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PubMed1996

Advanced mRNA differential display: isolation of a new differentially regulated myosin heavy chain-encoding gene in amphibian limb regeneration.

In an effort to make mRNA differential display more amenable as a molecular screen, we have optimized the technology for the isotopic and non-isotopic detection of differentially regulated mRNAs. The number of amplification rounds in the displays was significantly reduced, resulting in the semi-quantitative detection of expression patterns of both low- and high-abundance transcripts. Moreover, we extended the method beyond the display of mRNAs by introducing a direct sequencing approach for the fast molecular analysis of isolated cDNAs. Applying this improved technique to the regenerating amphibian limb system, we have identified cDNA PCR products with a temporal difference in expression. This differential regulation was confirmed by Northern analysis, and DNA sequencing uncovered a novel newt differentiation-specific transcript encoding a skeletal myosin heavy chain (MHC).

Amino Acid Sequence↗
PubMed1991

BDNF mRNA is decreased in the hippocampus of individuals with Alzheimer's disease.

In recent years, nerve growth factor (NGF) has gained attention as a potential therapeutic agent for Alzheimer's disease (AD). To study the expression of NGF and its homologs, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), postmortem samples of hippocampus from AD and control donors were examined by in situ hybridization. Hybridization signal for BDNF, but not NGF or NT-3, was decreased in samples of hippocampus from donors with AD. Decreased transcript abundance of BDNF mRNA in hippocampi of individuals with AD was verified by an RNAase protection assay. These results suggest the possibility that decreased expression of BDNF may contribute to the progression of cell death in AD.

Alzheimer Disease↗
PubMed2004

Mouse striatal transcriptome analysis: effects of oral self-administration of alcohol.

Results of recent studies support the notion that substance self-administration is partially a genetically controlled component of addiction tied to habit formation and cellular modification of the striatum. Aiming to define pathways among genomic, neural, and behavioral determinants of addiction, we investigated global striatal gene expression in a paradigm of oral self-administration of alcohol by using genomically very similar alcohol-nonpreferring B6.Cb(5)i(7)-alpha 3/Vad (C5A3) and alcohol-preferring B6.Ib(5)i(7)-beta 25A/Vad (I5B25A) quasi-congenic mouse strains and their progenitors, C57BL/6By (B6By) and BALB/cJ. Expression of 12,488 genes and expressed sequence tags (ESTs) was studied by using 24 high-density oligonucleotide microarrays. Transcript signal intensity differences were analyzed with z test after iterative median normalization across groups and Hochberg step-down Bonferroni procedure. As expected, striatal transcriptome differences were far more extensive between the independently derived progenitor strains than between the quasi-congenic strains and their background partner, B6By. However, the genes, which were differentially expressed between the quasi-congenic strains and their background partner, were not subsets of the progenitorial differences and were not located on the chromosome segments introgressed into the quasi-congenic strains from the donor BALB/cJ strain that have been so far defined. Although 25 transcripts showed significantly different expression between the progenitor strains, only two transcripts, phosphatidylserine decarboxylase and a hypothetical 21.2-kDa protein, and one transcript, molybdenum co-factor synthesis 2, showed significantly different expression between C5A3 and I5B25A, and between B6By and I5B25A, respectively. The latter three transcripts are not located on previously identified chromosome segments introgressed from the donor BALB/cJ strain, supporting the suggestion of trans-acting regulatory variations among strains. Exposure to alcohol did not induce statistically significant striatal gene expression changes in any of the mouse strains. In conclusion, the results support the hypothesis that in functional genomic studies the chance of detecting function-relevant genes can be increased by the comparative analysis of quasi-congenic and background strains because the number of functionally irrelevant, differentially expressed genes between genomically similar strains is reduced. Lack of statistically significant alcohol-induced changes in transcript abundance indicated that oral self-administration had subtle effects on striatal gene expression and directed attention to important implications for the experimental design of future microarray gene expression studies on complex behaviors.

Administration, Oral↗
PubMed2005

Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants.

Chlorophyllide a oxygenase (CAO) that converts chlorophyllide a to chlorophyllide b was overexpressed in tobacco to increase chlorophyll (Chl) b biosynthesis and alter the Chl a/b ratio. Transgenic plants along with their wild-type cultivars were grown in low and high light intensities. In low light there was 20% increase in chlorophyll b contents in transgenic plants, which resulted in 16% reduction in the Chl a/b ratio. In high light, total Chl contents were 31% higher in transgenic plants than those of wild type. The increase in Chl a was 19% and that of Chl b was 72% leading to 31% decline of Chl a/b ratio. The increase in Chl b contents was accompanied by enhanced CAO expression that was highly pronounced in low light. As compared to low light, in high light Lhcb1 and Chl a/b transcripts abundance was significantly increased in transgenic plants suggesting a close relationship between Chl b synthesis and cab gene expression. However, there was a small increase in expression of LHCII proteins, which did not correspond to 72% increase in Chl b content in transgenic line, implying that LHCPII has the ability to bind more Chl b molecules.

Arabidopsis↗
PubMed2006

Structural and functional properties of lengsin, a pseudo-glutamine synthetase in the transparent human lens.

Lengsin (LGS) is an abundant transcript in the human lens, encoding a predicted polypeptide similar to glutamine synthetase (GS). We show that a major alternatively spliced product of LGS codes for a 57kDa polypeptide that assembles into a catalytically inactive dodecamer, cross-reacts with anti-GS antibodies, and is expressed at high levels in transparent, but not cataractous, human lenses. Based on this characteristic oligomeric organization, preferential expression in the transparent lens, and amyloid-beta association previously reported for GS, a potential chaperone-like role of LGS has been investigated. We find that LGS has six binding sites for the hydrophobic surface probe bis-ANS and relieves cellular toxicity caused by amyloid-beta expression in a folding-impaired yeast mutant. While documenting the structural similarity between LGS and prokaryotic GS-I, the data rule out any involvement of lengsin in glutamine biosynthesis and suggest an unrelated role that may be important for lens homeostasis and transparency.

Alternative Splicing↗
PubMed2006

Isolation of erythroid cells from the mouse embryonic yolk sac by laser capture microdissection and subsequent microarray hybridization.

Erythropoietic tissues are complex, containing both erythroid and other cells. The embryonic yolk sac in particular contains primitive erythroid cells in low abundance. Laser capture microdissection (LCM) was performed to isolate erythroid cells, and epithelial cells, from mouse embryonic day 10 (E10) yolk sac. Quantitative RT-PCR was performed to confirm that enriched cell populations were obtained. epsilony- and betaH1-globin mRNAs were enriched in the erythroid compared to the epithelial fraction, and villin mRNA was enriched in the epithelial compared to the erythroid fraction. RNA isolated from the microdissected erythroid cells was of high quality as indicated by capillary electrophoresis. The RNA from the LCM erythroid fraction was linearly amplified with T7 RNA polymerase and hybridized to a Mouse 430A 2.0 Affymetrix array. Forty-eight percent of genes were present in the microarray assays, including low abundance transcripts such as erythroid transcription factors and enzymes involved in heme synthesis. With the LCM/microarray strategy, it will be possible to identify genes that are differentially regulated in native primitive and definitive erythroid cells.

Animals↗
PubMed2004

Application and optimization of microarray technologies for human postmortem brain studies.

A number of microarray investigations using human postmortem brain tissue have been published recently, exploring a multitude of human brain disorders with the aim of unraveling the underlying pathologies. Although the technology is still developing and lacks sufficient sensitivity with regard to detecting splice variants and low abundance transcripts, microarrays are becoming the prominent method for candidate gene screening in complex neuropsychiatric disorders. The use of postmortem tissue harbors a variety of potential pitfalls, however, which could result in unreliable or, at worst, meaningless results. During the course of our large-scale gene expression study on 150 human postmortem brain samples, using more than 200 Affymetrix GeneChips, we have identified several aspects within microarray experimental procedure that allows for the early identification of potentially unreliable samples. The general application of the guidelines and technical tips described here increase the efficiency, reliability, and amount of data generated by this powerful screening technology while reducing superfluous consumption of time and resources.

Brain Chemistry↗
PubMed2026

Genome-wide characterization of heat shock protein genes reveals thermal stress-responsive candidates in Litopenaeus vannamei.

Heat shock proteins (HSPs) are conserved molecular chaperones involved in protein folding, refolding, aggregation prevention, and degradation of damaged proteins. However, the genomic organization and thermal responsiveness of HSP genes in the Pacific white shrimp (Litopenaeus vannamei) remain incompletely understood. Here, we performed a genome-wide analysis of the HSP gene family and examined its phylogenetic relationships, structural features, duplication patterns, sequence variation, interaction networks, and transcriptional responses to acute heat stress. A total of 34 HSP genes were identified and classified into the HSP90, HSP70, HSP40/DNAJ, HSP60, and small HSP families. Phylogenetic, motif, gene structure, synteny, and subcellular localization analyses revealed evolutionary conservation and structural diversification among family members. Three duplicated gene pairs were identified, comprising two segmental duplications and one tandem duplication. All pairs exhibited Ka/Ks ratios below 1, consistent with purifying selection of varying strength. Sequence analysis identified 295 nonsynonymous single-nucleotide polymorphisms, of which 12 were consistently predicted to be deleterious by multiple algorithms. Protein-protein interaction analysis indicated enrichment of protein-folding and cellular stress-response functions. RT-qPCR analysis showed significant induction of HSPA4, HSP90AA1, TRAP1, BiP, and DNAJA1 after 6, 12, and 24 h of exposure to 34 °C, whereas DNAJC3 was significantly induced only at 12 h. All six genes reached their highest transcript abundance at 12 h. These findings may provide a genomic framework for HSP genes in L. vannamei and identify candidate genes and variants associated with thermal stress responses.

Animals↗
PubMed2026

PRDX1 facilitates USP7-dependent stabilization of SCD1 and promotes bladder cancer progression.

Bladder cancer is characterized by redox adaptation and metabolic plasticity, but the mechanisms linking these processes remain incompletely understood. Integrating bulk, single-cell, and spatial transcriptomic analyses, we identified PRDX1 as a malignant epithelial cell-associated factor linked to adverse outcome. Genetic gain- and loss-of-function studies showed that PRDX1 promoted proliferation, motility, and xenograft growth while limiting reactive oxygen species accumulation and mitochondrial apoptosis. Proteomic and biochemical analyses identified an association between PRDX1 and SCD1. PRDX1 prolonged the SCD1 protein half-life without detectably altering SCD1 transcript abundance and increased USP7-SCD1 co-precipitation. USP7 removed K48-linked polyubiquitin chains from SCD1 and prevented its proteasomal degradation, whereas catalytically inactive USP7 failed to deubiquitinate SCD1. Deletion of PRDX1 residues 157-199 weakened its association with SCD1 and reduced USP7-SCD1 co-precipitation. Depletion of SCD1 or USP7 suppressed PRDX1-dependent growth in vitro and in xenografts. These findings support a model in which PRDX1 facilitates USP7-dependent stabilization of SCD1 and promotes bladder cancer progression.

Bladder cancer↗
PubMed2005

Real-time PCR quantification of the in vitro effects of crustacean immunostimulants on gene expression in lobster (Homarus gammarus) granular haemocytes.

This paper presents data from an investigation of the mode of action of five different crustacean immunostimulants presented to European lobster (Homarus gammarus) granulocytes cultured in vitro. The experiments were designed to test whether or not the immunostimulants could cause the short-term up-regulation of genes coding for immune proteins without causing the cells to degranulate. Quantitative measurements of mRNA transcript abundance were made using real-time PCR and it was first necessary to isolate the complete gene sequences coding for the proteins prophenoloxidase (proPO), beta-1,3-glucan binding protein (betaGBP) and beta-actin (beta-act) in the lobster. These sequences were used to design TaqMan primer and fluorescent probe sets. The presented data indicated that the majority of the tested immunostimulants did not induce the up-regulation of immune-related gene expression in the granulocytes in isolation. Alternative modes of action, including the in vivo up-regulation of gene expression in haemopoetic tissues, are discussed.

Actins↗
PubMed2004

Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos.

A comprehensive analysis of transposable element (TE) expression in mammalian full-grown oocytes reveals that LTR class III retrotransposons make an unexpectedly high contribution to the maternal mRNA pool, which persists in cleavage stage embryos. The most abundant transcripts in the mouse oocyte are from the mouse transcript (MT) retrotransposon family, and expression of this and other TE families is developmentally regulated. Furthermore, TEs act as alternative promoters and first exons for a subset of host genes, regulating their expression in full-grown oocytes and cleavage stage embryos. To our knowledge, this is the first example of TEs initiating synchronous, developmentally regulated expression of multiple genes in mammals. We propose that differential TE expression triggers sequential reprogramming of the embryonic genome during the oocyte to embryo transition and in preimplantation embryos.

Animals↗
PubMed2006

Meloidogyne incognita: molecular and biochemical characterisation of a cathepsin L cysteine proteinase and the effect on parasitism following RNAi.

RNA interference has been used to investigate the function of a cathepsin L cysteine proteinase Mi-cpl-1, in the plant-parasitic nematode Meloidogyne incognita. A reduction in gene transcript was observed and the number of nematodes infecting plants was reduced by almost 60% as was the number of established females producing eggs at 21 days post-infection. The cysteine proteinase activity of M. incognita, reported by the substrate GLUpNA, was inhibited by the cysteine proteinase inhibitor Oc-IDeltaD86. A reduction in cysteine proteinase activity was also seen following RNAi of Mi-cpl-1 in J2 stage nematodes. In situ hybridization analysis in young and mature female nematodes has shown that Mi-cpl-1 is expressed in the intestine, which suggests that its product is a digestive enzyme. The effects of knocking-out Mi-cpl-1gene function were consistent with a reduction in feeding efficiency. Here, we have shown a correlation between transcript abundance proteinase activity and parasitic success of M. incognita.

Animals↗
PubMed2004

Function of alternative splicing.

Alternative splicing is one of the most important mechanisms to generate a large number of mRNA and protein isoforms from the surprisingly low number of human genes. Unlike promoter activity, which primarily regulates the amount of transcripts, alternative splicing changes the structure of transcripts and their encoded proteins. Together with nonsense-mediated decay (NMD), at least 25% of all alternative exons are predicted to regulate transcript abundance. Molecular analyses during the last decade demonstrate that alternative splicing determines the binding properties, intracellular localization, enzymatic activity, protein stability and posttranslational modifications of a large number of proteins. The magnitude of the effects range from a complete loss of function or acquisition of a new function to very subtle modulations, which are observed in the majority of cases reported. Alternative splicing factors regulate multiple pre-mRNAs and recent identification of physiological targets shows that a specific splicing factor regulates pre-mRNAs with coherent biological functions. Therefore, evidence is now accumulating that alternative splicing coordinates physiologically meaningful changes in protein isoform expression and is a key mechanism to generate the complex proteome of multicellular organisms.

Alternative Splicing↗
PubMed2005

Characterization of the origin recognition complex (ORC) from a higher plant, rice (Oryza sativa L.).

The origin recognition complex (ORC) protein plays a critical role in DNA replication through binding to sites (origins) where replication commences. The protein is composed of six subunits (ORC1 to 6) in animals and yeasts. Our knowledge of the ORC protein in plants is, however, much less complete. We have performed cDNA cloning and characterization of ORC subunits in rice (Oryza sativa L. cv. Nipponbare) in order to facilitate study of plant DNA replication mechanisms. Our previous report provided a description of a gene, ORC1 (OsORC1), that encodes one of the protein subunits. The present report extends this initial analysis to include the genes that encode four other rice ORC subunits, OsORC2, 3, 4 and 5. Northern hybridization analyses demonstrated the presence of abundant transcripts for all OsORC subunits in shoot apical meristems (SAM) and cultured cells, but not in mature leaves. Interestingly, only OsORC5 showed high levels of expression in organs in which cell proliferation is not active, such as flag leaves, the ears and the non-tip roots. The pattern of expression of OsORC2 also differed from other OsORC subunits. When cell proliferation was temporarily halted for 6-10 days by removal of sucrose from the growth medium, expression of OsORC1, OsORC3, OsORC4 and OsORC5 was substantially reduced. However, the level of expression of OsORC2 remained constant. We suggest from these results that expression of OsORC1, 3, 4 and 5 are correlated with cell proliferation, but the expression of OsORC2 is not.

Biolistics↗
PubMed2005

Characterization of the 3' untranslated region of the human mu-opioid receptor (MOR-1) mRNA.

The mu-opioid receptor (MOR) plays a mandatory role in the action of most opioid drugs, such as morphine, fentanyl, and heroin. It has been revealed that a deficiency in the MOR gene (Oprm1) or a difference in the 3' noncoding region of the gene markedly affects the sensitivity of mice to opioids. As the 3' noncoding region of the human OPRM1 gene had not yet been characterized, in the present study we conducted 3'-rapid amplification of cDNA ends (3'RACE)-PCR and identified the 3' end of the human MOR-1 mRNA, the most abundant transcript among OPRM1 gene transcripts. The poly(A) signal was located at 13612-13617 nucleotides downstream from the stop codon in the OPRM1 gene. Reverse transcription PCR analyses showed that the region from the stop codon to the poly(A) signal was transcribed. In the 3'UTR, we identified 33 AU-rich regions and more than 300 putative transcription factor-binding sites. Furthermore, we compared the 3' noncoding regions of the human and mouse OPRM1/Oprm1 genes and found apparent homology. In Northern blotting with mouse brain mRNAs, a same-size band was detected by a probe for the MOR-1 coding region and by a probe for a mouse genome region corresponding to the human MOR-1 3'UTR. Since 3'UTRs affect gene expression, the present characterization of the 3' noncoding region in the human OPRM1 gene should lead to a better understanding of the mechanisms underlying OPRM1 gene regulation and individual differences in sensitivity to opioids.

3' Untranslated Regions↗
PubMed2005

Regulatory mechanisms of chitin biosynthesis and roles of chitin in peritrophic matrix formation in the midgut of adult Aedes aegypti.

In mosquitoes, the peritrophic matrix is formed in response to blood feeding and can be a physical barrier when pathogens ingested with blood meal attempt to reach and transverse the midgut epithelium. The main components of the peritrophic matrix are chitin-biding-domain containing proteins, glycosylated proteins, and chitin fibrils. Chitin is synthesized from fructose-6-phosphate by a series of five enzymatic reactions. We previously found that blood feeding induces transcriptional up-regulation of glutamine: fructose-6-phosphate amidotransferase-1 (AeGfat-1) and chitin synthase (AeCs), the first and last enzymes of the biosynthetic pathway, respectively, in the midgut of Aedes aegypti. In this study, we demonstrated that formation of the peritrophic matrix is disrupted when the transcript abundance of either gene is knocked-down using RNAi methodologies. We also have shown that enzymatic activity of recombinant AeGFAT-1 is sensitive to feedback inhibition by UDP-N-acetylglucosamine, a substrate of chitin synthase. These findings demonstrate that in the midgut of adult Ae. aegypti, (1) chitin is synthesized de novo in response to blood feeding and is an essential component of the peritrophic matrix, and (2) chitin biosynthesis is negatively regulated, in part, by inhibitory sensitivity of AeGFAT-1 to UDP-N-acetylglucosamine.

Aedes↗
PubMed2006

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy.

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion.

Amino Acid Sequence↗
PubMed2006

Identification and analysis of genes expressed in the adult filarial parasitic nematode Dirofilaria immitis.

The heartworm Dirofilaria immitis is a filarial parasitic nematode infecting dogs and other mammals worldwide causing fatal complications. Here, we present the first large-scale survey of the adult heartworm transcriptome by generation and analysis of 4005 expressed sequence tags, identifying about 1800 genes and expanding the available sequence information for the parasite significantly. Brugia malayi genomic data offered the most valuable information to interpret heartworm genes, with about 70% of D. immitis genes showing significant similarities to the assembly. Comparative genomic analyses revealed both genes common to metazoans or nematodes and genes specific to filarial parasites that may relate to parasitism. Characterization of abundant transcripts suggested important roles for genes involved in energy generation and antioxidant defense in adults. In particular, we proposed that adult heartworm likely adopted an anaerobic electron transfer-based energy generation system distinct from the aerobic pathway utilized by its mammalian host, making it a promising target in developing next generation macrofilaricides and other treatments. Our survey provided novel insights into the D. immitis transcriptome and laid a foundation for further comparative studies on biology, parasitism and evolution within the phylum Nematoda.

Animals↗