited as a therapeutic strategy to fight these devastating cancers.

Yeast polyubiquitin gene UBI4 deficiency leads to early induction of apoptosis and shortened replicative lifespan.

Ubiquitin is a 76-amino acid protein that is highly conserved among higher and lower eukaryotes. The polyubiquitin gene UBI4 encodes a unique precursor protein that contains five ubiquitin repeats organized in a head-to-tail arrangement. Although the involvement of the yeast polyubiquitin gene UBI4 in the stress response was reported long ago, there are no reports regarding the underlying mechanism of this involvement. In this study, we used UBI4-deletion and UBI4-overexpressing yeast strains as models to explore the potential mechanism by which UBI4 protects yeast cells against paraquat-induced oxidative stress. Here, we show that ubi4Δ cells exhibit oxidative stress, an apoptotic phenotype, and a decreased replicative lifespan. Additionally, the reduced resistance of ubi4Δ cells to paraquat that was observed in this study was rescued by overexpression of either the catalase or the mitochondrial superoxide dismutase SOD2. We also demonstrated that only SOD2 overexpression restored the replicative lifespan of ubi4Δ cells. In contrast to the case of ubi4Δ cells, UBI4 overexpression in wild-type yeast increases the yeast's resistance to paraquat, and this overexpression is associated with large pools of expressed ubiquitin and increased levels of ubiquitinated proteins. Collectively, these findings highlight the role of the polyubiquitin gene UBI4 in apoptosis and implicate UBI4 as a modulator of the replicative lifespan.

Variable repeats in the eukaryotic polyubiquitin gene ubi4 modulate proteostasis and stress survival

Ubiquitin conjugation signals for selective protein degradation by the proteasome. In eukaryotes, ubiquitin is encoded both as a monomeric ubiquitin unit fused to a ribosomal gene and as multiple ubiquitin units in tandem. The polyubiquitin gene is a unique, highly conserved open reading frame composed solely of tandem repeats, yet it is still unclear why cells utilize this unusual gene structure. Using the Saccharomyces cerevisiae UBI4 gene, we show that this multi-unit structure allows cells to rapidly produce large amounts of ubiquitin needed to respond to sudden stress. The number of ubiquitin units encoded by UBI4 influences cellular survival and the rate of ubiquitin-proteasome system (UPS)-mediated proteolysis following heat stress. Interestingly, the optimal number of repeats varies under different types of stress indicating that natural variation in repeat numbers may optimize the chance for survival. Our results demonstrate how a variable polycistronic transcript provides an evolutionary alternative for gene copy number variation.

Regulation of REST levels overcomes dysregulation of neural stem cell differentiation caused by disruption of polyubiquitin gene Ubb

Reduced levels of cellular ubiquitin (Ub) caused by disruption of the polyubiquitin gene Ubb lead to dysregulated differentiation of neural stem/progenitor cells (NSCs) and apoptosis in cells cultured in vitro. However, the underlying mechanisms responsible for these phenotypes in Ub-deficient cells have not been studied extensively. In the present study, we found that levels of repressor element-1 silencing transcription factor (REST) are elevated in Ubb−/− cells. To determine whether dysregulation of NSC differentiation is caused by the increased REST levels, we investigated the effect of reduced REST levels in Ubb−/− cells. Rest knockdown was found to increase the expression of the neuronal marker βIII-tubulin (TUJ1) and restore the expression pattern of the early neuronal marker α-internexin (α-INX) in Ubb−/− cells. Furthermore, Rest knockdown reduced Ub deficiency-induced apoptosis in cells cultured in vitro. Therefore, our study validates that cellular Ub levels are crucial for precise control of the levels of regulatory proteins such as REST during neurogenesis. We propose that regulation of Rest levels is a promising approach to overcome dysregulation of NSC differentiation caused by disruption of the polyubiquitin gene Ubb.

Effect of cellular ubiquitin levels on the regulation of oxidative stress response and proteasome function via Nrf1

The polyubiquitin genes Ubb and Ubc are upregulated under oxidative stress induced by arsenite [As(III)]. However, the role of ubiquitin (Ub) under As(III) exposure is not known in detail. In a previous study, we showed that the reduced viability observed in Ubc−/− mouse embryonic fibroblasts under As(III) exposure was not due to dysregulation of the Nrf2–Keap1 pathway, which prompted us to investigate another NFE2 family protein, nuclear factor erythroid 2-related factor 1 (Nrf1). In this study, we found that Ub deficiency due to Ubc knockdown in N2a cells reduced cell viability and proteasome activity under As(III) exposure. Furthermore, mRNA levels of the proteasome subunit Psma1 were also reduced. In addition, Ub deficiency led to the nuclear accumulation of the p65 isoform of Nrf1 under As(III) exposure. Interestingly, the overexpression of p65-Nrf1 recapitulated the phenotypes of Ub-deficient N2a cells under As(III) exposure. On the other hand, Nrf1 knockdown suppressed the death of Ub-deficient N2a cells upon exposure to As(III). Therefore, the levels of p65-Nrf1 may play an important role in the maintenance of cell viability under oxidative stress induced by As(III).

Synthetic introns help identify sequences in the 5' UTR intron of the Glycine max polyubiquitin (Gmubi) promoter that give increased promoter activity.

Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.

Genome-Wide Survey of Gut Fungi (Harpellales) Reveals the First Horizontally Transferred Ubiquitin Gene from a Mosquito Host.

Harpellales, an early-diverging fungal lineage, is associated with the digestive tracts of aquatic arthropod hosts. Concurrent with the production and annotation of the first four Harpellales genomes, we discovered that Zancudomyces culisetae, one of the most widely distributed Harpellales species, encodes an insect-like polyubiquitin chain. Ubiquitin and ubiquitin-like proteins are universally involved in protein degradation and regulation of immune response in eukaryotic organisms. Phylogenetic analyses inferred that this polyubiquitin variant has a mosquito origin. In addition, its amino acid composition, animal-like secondary structure, as well as the fungal nature of flanking genes all further support this as a horizontal gene transfer event. The single-copy polyubiquitin gene from Z. culisetae has lower GC ratio compared with homologs of insect taxa, which implies homogenization of the gene since its putatively ancient transfer. The acquired polyubiquitin gene may have served to improve important functions within Z. culisetae, by perhaps exploiting the insect hosts’ ubiquitin-proteasome systems in the gut environment. Preliminary comparisons among the four Harpellales genomes highlight the reduced genome size of Z. culisetae, which corroborates its distinguishable symbiotic lifestyle. This is the first record of a horizontally transferred ubiquitin gene from disease-bearing insects to the gut-dwelling fungal endobiont and should invite further exploration in an evolutionary context.

Dynamic transcription of ubiquitin genes under basal and stressful conditions and new insights into the multiple UBC transcript variants

Ubiquitin (Ub) is a small 76-amino acid protein that is engaged in many different pathways within the cell, including protein turnover. During proteotoxic stress, when the demand of clearing damaged/misfolded proteins strongly increases, cells activate Ub gene transcription to face the need of extra ubiquitin. This paper shows the contribution of the four ubiquitin coding genes (UBB, UBC, UBA52, RPS27A) to the ubiquitin RNA pool under basal and stressful conditions. Our results reveal that UBC and RPS27A represent the major fraction of the Ub transcriptome in different cell lines, but when converted to the coding potential, polyubiquitin genes UBC and UBB mainly contribute to determine the intracellular ubiquitin content under basal conditions. Both the polyubiquitin genes UBB and UBC are upregulated upon proteasome inhibition and oxidative stress, with markedly higher responses from the UBC promoter. A similar output, with lower fold-inductions, is detected in heat-stressed cells, with UBC acting as the main contributor to thermotolerance. By contrast, upon these stressors, the levels of UBA52 and RPS27A mRNAs remain unchanged. Remarkably, UV irradiation fails to induce Ub gene transcription, but rather seems to act at the post-transcriptional level, by stabilizing ubiquitin mRNAs at UV doses which induce rapid degradation of other RNA molecules. Moreover, the evidence that the UBC core promoter contains multiple transcription start sites and their responsiveness to stress, is here reported for the first time.

Disruption of polyubiquitin gene Ubc leads to attenuated resistance against arsenite-induced toxicity in mouse embryonic fibroblasts

The polyubiquitin gene Ubc is upregulated under oxidative stress induced by arsenite [As(III)]. However, the detailed mechanism of Ubc upregulation and the exact role of ubiquitin (Ub) to protect cells against As(III)-induced toxicity remain unknown. Here, we found that Ubc−/− mouse embryonic fibroblasts (MEFs) exhibited reduced viability under As(III) exposure, although the Nrf2–Keap1 pathway was activated as a cytoprotective response. Intriguingly, due to the reduced polyubiquitination and delayed onset of degradation of Nrf2 in Ubc−/− MEFs, the basal expression levels of Nrf2 target genes were elevated. As(III)-induced accumulation of Ub conjugates occurred in an Nrf2-independent manner, probably due to cellular stress conditions, including reduced proteasomal activity. Increased cellular Ub levels were essential to polyubiquitinate misfolded proteins generated under As(III) exposure and to degrade them by the proteasome. However, when cellular Ub levels decreased, these misfolded proteins were not efficiently polyubiquitinated, but rather accumulated as large protein aggregates inside the cells, causing cytotoxicity. Furthermore, increased activity of the autophagic pathway to clear these aggregates was not observed in Ubc−/− MEFs. Therefore, reduced viability of Ubc−/− MEFs under As(III) exposure may not be due to dysregulation of the Nrf2–Keap1 pathway, but mostly to reduced efficacy to polyubiquitinate and degrade misfolded protein aggregates.

Ubiquitin homeostasis: from neural stem cell differentiation to neuronal development.

Ubiquitin (Ub) is a well-known eukaryotic protein that exerts diverse signaling functions inside the cells, including targeting its substrates for proteasomal degradation (Hershko and Ciechanover, 1998). Tagging substrates with Ub or ubiquitination occurs via the action of three sequential enzymes called Ub-activating enzymes (E1), Ub conjugating enzymes (E2), and Ub ligases (E3). This entire process is called the Ub conjugation reaction and the substrate can be either monoubiquitinated or polyubiquitinated, which results in different outcomes for the target substrate. Inside the cells, Ub is in dynamic equilibrium between “free Ub” and “conjugated Ub” (Komander et al., 2009; Park and Ryu, 2014). Free Ub refers to a readily available form of Ub for the conjugation reaction, while conjugated Ub refers to single or multiple Ub attached to target substrates. Thus, it needs to be deconjugated or removed from the substrate (i.e., Ub conjugates) to be used for another conjugation reaction. The free Ub pool is generally maintained by de novo Ub synthesis via expression of ubiquitin genes and by Ub removal or recycling from Ub conjugates via various deubiquitinating enzymes (Komander et al., 2009; Park and Ryu, 2014). In mammals, there are two different classes of ubiquitin genes, Ub-ribosomal fusion genes (Uba52 and Uba80) and polyubiquitin genes (Ubb and Ubc). The resulting Ub-ribosomal subunit or (Ub)n fusion proteins are rapidly cleaved by deubiquitinating enzymes. Thus, all four ubiquitin genes are eventually responsible for the generation of free Ub, which is virtually indistinguishable, regardless of the original encoding gene. The contribution of the four different ubiquitin genes toward the constitution of the free Ub pool varies depending on cell types, the tissue origin, or environmental conditions. Although Ub is an abundant protein inside the cells, because of its dynamic nature and the abundance of its target substrates, the maintenance of free Ub above threshold levels or Ub homeostasis is important for the ubiquitination of target substrates in a timely manner. About a decade ago, it was shown that free Ub depletion by cycloheximide treatment in yeast resulted in reduced viability with increased toxicity, which was ameliorated by Ub overexpression (Hanna et al., 2003). Since then, the cellular maintenance of the free Ub pool has been of great interest because the maintenance of free Ub is expected to be important for cellular function and survival. In 2009, the presence of unanchored free Ub chains, which are converted to monomeric free Ub by a deubiquitinating enzyme to maintain the free Ub pool, was proposed (Kimura et al., 2009). An inhibitor of the deubiquitinating enzyme also plays a role in the maintenance of adequate levels of free Ub. The overexpression of the inhibitor was shown to reduce free Ub levels and disrupt Ub homeostasis, which reduced the viability of yeast under stress conditions (Kimura et al., 2009).

Disruption of polyubiquitin gene Ubb causes dysregulation of neural stem cell differentiation with premature gliogenesis.

Disruption of polyubiquitin gene Ubb leads to early-onset reactive gliosis and adult-onset hypothalamic neurodegeneration in mice. However, it remains unknown why reduced levels of ubiquitin (Ub) due to loss of Ubb lead to these neural phenotypes. To determine whether or not the defects in neurons or their progenitors per se, but not in their cellular microenvironment, are the cause of the neural phenotypes observed in Ubb−/− mice, we investigated the properties of cultured cells isolated from Ubb−/− mouse embryonic brains. Although cells were cultured under conditions promoting neuronal growth, Ubb−/− cells underwent apoptosis during culture in vitro, with increased numbers of glial cells and decreased numbers of neurons. Intriguingly, at the beginning of the Ubb−/− cell culture, the number of neural stem cells (NSCs) significantly decreased due to their reduced proliferation and their premature differentiation into glial cells. Furthermore, upregulation of Notch target genes due to increased steady-state levels of Notch intracellular domain (NICD) led to the dramatic reduction of proneuronal gene expression in Ubb−/− cells, resulting in inhibition of neurogenesis and promotion of gliogenesis. Therefore, our study suggests an unprecedented role for cellular Ub pools in determining the fate and self-renewal of NSCs.

Restoration of cellular ubiquitin reverses impairments in neuronal development caused by disruption of the polyubiquitin gene Ubb

Disruption of the polyubiquitin gene Ubb leads to hypothalamic neurodegeneration and metabolic disorders, including obesity and sleep abnormalities, in mice. However, it has yet to be determined whether or not these neural phenotypes in Ubb−/− mice are directly caused by cell autonomous defects in maintaining proper levels of ubiquitin (Ub). To directly demonstrate that reduced levels of Ub are sufficient to cause neuronal abnormalities, we investigated the characteristics of cultured neurons isolated from Ubb−/− mouse embryonic brains. We found that neuronal morphology, neurite outgrowth, and synaptic development were significantly impaired in Ubb−/− neurons. Furthermore, we observed the growth of astrocytes in Ubb−/− cell cultures despite the fact that cells were cultured under conditions promoting neuronal growth. When the reduced levels of free Ub, but not Ub conjugates, in Ubb−/− cells were restored to those of wild-type cells by providing exogenous Ub via lentivirus-mediated delivery, the increased apoptosis observed in Ubb−/− cells was almost completely abolished. Ectopic expression of Ub also improved neuronal and glial phenotypes observed in Ubb−/− cells. Therefore, our study suggests that Ub homeostasis, or the maintenance of cellular free Ub above certain threshold levels, is essential for proper neuronal development and survival.

Cellular ubiquitin pool dynamics and homeostasis.

Ubiquitin (Ub) is a versatile signaling molecule that plays important roles in a variety of cellular processes. Cellular Ub pools, which are composed of free Ub and Ub conjugates, are in dynamic equilibrium inside cells. In particular, increasing evidence suggests that Ub homeostasis, or the maintenance of free Ub above certain threshold levels, is important for cellular function and survival under normal or stress conditions. Accurate determination of various Ub species, including levels of free Ub and specific Ub chain linkages, have become possible in biological specimens as a result of the introduction of the proteomic approach using mass spectrometry. This technology has facilitated research on dynamic properties of cellular Ub pools and has provided tools for in-depth investigation of Ub homeostasis. In this review, we have also discussed the consequences of the disruption of Ub pool dynamics and homeostasis via deletion of polyubiquitin genes or mutations of deubiquitinating enzymes. The common consequence was a reduced availability of free Ub and a significant impact on the function and viability of cells. These observations further indicate that the levels of free Ub are important determinants for cellular protection. [BMB Reports 2014; 47(9): 475-482]

Ubiquitin code assembly and disassembly

Ubiquitin, a 76 amino-acid polypeptide, presents a compact three-dimensional structure, utilising a fold that recurs within larger polypeptides and in other protein modifiers, such as NEDD8 and SUMO. Ubiquitylation was initially recognised as a signal for proteasome-mediated degradation. We shall consider here how this view has evolved to appreciate that the dynamic appendage of different types of ubiquitin chains represents a versatile, three-dimensional code, fundamental to the control of many cellular processes.

Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention

Ubiquitin is involved in almost every cellular process, and it is also known to be a stress-inducible protein. Based on previous reports that many types of cancer display an elevated level of ubiquitin, we hypothesized that this increased amount of ubiquitin is essential for the growth of cancer cells and that, consequently, the downregulation of ubiquitin may be a potential anti-cancer treatment. We first found that the level of ubiquitin can be effectively downregulated via knockdown of a polyubiquitin gene, Ubb, with siRNA (Ubb-KD) and then demonstrated its anti-cancer effects in several cancer cell lines and xenograft mice. Ubb-KD resulted in the attenuation of TNFα-induced NF-κB activation, the stabilization of the tumor suppressor p53, and stress-sensitization. Taken together, downregulation of ubiquitin through Ubb-KD is a potential anti-cancer treatment by inhibiting ubiquitination at multiple sites related to oncogenic pathways and by weakening the ability of cancer cells to overcome increased stress.

The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila

The ubiquitin proteasome system (UPS) regulates many biological pathways by post-translationally ubiquitylating proteins for degradation. Although maintaining a dynamic balance between free ubiquitin and ubiquitylated proteins is key to UPS function, the mechanisms that regulate ubiquitin homeostasis in different tissues through development are not clear. Here we show, via analysis of the magellan (magn) complementation group, that loss of function of the Drosophila polyubiquitin Ubi-p63E results specifically in meiotic arrest sterility in males. Ubi-p63E contributes predominantly to maintaining the free ubiquitin pool in testes. The function of Ubi-p63E is required cell-autonomously for proper meiotic chromatin condensation, cell cycle progression and spermatid differentiation. magn mutant germ cells develop normally to the spermatocyte stage but arrest at the G2/M transition of meiosis I, with lack of protein expression of the key meiotic cell cycle regulators Boule and Cyclin B. Loss of Ubi-p63E function did not strongly affect the spermatocyte transcription program regulated by the testis TBP-associated factor (tTAF) or meiosis arrest complex (tMAC) genes. Knocking down proteasome function specifically in spermatocytes caused a different meiotic arrest phenotype, suggesting that the magn phenotype might not result from general defects in protein degradation. Our results suggest a conserved role of polyubiquitin genes in male meiosis and a potential mechanism leading to meiosis I maturation arrest.

The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila

The ubiquitin proteasome system (UPS) regulates many biological pathways by post-translationally ubiquitylating proteins for degradation. Although maintaining a dynamic balance between free ubiquitin and ubiquitylated proteins is key to UPS function, the mechanisms that regulate ubiquitin homeostasis in different tissues through development are not clear. Here we show, via analysis of the magellan (magn) complementation group, that loss of function of the Drosophila polyubiquitin Ubi-p63E results specifically in meiotic arrest sterility in males. Ubi-p63E contributes predominantly to maintaining the free ubiquitin pool in testes. The function of Ubi-p63E is required cell-autonomously for proper meiotic chromatin condensation, cell cycle progression and spermatid differentiation. magn mutant germ cells develop normally to the spermatocyte stage but arrest at the G2/M transition of meiosis I, with lack of protein expression of the key meiotic cell cycle regulators Boule and Cyclin B. Loss of Ubi-p63E function did not strongly affect the spermatocyte transcription program regulated by the testis TBP-associated factor (tTAF) or meiosis arrest complex (tMAC) genes. Knocking down proteasome function specifically in spermatocytes caused a different meiotic arrest phenotype, suggesting that the magn phenotype might not result from general defects in protein degradation. Our results suggest a conserved role of polyubiquitin genes in male meiosis and a potential mechanism leading to meiosis I maturation arrest.

Disruption of polyubiquitin gene Ubc leads to defective proliferation of hepatocytes and bipotent fetal liver epithelial progenitor cells

We have previously demonstrated that disruption of polyubiquitin gene Ubc leads to mid-gestation embryonic lethality most likely due to a defect in fetal liver development, which can be partially rescued by ectopic expression of Ub. In a previous study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We confirmed that Ubc−/− embryonic lethality could not be attributed to impaired function of hematopoietic stem cells, which raises the question of whether or not FLECs such as hepatocytes and bile duct cells, the most abundant cell types in the liver, are affected by disruption of Ubc and contribute to embryonic lethality. To answer this, we isolated FLCs from E13.5 embryos and cultured them in vitro. We found that proliferation capacity of Ubc−/− cells was significantly reduced compared to that of control cells, especially during the early culture period, however we did not observe the increased number of apoptotic cells. Furthermore, levels of Ub conjugate, but not free Ub, decreased upon disruption of Ubc expression in FLCs, and this could not be compensated for by upregulation of other poly- or mono-ubiquitin genes. Intriguingly, the highest Ubc expression levels throughout the entire culture period were observed in bipotent FLEPCs. Hepatocytes and bipotent FLEPCs were most affected by disruption of Ubc, resulting in defective proliferation as well as reduced cell numbers in vitro. These results suggest that defective proliferation of these cell types may contribute to severe reduction of fetal liver size and potentially mid-gestation lethality of Ubc−/− embryos.

Isolation and Characterization of a Polyubiquitin Gene and Its Promoter Region fromMesembryanthemum crystallinum

Transcript levels of the polyubiquitin gene McUBI1 had been reported to be constant during Crassulacean acid metabolism (CAM) induction in the facultative CAM plant, Mesembryanthemum crystallinum. Here, we report the sequences of the full-length cDNA of McUBI1 and its promoter, and validation of the McUBI1 promoter as an internal control driving constitutive expression in transient assays using the dual-luciferase system to investigate the regulation of CAM-related gene expression. The McUBI1 promoter drove strong, constitutive expression during CAM induction. We compared the activities of this promoter with those of the cauliflower mosaic virus (CaMV) 35S promoter in detached C3- and CAM-performing M. crystallinum and tobacco leaves. We confirmed stable expression of the genes controlled by the McUBI1 promoter with far less variability than under the CaMV 35S promoter in M. crystallinum, whereas both promoters worked well in tobacco. We found the McUBI1 promoter more suitable than the CaMV 35S promoter as an internal control for transient expression assays in M. crystallinum.