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/ http://www.biomedcentral.com/bmcmolbiol/ The latest articles from BMC Molecular Biology (ISSN 1471-2199) published by BioMed Central Background: Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference genes are generally used to normalize RT qPCR data. Given that expression of some commonly used reference genes is altered in certain conditions, this study aimed to establish which reference genes were stably expressed in post mortem brain tissue from individuals with AD, PD or DLB. Results: The present study investigated the expression stability of 8 candidate reference genes, (ubiquitin C [UBC], tyrosine-3-monooxygenase [YWHAZ], RNA polymerase II polypeptide [RP II], hydroxymethylbilane synthase [HMBS], TATA box binding protein [TBP], beta-2-microglobulin [B2M], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], and succinate dehydrogenase complex-subunit A, [SDHA]) in cerebellum and medial temporal gyrus of 6 AD, 6 PD, 6 DLB subjects, along with 5 matched controls using RT qPCR (TaqMan Gene Expression Assays). Gene expression stability was analysed using geNorm to rank the candidate genes in order of decreasing stability in each disease group. The optimal number of genes recommended for accurate data normalization in each disease state was determined by pairwise variation analysis. Conclusions: This study identified validated sets of mRNAs which would be appropriate for the normalization of RT qPCR data when studying gene expression in brain tissue of AD, PD, DLB and control subjects. http://www.biomedcentral.com/1471-2199/9/46 David T R Coulson, Simon Brockbank, Joseph G Quinn, Suzanne Murphy, Rivka Ravid, G BRENT Irvine and Janet A Johnston BMC Molecular Biology 2008, 9:46 2008-05-06 doi:10.1186/1471-2199-9-46 BMC Molecular Biology 1471-2199 9 46 2008-05-06 Background: NKX3.1 and PCAN1 are both prostate-specific genes related to prostate development and prostate cancer. So far, little is known about the regulatory mechanisms of the expression of these two genes. In the present study, we found that NKX3.1 upregulated PCAN1 gene transcription in LNCaP prostate cancer cells. To understand the regulatory mechanisms, our work focused on identifying the functional NKX3.1 binding sites upstream of the PCAN1 gene, which might be involved in the positive regulation of PCAN1 expression by NKX3.1. Results: We cloned and characterized a 2.6 kb fragment upstream of the PCAN1 gene. Analysis of the 2.6 kb sequence with MatInspector 2.2 revealed five potential binding sites of NKX3.1 transcription factor. Luciferase reporter assays, electrophoretic mobility shift assays and chromatin immunoprecipitation were performed to study the effects of NKX3.1 on PCAN1 gene expression in prostate cancer cells. Our results showed that PCAN1 promoter activity and mRNA expression were increased by transfection with the NKX3.1 containing plasmid (pcDNA3.1-NKX3.1) in prostate cancer cells. The results of electrophoretic mobility shift assays and chromatin immunoprecipitation showed that NKX3.1 bound to NBS1 (-1848 to -1836) and NBS3 (-803 to -791) upstream of the PCAN1 gene. The luciferase reporter assays showed that NBS1 and NBS3 enhanced the promoter activity in pGL3-promoter vector with cotransfection of the NKX3.1 containing plasmid. Furthermore, the deletion of NBS1 or both NBS1 and NBS3 reduced PCAN1 promoter activity and abolished the positive regulation of PCAN1 expression by NKX3.1. Conclusions: Our results suggested that two functional NKX3.1 binding sites located at -1848 to -1836 and -803 to -791 upstream of the PCAN1 gene were involved in the positive regulation of PCAN1 gene transcription by NKX3.1. http://www.biomedcentral.com/1471-2199/9/45 Wenwen Liu, Pengju Zhang, Weiwen Chen, Chunxiao Yu, Fuai Cui, Feng Kong, Jianye Zhang and Anli Jiang BMC Molecular Biology 2008, 9:45 2008-05-04 doi:10.1186/1471-2199-9-45 BMC Molecular Biology 1471-2199 9 45 2008-05-04 Background: In mammals, sex determination is genetically controlled. The SRY gene, located on Y chromosome, functions as a dominant genetic switch for testis development. The SRY gene is specifically expressed in a subpopulation of somatic cells (pre-Sertoli cells) of the developing urogenital ridge for a brief period during gonadal differentiation. Despite this tight spatiotemporal expression pattern, the molecular mechanisms that regulate SRY transcription remain poorly understood. Sry expression has been shown to be markedly reduced in transgenic mice harboring a mutant GATA4 protein (a member of the GATA family of transcription factors) disrupted in its ability to interact with its transcriptional partner FOG2, suggesting that GATA4 is involved in SRY gene transcription. Results: Although our results show that GATA4 directly targets the pig SRY promoter, we did not observe similar action on the mouse and human SRY promoters. In the mouse, Wilms' tumor 1 (WT1) is an important regulator of both Sry and Mullerian inhibiting substance (Amh/Mis) expression and in humans, WT1 mutations are associated with abnormalities of sex differentiation. GATA4 transcriptionally cooperated with WT1 on the mouse, pig, and human SRY promoters. Maximal GATA4/WT1 synergism was dependent on WT1 but not GATA4 binding to the SRY promoter and required both the zinc finger and C-terminal regions of the GATA4 protein. Although both isoforms of WT1 synergized with GATA4, synergism was stronger with the +KTS rather than the -KTS isoform. WT1/GATA4 synergism was also observed on the AMH promoter. In contrast to SRY, WT1/GATA4 action on the mouse Amh promoter was specific for the -KTS isoform and required both WT1 and GATA4 binding. Conclusions: Our data therefore provide new insights into the molecular mechanisms that contribute to the tissue-specific expression of the SRY and AMH genes in both normal development and certain syndromes of abnormal sex differentiation. http://www.biomedcentral.com/1471-2199/9/44 Yoko Miyamoto, Hiroaki Taniguchi, Frederic Hamel, David W Silversides and Robert S Viger BMC Molecular Biology 2008, 9:44 2008-04-29 doi:10.1186/1471-2199-9-44 BMC Molecular Biology 1471-2199 9 44 2008-04-29 Background: Senescence marker protein-30 (SMP30), whose expression declines during aging in rat liver, has been proposed as an important aging marker. Besides apoptosis, SMP30 also protects cells against various other injuries by enhancement of membrane calcium-pump activity. The mechanism of this differential gene expression mechanism is not known. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed to elucidate the mechanism of transcriptional regulation of SMP30 gene. Results: We have characterized up to -2750 bp of the promoter by DNA-protein interactions studies. 28 transcription factor binding sites have been identified by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Transient transfection of 5 and 3-deleted promoter-reporter constructs and luciferase assay illustrated the region between -128/ +157 bp is sufficient to drive promoter activity. We have mapped an essential regulatory region between -513 to -352 bp which causes a drastic decline of reporter activity. This region contains CdxA, GATA2 and SRY transcription factor binding sites. Individual mutation of these three sites showed increase in reporter activity. Mutation in SRY site (-403/ -368) showed maximum increase in reporter activity among these three sites. Therefore, we suggest that SRY like protein may be acting as a strong repressor of SMP30 gene along with CdxA and GATA-2. We also report that mutation of both Sp1 (172/ -148 bp) and a C/EBPbeta (-190/ -177bp) transcription binding site located adjacent to each other on SMP30 gene promoter, causes a significant enhancement in reporter activity than individual mutation, thus may be causing the repression of SMP30 promoter activity. Conclusions: These studies provide novel insights into the mechanism that regulate SMP30 gene expression. http://www.biomedcentral.com/1471-2199/9/43 Bandita Rath, Ravi S Pandey, Priya R Debata, Naoki Maruyama and Prakash C Supakar BMC Molecular Biology 2008, 9:43 2008-04-29 doi:10.1186/1471-2199-9-43 BMC Molecular Biology 1471-2199 9 43 2008-04-29 Background: Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory. Results: The co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana. Conclusions: We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants. http://www.biomedcentral.com/1471-2199/9/42 Karine Triques, Elodie Piednoir, Marion Dalmais, Julien Schmidt, Christine Le Signor, Mark Sharkey, Michel Caboche, Benedicte Sturbois and Abdelhafid Bendahmane BMC Molecular Biology 2008, 9:42 2008-04-23 doi:10.1186/1471-2199-9-42 BMC Molecular Biology 1471-2199 9 42 2008-04-23 Background: Circadian oscillation of clock-controlled gene expression is mainly regulated at the transcriptional level. Heterodimers of CLOCK and BMAL1 act as activators of target gene transcription; however, interactions of PER and CRY proteins with the heterodimer abolish its transcriptional activation capacity. PER and CRY are therefore referred to as negative regulators of the circadian clock. To further elucidate the mechanism how positive and negative components of the clock interplay, we characterized the interactions of PER2, CRY1 and CRY2 with BMAL1 and CLOCK using a mammalian two-hybrid system and co-immunoprecipitation assays. Results: Both PER2 and the CRY proteins were found to interact with BMAL1 whereas only PER2 interacts with CLOCK. CRY proteins seem to have a higher affinity to BMAL1 than PER2. Moreover, we provide evidence that PER2, CRY1 and CRY2 bind to different domains in the BMAL1 protein. Conclusions: The regulators of clock-controlled transcription PER2, CRY1 and CRY2 differ in their capacity to interact with each single component of the BMAL1-CLOCK heterodimer and, in the case of BMAL1, also in their interaction sites. Our data supports the hypothesis that CRY proteins, especially CRY1, are stronger repressors than PER proteins. http://www.biomedcentral.com/1471-2199/9/41 Sonja Langmesser, Tiziano Tallone, Alain Bordon, Sandro Rusconi and Urs Albrecht BMC Molecular Biology 2008, 9:41 2008-04-22 doi:10.1186/1471-2199-9-41 BMC Molecular Biology 1471-2199 9 41 2008-04-22 Background: DNA-based watermarks are helpful tools to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. In silico analyses showed that in coding regions synonymous codons can be used to insert encrypted information into the genome of living organisms by using the DNA-Crypt algorithm. Results: We integrated an authenticating watermark in the Vam7 sequence. For our investigations we used a mutant Saccharomyces cerevisiae strain, called CG783, which has an amber mutation within the Vam7 sequence. The CG783 cells are unable to sporulate and in addition display an abnormal vacuolar morphology. Transformation of CG783 with pRS314 Vam7 leads to a phenotype very similar to the wildtype yeast strain CG781. The integrated watermark did not influence the function of Vam7 and the resulting phenotype of the CG783 cells transformed with pRS314 Vam7-TB shows no significant differences compared to the CG783 cells transformed with pRS314 Vam7. Conclusion: From our experiments we conclude that the DNA watermarks produced by DNA-Crypt do not influence the translation from mRNA into protein. By analyzing the vacuolar morphology, growth rate and ability to sporulate we confirmed that the resulting Vam7 protein was functionally active. http://www.biomedcentral.com/1471-2199/9/40 Dominik Heider and Angelika Barnekow BMC Molecular Biology 2008, 9:40 2008-04-21 doi:10.1186/1471-2199-9-40 BMC Molecular Biology 1471-2199 9 40 2008-04-21 Background: The clinical course of cystic fibrosis (CF) is characterized by recurrent pulmonary infections and chronic inflammation. We have recently shown that decreased methylation of the toll-like receptor-2 (TLR2) promoter leads to an apparent CF-related up-regulation of TLR2. This up-regulation could be responsible, in part, for the CF-associated enhanced proinflammatory responses to various bacterial products in epithelial cells. However, the molecular mechanisms underlying DNA hypomethylation-dependent enhancement of TLR2 expression in CF cells remain unknown. Results: The present study indicates that there is a specific CpG region (CpG#18-20), adjacent to the SP1 binding site that is significantly hypomethylated in several CF epithelial cell lines. These CpGs encompass a minimal promoter region required for basal TLR2 expression, and suggests that CpG#18-20 methylation regulates TLR2 expression in epithelial cells. Furthermore, reporter gene analysis indicated that the SP1 binding site is involved in the methylation-dependent regulation of the TLR2 promoter. Inhibition of SP1 with mithramycin A decreased TLR2 expression in both CF and 5-azacytidine-treated non-CF epithelial cells. Moreover, even though SP1 binding was not affected by CpG methylation, SP1-dependent transcription was abolished by CpG methylation. Conclusions: This report implicates SP1 as a critical component of DNA demethylation-dependent up-regulation of TLR2 expression in CF epithelial cells. http://www.biomedcentral.com/1471-2199/9/39 Takashi Furuta, Tsuyoshi Shuto, Shogo Shimasaki, Yuko Ohira, Mary Ann Suico, Dieter C Gruenert and Hirofumi Kai BMC Molecular Biology 2008, 9:39 2008-04-21 doi:10.1186/1471-2199-9-39 BMC Molecular Biology 1471-2199 9 39 2008-04-21 Background: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts. Results: Cloning of two zebrafish β2 subunit genes (β2.1 and β2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced β2 transcript variants is detected in the embryonic heart compared to the adult. Conclusion: These studies refine our understanding of β2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of β2 subunit diversity in the embryonic heart. http://www.biomedcentral.com/1471-2199/9/38 Alicia M Ebert, Catherine A McAnelly, Ashok Srinivasan, Rachel Lockridge Mueller, David B Garrity and Deborah M Garrity BMC Molecular Biology 2008, 9:38 2008-04-17 doi:10.1186/1471-2199-9-38 BMC Molecular Biology 1471-2199 9 38 2008-04-17 Background: The display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. After uptake of the phage, their internal single-stranded DNA is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette. This opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low. Results: Here we demonstrate the display of an anti-CD30 single chain variable fragment fused to the major coat protein pVIII on the surface of bacteriophage. These phage particles showed an improved binding and transduction efficiency of CD30 positive Hodgkin-lymphoma cells, compared to bacteriophage with the anti-CD30 single chain variable fragment fused to the minor coat protein pIII. Conclusion: We can conclude from the results that the postulated multivalency of the anti-CD30-pVIII displaying bacteriophage combined with disseminated display of the anti-CD30 scFv on the whole particle surface is responsible for the improved gene transfer rate. These results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors. http://www.biomedcentral.com/1471-2199/9/37 Yoon-Suk A Chung, Katja Sabel, Martin Krönke and Alexander Klimka BMC Molecular Biology 2008, 9:37 2008-04-16 doi:10.1186/1471-2199-9-37 BMC Molecular Biology 1471-2199 9 37 2008-04-16