Polyubiquitin Gene Research Articles

Osmotic induction of stress-responsive gene expression in the lobster Homarus americanus.

The American lobster, Homarus americanus, encounters osmotic stress throughout its life cycle. To understand the molecular basis of osmotic stress responses in vivo, we used homologous cDNA probes to characterize the mRNA patterns of lobster HSP70 (=70-kDa heat-shock protein), HSP90 (=90-kDa heat-shock protein), and polyubiquitin during hypo- and hyper-osmotic stress in abdominal muscle and hepatopancreas (a digestive tissue) at 30, 60, and 120 min of osmotic stress. Hypo- and hyper-osmotic stress significantly increased the levels of the mRNAs encoding HSP70 and HSP90 in abdominal muscle. Hyper-osmotic stress increased HSP90 mRNA levels in hepatopancreas, but hypo-osmotic stress did not. Both abdominal muscle and hepatopancreas exhibited significant changes in polyubiquitin gene expression during osmotic stress. In abdominal muscle, polyubiquitin mRNA levels increased during both hypo- and hyper-osmotic stress. Hepatopancreas, however, showed a significant elevation in polyubiquitin mRNA only during hypo-osmotic stress.

Transgene expression from the Tribolium castaneum Polyubiquitin promoter.

The highly conserved Ubiquitin proteins are expressed from genes with strong, constitutively active promoters in many species, making these promoters attractive candidates for use in driving transgene expression. Here we report the cloning and characterization of the Tribolium castaneum Polyubiquitin (TcPUb) gene. We placed the TcPUb promoter upstream of the coding region of the T. castaneum eye-colour gene Tc vermilion (Tcv) and injected this construct into embryos from a Tcv-deficient strain. Transient expression of Tcv during embryogenesis resulted in complete rescue of the larval mutant phenotype. We then incorporated the TcPUb-Tcv chimera into a piggyBac donor. Resulting germline transformants were easily recognized by rescue of eye pigmentation, illustrating the potential of the TcPUb promoter for use in driving transgene expression.

Ubiquitin-proteasome-dependent proteolysis in rainbow trout (Oncorhynchus mykiss): effect of food deprivation.

The ubiquitin-proteasome proteolytic pathway is a major route of protein degradation and of particular importance in muscle proteolysis in mammals. In this study, the beta proteasome subunit N3 and polyubiquitin genes of the rainbow trout, Oncorhynchus mykiss, were sequenced and tissue distribution of gene expression was examined. The effects of 14-day food withdrawal were assessed on the N3 subunit and polyubiquitin gene expression in terms of mRNA, 20S proteasome proteolytic activity and ubiquitin protein abundance in trout liver and muscle. Both sequences are highly conserved, and the rainbow trout ubiquitin amino acid sequence is identical to the mammalian protein. The proteasome beta subunit N3 has 92% similarity to the Xenopus sequence. Starvation halved the polyubiquitin mRNA level in liver but had no effect on muscle levels. No significant effect of food withdrawal was observed on the proteasome mRNA in liver or muscle. Food withdrawal decreased the 20S proteasome proteolytic activity and the abundance of ubiquitin protein in both muscle and liver. Co-regulation of the proteasome and ubiquitin was indicated by the high correlation ( R=0.924) between 20S activity and ubiquitin abundance. Overall, this study demonstrates that starvation down-regulates the ubiquitin-proteasome pathway, possibly highlighting differences in the regulation of protein turnover in poikilothermic and endothermic animals.

Developmental expression of polyubiquitin genes and distribution of ubiquitinated proteins in generative and sperm cells

Polyubiquitin-encoding cDNA clones were isolated from the generative cells of lily (Lilium longiflorum) and the sperm cells of Plumbago zeylanica. The described genes encode identical amino acid sequences, with no homology outside the coding regions. This gene participates in ubiquitination of proteins, presumably enhancing protein turnover in the germline during male reproductive differentiation. In this paper we show that the gene encoding polyubiquitin is highly up-regulated in both Lilium generative cells and one of the Plumbago sperm cell types in particular.

Cloning and characterisation of a polyubiquitin gene from the ectomycorrhizal fungus Tuber borchii vittad.

Ubiquitin is a highly conserved 76-amino acid protein implicated in the function of quite different vital cellular processes. In the present study, we cloned and sequenced a polyubiquitin gene from Tuber borchii (Ubil) that is organised in four tandem repeats, with two C-terminal extension amino acids, serine and leucine. Two introns of 116 bp and 55 bp in length were detected in the first and second repeats, respectively. The Ubil gene is highly expressed in mycelium and is less expressed in the ripe fruiting body. Southern and Northern blot analyses revealed a second form of the ubiquitin gene.

Secretion of human proteins from yeast: stimulation by duplication of polyubiquitin and protein disulfide isomerase genes in Kluyveromyces lactis

The efficiency of secreted production of mammalian proteins from yeasts remains unpredictably variable, depending on each protein. On the hypothesis that the control of protein conformation during protein translocation is the bottleneck in many cases, we examined the effects of an increased dosage of the genes coding for protein disulfide isomerase and of polyubiquitin on the secretion of two human proteins, serumalbumin and interleukin 1β. The yeast Kluyveromyces lactis was used as a production host. Duplication of either one of these genes had a strong stimulating effect on the production of the highly disulfide-bonded serumalbumin, but not of interleukin 1β.

Quantitative RT-PCR Analysis of Polyubiquitin Gene Expression in Biomphalaria arabica after Exposure to Heat Stress and Cadmium

Quantitative RT-PCR Analysis of Polyubiquitin Gene Expression in Biomphalaria arabica after Exposure to Heat Stress and Cadmium

Evidence of Population Polymorphism of the Polyubiquitin Gene and Its Up-regulation During Thyroid Hormone Induced Program Cell Death In The Tail Muscle of Rana catesbeiana tadpoles

Evidence of Population Polymorphism of the Polyubiquitin Gene and Its Up-regulation During Thyroid Hormone Induced Program Cell Death In The Tail Muscle of <i>Rana catesbeiana</i> tadpoles

Interspecific Comparison in the Frequency of Concerted Evolution at the Polyubiquitin Gene Locus

The polyubiquitin gene, encoding tandemly repeated multiple ubiquitins, constitutes a uniquitin gene subfamily. It has been demonstrated that polyubiquitin genes are subject to concerted evolution; namely, the individual ubiquitin coding units contained within a polyubiquitin gene are more similar to one another than they are to the ubiquitin coding units in the orthologous gene from other species. However there has been no comprehensive study on the concerted evolution of polyubiquitin genes in a wide range of species, because the relationships (orthologous or paralogous) among multiple polyubiquitin genes from different species have not been extensively analyzed yet. In this report, we present the results of analyzing the nucleotide sequence of polyubiquitin genes of mammals, available in the DDBJ/EMBL/GenBank nucleotide sequence databases, in which we found that there are two groups of polyubiquitin genes in an orthologous relationship. Based on this result, we analyzed the concerted evolution of the polyubiquitin gene in various species and compared the frequency of concerted evolutionary events interspecifically by taking into consideration that the rate of synonymous substitution at the polyubiquitin gene locus may vary depending on species. We found that the concerted evolutionary events in polyubiquitin genes have been more frequent in rats and Chinese hamsters than those in humans, cows, and sheep. The guinea pig polyubiquitin gene was an intermediate example. The frequency of concerted evolution in the mouse gene was unexpectedly low compared to that of other rodent genes.

Structure, expression and promoter activity of two polyubiquitin genes from rice ( Oryza sativa L.)

We have isolated two rice polyubiquitin genes designated as RUBQ1 and RUBQ2 by screening a Bacterial Artificial Chromosome (BAC) genomic library with a 32P-labeled ubiquitin cDNA probe. DNA sequence data revealed that both genes contained an open reading frame encoding a hexameric precursor ubiquitin and an intron immediate upstream of the initiation codon. The deduced amino acid sequences of both genes were identical to each other and to other plant ubiquitin sequences. Several putative regulatory elements such as enhancer core and heat shock consensus sequences were found in the 5′-upstream regions of both genes. Northern blot analyses using the 3′-untranslated region as gene specific probes showed that both genes were actively expressed in all rice plant tissues tested. Differential expression was observed in roots where RUBQ2 appeared to be predominantly expressed. Chimeric genes containing the 5′-upstream region including the intron of RUBQ1 or RUBQ2 and the β-glucuronidase (GUS) coding region were constructed and transferred into rice suspension cells via particle bombardment. GUS activity from constructs containing RUBQ1 and RUBQ2 promoters in rice suspension cells was ten to 15-fold greater than those using the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, and two to threefold greater than constructs with the maize polyubiquitin Ubi1 promoter. The results demonstrate the potential usefulness of the two rice polyubiquitin promoters in rice or other monocot transformation systems.

Structure and expression of the Aplysia polyubiquitin gene

The ubiquitin–proteasome pathway, which is up-regulated in response to sensitizing treatments with serotonin (5-HT), plays a critical role in inducing long-term facilitation (LTF) of sensory-to-motor synapses in Aplysia. We characterized the structure of the polyubiquitin gene of Aplysia and studied its expression. At least six ubiquitin coding units exist in tandem, one of which encodes a protein with an amino acid sequence identical to human ubiquitin. Although the synthesis of polyubiquitin is induced by strong stimuli in many organisms, we found that the expression of ubiquitin in Aplysia is not affected by protocols that produce LTF.

TransientuidAgene expression in electroporated cotyledonary protoplasts ofPinus nigrassp.salzmanniiand in bombarded cotyledons

We have developed a method for the routine isolation of Pinus nigra Arn. ssp. salzmannii (Dunal) Franco protoplasts. The optimized electroporation conditions for uidA gene (gene for beta-glucuronidase) expression in protoplasts from cotyledons excised from 8-day-old seedlings were determined using an exponential pulse wave generator for gene transfer. The protoplasts were electroporated with plasmids containing the chimeric uidA gene under the control of several promoters, and in parallel, cotyledons were bombarded with the same constructs using a biolistic gun. Both techniques confirmed that gene expression was higher when controlled by the sunflower polyubiquitin gene promoter than by the cauliflower mosaic virus CaMV35S promoter, whereas the rice actin and maize alcohol dehydrogenase promoters resulted in lower uidA expression levels, as determined fluorometrically. In this study, the electroporation procedure has been more effective than the particle bombardment procedure to determine the promoter strength on transient gene expression in P. nigra.

Molecular characterization of ubiquitin genes from Aspergillus nidulans: mRNA expression on different stress and growth conditions

We are interested in studying the ubiquitin (UBI) gene expression during different stress and growth conditions in the filamentous fungus Aspergillus nidulans. Here, we report the cloning of a cDNA clone that corresponds to a gene, ubi1, that encodes a carboxyl extension protein from A. nidulans. This cDNA corresponds to a gene that encodes a protein that showed high homology to other polyubiquitin and CEP-80 genes at the N- and C-terminus, respectively. We characterize the mRNA expression of the CEP and polyubiquitin genes during several growth and stress conditions. Expression of the ubi1 and ubi4 genes was correlated with cell growth in most of the carbon sources used, except maltose. Both ubi1 and ubi4 genes were induced upon heat-shock, although the levels of expression were raised quicker for ubi4 than for ubi1. The ubi1 and ubi4 genes displayed a very complex expression pattern in presence of drugs with a different mechanism of action suggesting that the regulatory processes controlling UBI gene expression discriminate between different stresses and can affect individually each UBI gene. The ubi1 gene was highly expressed in presence of hydrogen peroxide while the ubi4 mRNA level was not affected; several metals in our experimental conditions were not able to induce either ubi1 nor ubi4 genes.

Proteasome Inhibition in Neuronal Cells Induces a Proinflammatory Response Manifested by Upregulation of Cyclooxygenase-2, Its Accumulation as Ubiquitin Conjugates, and Production of the Prostaglandin PGE2

Inclusions containing ubiquitin-protein aggregates appear in neurons of patients with neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. The relationship between inclusion production and cell viability is not understood. To address this issue, we investigated the response of an established mouse neuronal cell line and of embryonic rat mesencephalic cultures to inhibition of the ubiquitin/proteasome pathway. Two proteasome inhibitors, a peptidyl aldehyde and an epoxy ketone, which cause accumulation of ubiquitinated proteins, were found to enhance expression of stress-inducible genes, including HSP70i and the polyubiquitin genes UbB and UbC. Under these conditions, mRNA and protein levels of the inducible form of cyclooxygenase (COX-2) were upregulated together with its product, PGE2, a proinflammatory prostaglandin. Proteasomal inhibition also led to stabilization of COX-2 as ubiquitin conjugates, suggesting that the ubiquitin/proteasome pathway contributes to the regulation of COX-2 protein levels. Treatment with antioxidants known to inhibit NFκB and AP-1 transcriptional activation failed to abrogate COX-2 upregulation. Instead, these inhibitors exacerbated the stress response by potentiating HSP70i levels while eliciting a decrease in PGE2 production. These findings suggest that the accumulation of ubiquitinated proteins resulting from proteasome inhibition in neuronal cells is associated with a proinflammatory response that may be an important contributor to neurodegeneration.

The Polyubiquitin Gene Is Essential for Meiosis in Fission Yeast

We isolated a novel sporulation-deficient mutant of Schizosaccharomyces pombe. The mutant did not have a mitotic growth defect but aborted meiosis at the first or the second division with condensed chromosomes that failed to separate, abnormal spindle(s), and disintegrated spindle pole bodies (SPBs). During the first division, the centromeres were pulled to near the spindle poles but condensed divalent chromosomes remained at the center. The failure to proceed to anaphase was also observed during a time-lapse recording of a SPB protein tagged with green fluorescent protein. The polyubiquitin gene ubi4+, which encoded eight ubiquitins fused in tandem, complemented this mutant. The mutation, an A to G substitution, was identified within the ubi4+ gene at the ATG initiation codon. Disruption of the ubi4+ gene produced the same phenotypes. The ubi4+ mRNA was strongly induced for meiosis. However, ubiquitin increases only slightly, suggesting that the role of the polyubiquitin gene is to supply ubiquitin that is consumed by unidentified mechanisms. Before the ubi4 mutant cells entered meiosis, ubiquitin was greatly decreased indicating that shortage of ubiquitin caused abortion of meiosis. This work provides insights for the role of polyubiquitin gene and importance of ubiquitination in SPB integrity at the meiotic divisions.

Molecular cloning and characterization of the Candida albicans UBI3 gene coding for a ubiquitin-hybrid protein.

Using a polyubiquitin cDNA as a probe, we have isolated a clone (pPR3, a pEMBLYe23 derivative plasmid) containing the Candida albicans UBI3 gene coding for a fusion protein. This protein is formed by one ubiquitin subunit fused, at its C-terminus, to an unrelated peptide which is similar to the ribosomal protein encoded by the 3' tail of the Saccharomyces cerevisiae UBI3 gene. Southern blot analysis of chromosomal DNA probed with the 3' non-ubiquitin tail of UBI3 indicated that only one homologous gene is present in the C. albicans genome. Heterelogous expression of pPR3 in a S. cerevisiae ubi3 mutant strain complements the mutant phenotype (slow growth) conferred by the ubi3 defect; this provides direct evidence indicating that the clone contains the C. albicans UBI3 gene Northern blot analysis showed that UBI3 gene is expressed in yeast and germ-tube cells of C. albicans, although the UBI3 mRNA levels in starved yeast cells are below the detection limit; UBI3 mRNA drops to undetectable levels on shifting the temperature of growing yeast cells from 28 degrees C to 42 degrees C. When Northern blot analysis was performed using a specific probe for the polyubiquitin (UBI4) gene, no drop in the mRNA levels was detected following thermal upshift or in starved cells. These results indicate that stress conditions (starvation or thermal upshift) negatively regulate UBI3 expression (transcriptional arrest and/or enhanced mRNA decay), and suggest that UBI4 gene provides ubiquitin during the stress response. In addition, we failed to obtain C. albicans UBI3 null mutant cells by sequential disruption of both alleles using the hisG::URA3::hisG ('ura-blaster') cassette, suggesting that null mutants cells may be unable to grow on selective media after transformation.

Transient uidA gene expression in electroporated cotyledonary protoplasts of Pinus nigra ssp. salzmannii and in bombarded cotyledons

We have developed a method for the routine isolation of Pinus nigra Arn. ssp. salzmannii (Dunal) Franco protoplasts. The optimized electroporation conditions for uidA gene (gene for beta-glucuronidase) expression in protoplasts from cotyledons excised from 8-day-old seedlings were determined using an exponential pulse wave generator for gene transfer. The protoplasts were electroporated with plasmids containing the chimeric uidA gene under the control of several promoters, and in parallel, cotyledons were bombarded with the same constructs using a biolistic gun. Both techniques confirmed that gene expression was higher when controlled by the sunflower polyubiquitin gene promoter than by the cauliflower mosaic virus CaMV35S promoter, whereas the rice actin and maize alcohol dehydrogenase promoters resulted in lower uidA expression levels, as determined fluorometrically. In this study, the electroporation procedure has been more effective than the particle bombardment procedure to determine the promoter str...

Isolation and characterization of a cDNA clone encoding polyubiquitin from the root nodule ofElaeagnus umbellata

A cDNA clone encoding polyubiquitin was isolated from a root nodule cDNA library of Elaeagnus umbellata Thunb. by differential hybridization, and its molecular aspects were characterized. The polyubiquitin clone pEuNOD-PUB1 has an insert size of 1642 bp and has the capacity to code for a 458 amino acid residue polyubiquitin protein. The derived amino acid sequence indicates that pEuNOD-PUB1 encodes a polyprotein consisting of six repeats of ubiquitin monomer, except for the last repeat, which has two additional amino acids (Asp-Phe) to be removed in the course of polyubiquitin processing into monomer. The molecular mass of ubiquitin monomer, consisting of 76 amino acids, was predicted to be 8524 Da, and the pI value was predicted to be 7.57. The nucleotide sequence of ubiquitin monomers from pEuNOD-PUB1 showed 73.1-86.0% sequence similarity with those from other organisms. The polyubiquitin mRNA content was four to six times higher in the root nodules than in the leaves and roots. In situ hybridization results showed polyubiquitin transcripts were strongly detected in the meristem zone, in infected cells of the fixation zone, and in the central vascular system. Genomic Southern hybridization revealed that polyubiquitin genes are present as a small multigene family in the genome of E. umbellata.Key words: Elaeagnus umbellata, root nodule, cDNA, polyubiquitin, Northern hybridization, in situ hybridization.

Intraerythrocytic polyubiquitin expression in Plasmodium falciparum is subjected to developmental and heat-hock control

The polyubiquitin gene of the human protozoan parasite Plasmodium falciparum ( PfpUB) was cloned and shown to be comprised of five tandem repeats of the ubiquitin open reading frame, present as a single copy on chromosome 12. The 1672 bp of PfpUB is interrupted at the 5′ end by a single intron of 526 bp. PfpUB expression is developmentally regulated in intraerythrocytic stages with a marked increase in both steady-state transcript and polyubiquitin protein levels in late trophozoite stages. On response to heat shock, late stage parasites (late trophozoites and schizonts) have a slightly elevated PfpUB transcript level as well as readily observable increases in the amount of polyubiquitin and ubiquitin-conjugated proteins.

Differential Expression of Sugarcane Polyubiquitin Genes and Isolation of Promoters from two Highly-Expressed Members of the Gene Family

Summary To identify potential strong constitutive promoters, accumulation of polyubiquitin mRNA in sugarcane ( Saccharum hybrid) was studied using gene-specific probes from five sugarcane polyubiquitin cDNA clones. Based on comparisons of 3′ untranslated regions, these cDNAs can be grouped into four sub-groups within the sugarcane polyubiquitin gene family. RNA gel blot experiments indicated that these sub-groups differ substantially in constitutive expression levels and response to heat shock. Reverse northern experiments confirmed the relative magnitudes of constitutive expression in young leaves. One sub-group, homologous to cDNAs scubi241 and scubi511, was found to have the highest level of constitutive expression and to be little affected by heat shock. Genomic clones belonging to this sub-group were isolated; two of these clones, ubi4 and ubi9, were found to differ in the number of protein coding repeats but were highly similar in overall structure and in the sequence of DNA flanking the transcribed region. Both genes contain a single large intron located immediately upstream of the translation start codon. Transient expression experiments showed that the cloned promoters are sufficient to drive high levels of GUS expression in sugarcane suspension cells and in tobacco leaves.