Showing papers on "Complementary DNA published in 2014"

c-kit + cells minimally contribute cardiomyocytes to the heart

Abstract: If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit(+) cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit(+) cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit(+) cells amply generated cardiac endothelial cells. Thus, endogenous c-kit(+) cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

RNA-Seq transcriptome analysis to identify genes involved in metabolism-based diclofop resistance in Lolium rigidum.

Abstract: Summary Weed control failures due to herbicide resistance are an increasing and worldwide problem that significantly affect crop yields. Metabolism-based herbicide resistance (referred to as metabolic resistance) in weeds is not well characterized at the genetic level. An RNA-Seq transcriptome analysis was used to find candidate genes that conferred metabolic resistance to the herbicide diclofop in a diclofop-resistant population (R) of the major global weed Lolium rigidum. A reference cDNA transcriptome (19 623 contigs) was assembled and assigned putative annotations. Global gene expression was measured using Illumina reads from untreated control, adjuvant-only control, and diclofop treatment of R and susceptible (S). Contigs that showed constitutive expression differences between untreated R and untreated S were selected for further validation analysis, including 11 contigs putatively annotated as cytochrome P450 (CytP450), glutathione transferase (GST), or glucosyltransferase (GT), and 17 additional contigs with annotations related to metabolism or signal transduction. In a forward genetics validation experiment, nine contigs had constitutive up-regulation in R individuals from a segregating F2 population, including three CytP450, one nitronate monooxygenase (NMO), three GST, and one GT. Principal component analysis using these nine contigs differentiated F2-R from F2-S individuals. In a physiological validation experiment in which 2,4-D pre-treatment induced diclofop protection in S individuals due to increased metabolism, seven of the nine genetically validated contigs were induced significantly. Four contigs (two CytP450, NMO, and GT) were consistently highly expressed in nine field-evolved metabolic resistant L. rigidum populations. These four contigs were strongly associated with the resistance phenotype and are major candidates for contributing to metabolic diclofop resistance.

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing–RNA affinity profiling

Abstract: RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

DNA polymerases as useful reagents for biotechnology - the history of developmental research in the field.

Abstract: DNA polymerase is a ubiquitous enzyme that synthesizes complementary DNA strands according to the template DNA in living cells. Multiple enzymes have been identified from each organism, and the shared functions of these enzymes have been investigated. In addition to their fundamental role in maintaining genome integrity during replication and repair, DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, labeling, mutagenesis, and other purposes. The fundamental ability of DNA polymerases to synthesize a deoxyribonucleotide chain is conserved. However, the more specific properties, including processivity, fidelity (synthesis accuracy), and substrate nucleotide selectivity, differ among the enzymes. The distinctive properties of each DNA polymerase may lead to the potential development of unique reagents, and therefore searching for novel DNA polymerase has been one of the major focuses in this research field. In addition, protein engineering techniques to create mutant or artificial DNA polymerases have been successfully developing powerful DNA polymerases, suitable for specific purposes among the many kinds of DNA manipulations. Thermostable DNA polymerases are especially important for PCR-related techniques in molecular biology. In this review, we summarize the history of the research on developing thermostable DNA polymerases as reagents for genetic manipulation and discuss the future of this research field.

Nuclease-mediated gene editing by homologous recombination of the human globin locus

Abstract: Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and β-thalassemia are common genetic disorders caused by mutations in β-globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human β-globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous β-globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human γ-globin, a pharmacologic target in sickle cell disease therapy. Using the β-globin and γ-globin TALENs, we generated cell lines that express GFP under the control of the endogenous β-globin promoter and tdTomato under the control of the endogenous γ-globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous β- and γ-globin genes and identified several that preferentially upregulate γ-globin expression.

A Chrysanthemum Heat Shock Protein Confers Tolerance to Abiotic Stress

Abstract: Heat shock proteins are associated with protection against various abiotic stresses. Here, the isolation of a chrysanthemum cDNA belonging to the HSP70 family is reported. The cDNA, designated CgHSP70, encodes a 647-residue polypeptide, of estimated molecular mass 70.90 kDa and pI 5.12. A sub-cellular localization assay indicated that the cDNA product is deposited in the cytoplasm and nucleus. The performance of Arabidopsis thaliana plants constitutively expressing CgHSP70 demonstrated that the gene enhances tolerance to heat, drought and salinity. When CgHSP70 was stably over-expressed in chrysanthemum, the plants showed an increased peroxidase (POD) activity, higher proline content and inhibited malondialdehyde (MDA) content. After heat stress, drought or salinity the transgenic plants were better able to recover, demonstrating CgHSP70 positive effect.

Characterization of mice expressing Ins1 gene promoter driven CreERT recombinase for conditional gene deletion in pancreatic β-cells.

Abstract: Gene manipulation using Cre-loxP recombination has proven to be an important approach for studying the impact of gene expression on pancreatic β-cell biology. We report the generation of a transgenic mouse line that enables a highly specific system for conditional gene manipulation within β-cells and achieve tissue specific and temporally regulated deletion of the Ctnnb1 (β-catenin) gene in pancreatic β-cells. cDNA encoding Cre recombinase fused to modified estrogen receptor (CreERT) under control of mouse insulin 1 gene promoter (Ins1) was used to construct the mouse line Tg(Ins1-Cre/ERT)1Lphi, also termed MIP1-CreERT. In a cross of MIP1-CreERT with a ROSA26/LacZ reporter strain, tamoxifen [Tmx] - dependent β-galactosidase expression occurred within pancreatic β-cells but not in other organ systems. Intraperitoneal glucose tolerance tests and glucose-stimulated changes in β-cell cytoplasmic calcium concentration were not adversely affected in adult MIP1-CreERT. A mouse line with floxed Ctnnb1 gene (Ctnnb1f/f) was crossed with the MIP1-CreERT line to generate a mouse model for inducible β-cell specific deletion of β-catenin gene (Ctnnb1f/f:MIP1-CreERT). Ctnnb1f/f:MIP1-CreERT mice and Ctnnb1f/f littermate controls, were injected with Tmx as adults to knock down β-catenin production in the majority of pancreatic β-cells. These mice showed normal glucose tolerance, islet cyto-architecture and insulin secretion. A novel protein fraction of 50Kd, immunoreactive with anti-β-catenin was observed in islet extracts from Ctnnb1f/f:MIP1-CreERT[Tmx] mice but not MIP1-CreERT-negative Ctnnb1f/f[Tmx] controls, indicating possible presence of a cryptic protein product of recombined Ctnnb1 gene. The MIP1-CreERT mouse line is a powerful tool for conditional manipulation of gene expression in β-cells.

Quantitative T cell repertoire analysis by deep cDNA sequencing of T cell receptor α and β chains using next-generation sequencing (NGS)

Abstract: Immune responses play a critical role in various disease conditions including cancer and autoimmune diseases. However, to date, there has not been a rapid, sensitive, comprehensive, and quantitative analysis method to examine T-cell or B-cell immune responses. Here, we report a new approach to characterize T cell receptor (TCR) repertoire by sequencing millions of cDNA of TCR α and β chains in combination with a newly-developed algorithm. Using samples from lung cancer patients treated with cancer peptide vaccines as a model, we demonstrate that detailed information of the V-(D)-J combination along with complementary determining region 3 (CDR3) sequences can be determined. We identified extensive abnormal splicing of TCR transcripts in lung cancer samples, indicating the dysfunctional splicing machinery in T lymphocytes by prior chemotherapy. In addition, we found three potentially novel TCR exons that have not been described previously in the reference genome. This newly developed TCR NGS platform can be applied to better understand immune responses in many disease areas including immune disorders, allergies, and organ transplantations.

Rhipicephalus ( Boophilus ) microplus aquaporin as an effective vaccine antigen to protect against cattle tick infestations

Abstract: Vaccination as a control method against the cattle tick, Rhipicephalus (Boophilus) microplus has been practiced since the introduction of two products in the mid-1990s. There is a need for a vaccine that could provide effective control of R. microplus in a more consistent fashion than existing products. During our transcriptome studies of R. microplus, several gene coding regions were discovered to encode proteins with significant amino acid similarity to aquaporins. A cDNA encoding an aquaporin from the cattle tick, Rhipicephalus microplus, was isolated from transcriptomic studies conducted on gut tissues dissected from fully engorged adult female R. microplus. Bioinformatic analysis indicates this aquaporin, designated RmAQP1, shows greatest amino acid similarity to the human aquaporin 7 family. Members of this family of water-conducting channels can also facilitate the transport of glycerol in addition to water. The efficacy of this aquaporin as an antigen against the cattle tick was explored in cattle vaccine trials conducted in Brazil. A cDNA encoding a significant portion of RmAQP1 was expressed as a recombinant protein in Pichia pastoris, purified under native conditions using a polyhistidine C-terminus tag and nickel affinity chromatography, emulsified with Montanide adjuvant, and cattle vaccinated intramuscularly. The recombinant protein provided 75% and 68% efficacy in two cattle pen trials conducted in Campo Grande, Brazil on groups of 6 one year old Holstein calves. The effectiveness of this vaccine in reducing the numbers of adult female ticks shows this aquaporin antigen holds promise as an active ingredient in cattle vaccines targeted against infestations of R. microplus.

Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity

Abstract: LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.

Uniquely tagged rearranged adaptive immune receptor genes in a complex gene set

Abstract: Compositions and methods are disclosed for uniquely tagging each rearranged gene segment that encodes a T cell receptor (TCR) and/or an immunoglobulin (Ig), in a DNA (or mRNA or cDNA reverse transcribed therefrom) sample from lymphoid cells. These and related embodiments permit accurate, high throughput quantification of distinct TCR and/or Ig encoding sequences. Also provided are compositions and methods for quantitatively sequencing the genes that encode both chains of a TCR or Ig heterodimer in a single cell, for example, to characterize the degree of T or B cell clonality in a sample.

Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

Abstract: Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer’s disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4′-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4′-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA

Abstract: Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1α (EF-1α) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoVU201F) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient’s stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1α mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions.

Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.).

Abstract: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses. In this study, we mined the available expressed sequence tags (ESTs) of B. napus and identified a total of 25 CPK genes, among which cDNA sequences of 23 genes were successfully cloned from a double haploid cultivar of canola. Phylogenetic analysis demonstrated that they could be clustered into four subgroups. The subcellular localization of five selected BnaCPKs was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of 21 BnaCPK genes in response to salt, drought, cold, heat, abscisic acid (ABA), low potassium (LK) and oxidative stress were studied by quantitative RT-PCR and were found to respond to multiple stimuli, suggesting that canola CPKs may be convergence points of different signaling pathways. We also identified and cloned five and eight Clade A basic leucine zipper (bZIP) and protein phosphatase type 2C (PP2C) genes from canola and, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC), determined the interaction between individual BnaCPKs and BnabZIPs or BnaPP2Cs (Clade A). We identified novel, interesting interaction partners for some of the BnaCPK proteins. We present the sequences and characterization of CPK gene family members in canola for the first time. This work provides a foundation for further crop improvement and improved understanding of signal transduction in plants.

TIGAR2: sensitive and accurate estimation of transcript isoform expression with longer RNA-Seq reads

Abstract: High-throughput RNA sequencing (RNA-Seq) enables quantification and identification of transcripts at single-base resolution. Recently, longer sequence reads become available thanks to the development of new types of sequencing technologies as well as improvements in chemical reagents for the Next Generation Sequencers. Although several computational methods have been proposed for quantifying gene expression levels from RNA-Seq data, they are not sufficiently optimized for longer reads (e.g. > 250 bp). We propose TIGAR2, a statistical method for quantifying transcript isoforms from fixed and variable length RNA-Seq data. Our method models substitution, deletion, and insertion errors of sequencers based on gapped-alignments of reads to the reference cDNA sequences so that sensitive read-aligners such as Bowtie2 and BWA-MEM are effectively incorporated in our pipeline. Also, a heuristic algorithm is implemented in variational Bayesian inference for faster computation. We apply TIGAR2 to both simulation data and real data of human samples and evaluate performance of transcript quantification with TIGAR2 in comparison to existing methods. TIGAR2 is a sensitive and accurate tool for quantifying transcript isoform abundances from RNA-Seq data. Our method performs better than existing methods for the fixed-length reads (100 bp, 250 bp, 500 bp, and 1000 bp of both single-end and paired-end) and variable-length reads, especially for reads longer than 250 bp.

The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5′-UTR Variant

Abstract: Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5'-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5'-untranslated regions (5'-UTR) of 28 and 234 nucleotides. The transcript containing the long 5'-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5'-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5'-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR. These findings emphasize how 5'-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5'-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.

Identification of the porcine G protein-coupled receptor 41 and 43 genes and their expression pattern in different tissues and development stages.

Abstract: Short-chain fatty acids (SCFAs) are not only an important energy source, but they also play a regulatory role in various physiological processes in humans and rodents. Current studies, mostly in humans and rodents, have revealed that SCFAs acted as endogenous ligands for G protein-coupled receptor GPR41 and GPR43. Whether proteins similar to human GPR41 and GPR43 mediate the regulatory effects of SCFAs in swine remains unclear to date. The aims of this study were to determine whether GPR41 and GPR43 genes are expressed in porcine different tissues; and whether the expression of GPR41 and GPR43 is tissue-specific and/or time-associated. The alignment results showed that pig chromosome 6 contained GPR41 and GPR43 genes. Reverse transcription polymerase chain reaction (RT-PCR) indicated that GPR41 and GPR43 were expressed in porcine various tissues. The 2218 bp and 1908 bp nucleotide sequence representing the full-length cDNA sequence of porcine GPR41 and GPR43 was obtained from the ileum and spleen using rapid amplification of cDNA ends (RACE), which were capable of encoding 335 and 329 amino acid sequences, respectively. The structure prediction revealed that porcine GPR41 and GPR43 proteins had seven putative trans-membrane domains. The real-time PCR results indicated that GPR41 and GPR43 were expressed throughout the developmental stages in a tissue-specific and time-associated manner. GPR41 and GPR43 were most highly expressed in the ileum (P<0.01) and the spleen (P<0.01), respectively. Western blot results showed that porcine GPR41 and GPR43 proteins were expressed in a variety of porcine tissues, including the spleen, ileum, colon, and adipose tissue. In situ GPR41 and GPR43 immunoreactivities were observed through immunohistochemistry in the spleen, ileum, colon, and adipose tissue. In conclusion, the pig genome encoded GPR41 and GPR43 genes, and these two genes were detected in a variety of porcine tissues and expressed in tissue-specific and time-associated manner.

Uromodulin, an Emerging Novel Pathway for Blood Pressure Regulation and Hypertension

Abstract: Tamm–Horsfall protein was discovered in 1950 by Igor Tamm and Frank Horsfall,1 using a salt precipitation procedure to isolate a potent inhibitor of viral hemagglutination from urine. Muchmore and Decker,2 in 1985, isolated a glycoprotein (calling it uromodulin) with in vitro immunosuppressive properties from urine of pregnant women. In 1987, Pennica et al3 confirmed by cDNA analysis that uromodulin and Tamm–Horsfall protein were identical proteins. Since the initial discovery and without any clear understanding of the function of uromodulin, it was only in 2002 when Hart et al4 identified causative uromodulin mutations in a subset of families having familial juvenile hyperuricemic nephropathy and medullary cystic kidney disease type 2 that interest in uromodulin biology and function was revived. Interest in uromodulin was further revitalized by genome-wide association studies (GWASs) in 2009/2010 showing an association between common single-nucleotide polymorphisms in the upstream region of the UMOD gene with renal function and hypertension.5–8 In 2013, 2 independent groups undertaking post-GWAS functional dissection of the UMOD loci provided molecular insights into a new pathway for hypertension and sodium homeostasis involving uromodulin and opening an exciting line of investigation that could enhance our understanding of renal tubule physiology, sodium homeostasis, blood pressure (BP) regulation, and potentially lead to novel therapies for hypertension.9,10 Uromodulin is a protein exclusively expressed by epithelial cells of the thick ascending limb of Henle’s loop (TAL; Figure 1). The 640 amino-acid precursor is cotranslationally translocated into the endoplasmic reticulum (ER), extensively glycosylated, glypiated, and glycosylphosphatidylinositol anchored to the apical tubular cell membrane. From here it is released by a specific, but as yet unidentified, serine protease(s). The released protein is excreted in the urine at a rate of 20 to 100 mg/d and represents the most abundant urinary protein in …

Characterization of Rubisco Activase Genes in Maize: An α-Isoform Gene Functions alongside a β-Isoform Gene

Abstract: Rubisco activase (RCA) catalyzes the activation of Rubisco in vivo and plays a crucial role in regulating plant growth. In maize (Zea mays), only β-form RCA genes have been cloned and characterized. In this study, a genome-wide survey revealed the presence of an α-form RCA gene and a β-form RCA gene in the maize genome, herein referred to as ZmRCAα and ZmRCAβ, respectively. An analysis of genomic DNA and complementary DNA sequences suggested that alternative splicing of the ZmRCAβ precursor mRNA (premRNA) at its 3' untranslated region could produce two distinctive ZmRCAβ transcripts. Analyses by electrophoresis and matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry showed that ZmRCAα and ZmRCAβ encode larger and smaller polypeptides of approximately 46 and 43 kD, respectively. Transcriptional analyses demonstrated that the expression levels of both ZmRCAα and ZmRCAβ were higher in leaves and during grain filling and that expression followed a specific cyclic day/night pattern. In 123 maize inbred lines with extensive genetic diversity, the transcript abundance and protein expression levels of these two RCA genes were positively correlated with grain yield. Additionally, both genes demonstrated a similar correlation with grain yield compared with three C₄ photosynthesis genes. Our data suggest that, in addition to the β-form RCA-encoding gene, the α-form RCA-encoding gene also contributes to the synthesis of RCA in maize and support the hypothesis that RCA genes may play an important role in determining maize productivity.

Isolation and characterization of a pathogenesis-related protein 10 gene (GmPR10) with induced expression in soybean (Glycine max) during infection with Phytophthora sojae.

Abstract: In previous study, a cDNA library enriched for mRNAs encoding ESTs that increased in abundance during infection with Phytophthora sojae was constructed by suppression subtractive hybridization from leaf tissues of a high resistant soybean, and an EST homologous to the class 10 of pathogenesis-related (PR) proteins was identified to be up-regulated by microarray and real-time PCR. Here, the full-length cDNA (termed GmPR10, GenBank accession number FJ960440; ADC31789.1) of the EST was isolated by rapid amplification of cDNA ends, and contains an open reading frame of 474 bp. The GmPR10 protein included a “P-loop’’ motif. The constitutive transcript abundance of GmPR10 in soybean was the highest in leaves, followed by roots and stems. Further analysis showed that GmPR10 mRNA abundance was increased during infection with P. sojae following leaf treatments with gibberellin (GA3), hydrogen peroxide (H2O2), salicylic acid (SA), and abscisic acid (ABA). The dialytically renatured GmPR10 protein significantly inhibited P. sojae hyphal growth and exhibited RNase activity. Transgenic tobacco and soybean plants overexpressing GmPR10 showed increased resistance to P. nicotianae Breda and P. sojae, respectively. These results suggest that the GmPR10 protein plays an important role in host defense against P. sojae infection. To the best of our knowledge, this is the first report on the functional characterization of a PR10 protein from soybean in defense against P. sojae.