http://www.biomedcentral.com/bmcmolbiol/ The latest research articles published by BMC Molecular Biology 2009-07-06T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ Background: Laser capture microdissection enables the isolation of single cells or small cell groups from histological sections under direct microscopic observation. Combined with quantitative PCR or microarray, it is a very powerful approach for studying gene expression profiles in discrete cell populations. The major challenge for such studies is to obtain good quality RNA from small amounts of starting material. Results: We have developed a simple, flexible, and low-cost method for simultaneously producing RNA from discrete cell groups in embryonic day 15 mouse brain. In particular, we have optimized the following key steps in the procedure: staining, cryosectioning, storage of sections and harvesting of microdissected cells. We obtained the best results when staining 20 mum-thick sections with 1% cresyl violet in 70% ethanol and harvesting the microdissected tissue in RNA stabilization solution. In addition, we introduced three stop-points in the protocol which makes the tedious process of laser capture microdissection more flexible, without compromising RNA quality. Conclusions: Using this optimized method, we have consistently obtained RNA of high quality from all four simultaneously microdissected cell groups. RNA integrity numbers were all above 8, and long cDNA fragments (> 1.2 kb) were successfully amplified by reverse transcription PCR from all four samples. We conclude that RNAs isolated by this method are well suited for downstream quantitative PCR or microarray studies. http://www.biomedcentral.com/1471-2199/10/69 Wei-Zhi Wang Franziska Oeschger Sheena Lee Zoltan Molnar BMC Molecular Biology 2009, 10:69 2009-07-06T00:00:00Z doi:10.1186/1471-2199-10-69 BMC Molecular Biology 1471-2199 10 69 2009-07-06T00:00:00Z PDF Background: The choroid plexus consists of highly differentiated epithelium and functions as a barrier at the interface of the blood-cerebrospinal-fluid (CSF). This tissue may therefore determine the bioavailability and transport of drugs to the brain. Little is known about the expression of drug and xenobiotic metabolizing enzymes (DME) and of drug transporters in the human choroid plexus. Notably, the transcription factor and zinc finger protein HNF4alpha is a master regulator of DMEs and of drug transporters. As of today its activity in the blood-CSF barrier is unknown. Here we report our efforts in determining HNF4alpha activity in the regulation of ABC transporters in the human and rat choroid plexus. Results: We report expression of HNF4alpha by qRT-PCR and by immunohistochemistry and evidence transcript expression of the ATP-binding cassette transporters ABCB1, ABCB4, ABCC1-6 in choroid plexus. Additionally, HNF4alpha DNA binding activity at regulatory sequences of ABCB4 and ABCC1 was determined by EMSA bandshift assays with a specific antibody. We then performed siRNA mediated functional knock down of HNF4alpha in Caco-2 cells and found ABCC1 gene expression to be repressed in cell culture experiments. Conclusions: Our study evidences activity of HNF4alpha in human and rat choroid plexus. This transcription factor targets DMEs and drug transporters and may well determine availability of drugs at the blood-CSF barrier. http://www.biomedcentral.com/1471-2199/10/68 Monika Niehof Juergen Borlak BMC Molecular Biology 2009, 10:68 2009-07-03T00:00:00Z doi:10.1186/1471-2199-10-68 BMC Molecular Biology 1471-2199 10 68 2009-07-03T00:00:00Z PDF Background: Many mammalian genes are organized as bidirectional (head-to-head) gene pairs with the two genes separated only by less than 1 kb. The transcriptional regulation of these bidirectional gene pairs remains largely unclear, but a few studies have suggested that the two closely adjacent genes in divergent orientation can be co-regulated by a single transcription factor binding to a specific regulatory fragment. Here we report an evolutionarily conserved bidirectional gene pair, known as the PREPL-C2ORF34 gene pair, whose transcription relies on the synergic cooperation of two transcription factors binding to an intergenic bidirectional minimal promoter. Results: While PREPL is present primarily in brain and heart, C2ORF34 is ubiquitously and abundantly expressed in almost all tissues. Genomic analyses revealed that these two non-homologous genes are adjacent in a head-to-head configuration on human chromosome 2p21 and separated by only 405 bp. Within this short intergenic region, a 243-bp GC-rich segment was demonstrated to function as a bidirectional minimal promoter to initiate the transcription of both flanking genes. Two key transcription factors, NRF-2 and YY-1, were further identified to coordinately participate in driving both gene expressions in an additive manner. The functional cooperation between these two transcription factors, along with their genomic binding sites and some cis-acting repressive elements, are essential for the transcriptional activation and tissue distribution of the PREPL-C2ORF34 bidirectional gene pair. Conclusion: This study provides new insights into the complex transcriptional mechanism of a mammalian head-to-head gene pair which requires cooperative binding of multiple transcription factors to a bidirectional minimal promoter of the shared intergenic region. http://www.biomedcentral.com/1471-2199/10/67 Chien-Chang Huang Wun-Shaing Chang BMC Molecular Biology 2009, 10:67 2009-07-03T00:00:00Z doi:10.1186/1471-2199-10-67 BMC Molecular Biology 1471-2199 10 67 2009-07-03T00:00:00Z PDF Background: Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input. Results: Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked. Conclusions: RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP. http://www.biomedcentral.com/1471-2199/10/66 Jens Stern-Straeter Gabriel Bonaterra Karl Hormann Ralf Kinscherf Ulrich Goessler BMC Molecular Biology 2009, 10:66 2009-07-02T00:00:00Z doi:10.1186/1471-2199-10-66 BMC Molecular Biology 1471-2199 10 66 2009-07-02T00:00:00Z PDF Background: : Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae. Results: : We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae. Conclusions: : We have characterized vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression. http://www.biomedcentral.com/1471-2199/10/65 Philippos Papathanos Nikolai Windbichler Miriam Menichelli Austin Burt Andrea Crisanti BMC Molecular Biology 2009, 10:65 2009-07-02T00:00:00Z doi:10.1186/1471-2199-10-65 BMC Molecular Biology 1471-2199 10 65 2009-07-02T00:00:00Z PDF Background: Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells. Results: By RT PCR and western blot analysis we found that nucleolin is strongly down regulated in meiotic spermatocytes and haploid germ cells. We have identified a new nucleolin related protein (NRP) gene in the rat genome, which is over expressed in the testis and is up regulated several fold in meiotic spermatocytes and haploid germ cells. The NRP protein lacks the acidic stretches in its N terminal domain, and it is encoded in rat chromosome 15 having a different genomic organization as compared to nucleolin gene present on chromosome 9. We have also found NRP genes encoded in genomes of other mammalian species. We performed run-on transcription assay where we have observed that rDNA is transcribed at much lower level in meiotic spermatocytes and haploid spermatids as compared to diploid cells. By siRNA knock down experiments we could also demonstrate that NRP can support rDNA transcription in the absence of nucleolin. Conclusion: We have identified a new nucleolin variant over expressed in germ cells in rat and analyzed its domain structure. We attribute that the transcriptional activity of rDNA genes in the late spermatogenesis is due to the presence of this variant NRP. The expression of this variant in the germ cells in the absence of nucleolin, could have additional functions in the mammalian spermatogenesis which needs to be investigated further. http://www.biomedcentral.com/1471-2199/10/64 Keerthi Chathoth Gayatri Ganesan M Rao BMC Molecular Biology 2009, 10:64 2009-07-01T00:00:00Z doi:10.1186/1471-2199-10-64 BMC Molecular Biology 1471-2199 10 64 2009-07-01T00:00:00Z PDF Background: Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data. Results: Here, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes rpoC, alaS and map result in improved interpretation of gene expression profiles in A. ferrooxidans. Conclusions: This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems. http://www.biomedcentral.com/1471-2199/10/63 Pamela Nieto Paulo Covarrubias Eugenia Jedlicki David Holmes Raquel Quatrini BMC Molecular Biology 2009, 10:63 2009-06-25T00:00:00Z doi:10.1186/1471-2199-10-63 BMC Molecular Biology 1471-2199 10 63 2009-06-25T00:00:00Z PDF Background: Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its settlement behaviour. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR) data obtained from different developmental stages of this animal. Results: We generated a cDNA library containing expressed sequence tags (ESTs) from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled cluster and 530 singlets) were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being Cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. Conclusions: The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies. In order to minimize sample variability, we recommend that future molecular genetic studies of B. amphitrite that utilise RNA should pay particular attention to the RNA extraction step. http://www.biomedcentral.com/1471-2199/10/62 Tristano Bacchetti De Gregoris Marco Borra Elio Biffali Thomas Bekel J Burgess Richard Kirby Anthony Clare BMC Molecular Biology 2009, 10:62 2009-06-24T00:00:00Z doi:10.1186/1471-2199-10-62 BMC Molecular Biology 1471-2199 10 62 2009-06-24T00:00:00Z PDF Background: Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with Clostridium perfringens enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes. Results: Immunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE116-319 fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture made it possible to discriminate specific binding from non-specific association to GST and/or matrix material. Conclusions: CPE116-319 provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions. http://www.biomedcentral.com/1471-2199/10/61 Dorte Lohrberg Eberhard Krause Michael Schumann Jorg Piontek Lars Winkler Ingolf Blasig Reiner Haseloff BMC Molecular Biology 2009, 10:61 2009-06-22T00:00:00Z doi:10.1186/1471-2199-10-61 BMC Molecular Biology 1471-2199 10 61 2009-06-22T00:00:00Z PDF Background: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. While eRF1 recognizes nonsense codons, eRF3 facilitates polypeptide chain release from the ribosome in a GTP-dependent manner. Besides termination, both release factors have essential, but poorly characterized functions outside of translation. Results: To characterize further the functions of yeast eRF1 and eRF3, a genetic screen for their novel partner proteins was performed. As a result, the genes for γ (TEF4 and TEF3/CAM1) and α (TEF5/EFB1) subunits of the translation elongation factor eEF1B, known to catalyze the exchange of bound GDP for GTP on eEF1A, were revealed. These genes act as dosage suppressors of a synthetic growth defect caused by some mutations in the SUP45 and SUP35 genes encoding eRF1 and eRF3, respectively. Extra copies of TEF5 and TEF3 can also suppress the temperature sensitivity of some sup45 and sup35 mutants and reduce nonsense codon readthrough caused by these omnipotent suppressors. Besides, overproduction of eEF1Bα reduces nonsense codon readthrough in the strain carrying suppressor tRNA. Such effects were not shown for extra copies of TEF2, which encodes eEF1A, thus indicating that they were not due to eEF1A activation. Conclusion: The data obtained demonstrate involvement of the translation elongation factor eEF1B in modulating the functions of translation termination factors and suggest its possible role in GDP for GTP exchange on eRF3. http://www.biomedcentral.com/1471-2199/10/60 Igor Valouev Gleb Fominov Elizaveta Sokolova Vladimir Smirnov Michael Ter-Avanesyan BMC Molecular Biology 2009, 10:60 2009-06-22T00:00:00Z doi:10.1186/1471-2199-10-60 BMC Molecular Biology 1471-2199 10 60 2009-06-22T00:00:00Z XML