Transcription Start Point Research Articles

Identification of mutations resulting in derepression of the intermedilysin gene by sequential mutagenesis of its promoter region in Streptococcus intermedius.

Streptococcus intermedius secretes the human-specific cytolysin intermedilysin (ILY), a crucial factor in the pathogenicity of this bacterium. Previously, we reported that a lactose phosphotransferase repressor (LacR) represses ily expression, and that its mutation increases ILY production. Interestingly, UNS40, a strain isolated from a liver abscess, produces high levels of ILY despite the absence of mutations in the lacR promoter and coding regions. Our results showed that a G>A mutation at the-90th position from the transcription start point in the UNS40 ily promoter region increased hemolytic activity and decreased the binding ability to LacR. To elucidate the regions involved in the repression of ily expression, we generated mutant strains, in which point or deletion mutations were introduced into the ily promoter region, and then compared their hemolytic activity. Among the point mutations, -120 C>A and-90 G>A and their flanking mutations increased hemolytic activity. These results indicated that these mutations may increase the virulence of S. intermedius.

Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion.

Cerebral cavernous malformations are clusters of aberrant vessels that can lead to severe neurological complications. Pathogenic loss-of-function variants in the CCM1, CCM2, or CCM3 gene are associated with the autosomal dominant form of the disease. While interpretation of variants in protein-coding regions of the genes is relatively straightforward, functional analyses are often required to evaluate the impact of non-coding variants. Because of multiple alternatively spliced transcripts and different transcription start points, interpretation of variants in the 5′ untranslated and upstream regions of CCM1 is particularly challenging. Here, we identified a novel deletion of the non-coding exon 1 of CCM1 in a proband with multiple CCMs which was initially classified as a variant of unknown clinical significance. Using CRISPR/Cas9 genome editing in human iPSCs, we show that the deletion leads to loss of CCM1 protein and deregulation of KLF2, THBS1, NOS3, and HEY2 expression in iPSC-derived endothelial cells. Based on these results, the variant could be reclassified as likely pathogenic. Taken together, variants in regulatory regions need to be considered in genetic CCM analyses. Our study also demonstrates that modeling variants of unknown clinical significance in an iPSC-based system can help to come to a final diagnosis.

Examining Galectin Gene Regulation by Reporter Assays.

Matching their role as potent and versatile effectors in cellular homeostasis and disease processes, galectins are subject to a fine-tuned transcriptional regulation of gene expression. It can apparently even involve coregulation with certain elements of the enzymatic machinery for glycan biosynthesis/remodeling and/or functional carriers of galectin-binding glycans such as the α5β1-integrin. All this suggests not yet fully known combinatorial processes to reach the desired outcome. Identification of transcription start point(s), cloning of upstream promoter region, and the design of plasmids for luciferase-based reporter assays establish the platform to initiate a systematic search of regulatory sequences. Their elucidation is also a step toward rationally manipulating expression of galectin genes in pathogenesis.

Gene Networks Underlying the Resistance of Bifidobacterium longum to Inflammatory Factors.

As permanent residents of the normal gut microbiota, bifidobacteria have evolved to adapt to the host’s immune response whose priority is to eliminate pathogenic agents. The mechanisms that ensure the survival of commensals during inflammation and maintain the stability of the core component of the normal gut microbiota in such conditions remain poorly understood. We propose a new in vitro approach to study the mechanisms of resistance to immune response factors based on high-throughput sequencing followed by transcriptome analysis. This approach allowed us to detect differentially expressed genes associated with inflammation. In this study, we demonstrated that the presence of the pro-inflammatory cytokines IL-6 and TNFα to the growth medium of the B. longum subsp. longum GT15 strain changes the latter’s growth rate insignificantly while affecting the expression of certain genes. We identified these genes and performed a COG and a KEGG pathway enrichment analysis. Using phylogenetic profiling we predicted the operons of genes whose expression was triggered by the cytokines TNFα and IL-6 in vitro. By mapping the transcription start points, we experimentally validated the predicted operons. Thus, in this study, we predicted the genes involved in a putative signaling pathway underlying the mechanisms of resistance to inflammatory factors in bifidobacteria. Since bifidobacteria are a major component of the human intestinal microbiota exhibiting pronounced anti-inflammatory properties, this study is of great practical and scientific relevance.

RNA Characterization in Trichoderma reesei.

This chapter provides an overview on different methods for the characterization of RNAs in Trichoderma reesei. In the first section, protocols for the extraction of total RNA from fungal mycelia and the identification of 5' and 3' ends of certain RNAs of interest via rapid amplification of cDNA ends (RACE) are presented. In the next section, this knowledge on the transcriptional start and end points is used for in vitro synthesis and fluorescence labeling of the RNA of interest. The in vitro synthesized RNA can then be applied for in vitro analyses such as RNA electrophoretic mobility shift assays (RNA-EMSA) and RNA in vitro footprinting. RNA-EMSA is a method suitable for the identification and characterization of RNA-protein interactions or interactions of an RNA with other nucleic acids. RNA in vitro footprinting allows exact mapping of protein-binding sites on RNA molecules and also the determination of RNA secondary and tertiary structures at singe-nucleotide resolution. All protocols presented in this chapter are optimized for the analysis of noncoding RNAs (ncRNAs), especially long ncRNAs (lncRNAs) or other specific RNA species of more than 200nt in length.

Abstract 5202: Molecular mechanism of transcriptional regulation of REV7, a DNA damage response gene

Abstract REV7 is involved in several biological response after DNA damage, including the double strand break repair and translesion DNA synthesis. It has bee reported that high levels of REV7 expression are observed in several malignant tumors and are associated with resistance to chemotherapeutic agents such as cisplatin. In normal tissues, REV7 expression is extremely high in germ cells compared with the other normal cells, suggesting the cell type-specific expression of REV7. However, the mechanism of regulation of REV7 expression has not been understood. To assess the mechanism of regulation of REV7 expression, we analyzed the promotor region of the human REV7 locus to identify transcriptional regulators controlling REV7 expression. The transcriptional start point of the REV7 gene was determined by 5’-RACE using human testis cDNA. About 3 kb of the upstream region of the REV7 locus was isolated by genomic PCR and the promoter activity was analyzed by the luciferase reporter assay. Using several deletion mutants, we found that there are two transcriptional activation regions in the upstream of the REV7 locus. Bioinformatic analyses revealed several motifs of transcription factor binding sites in these regions, and we focused on one molecule as a candidate for the transcription factor controlling REV7 expression. We will present and discuss our further analyses. Note: This abstract was not presented at the meeting. Citation Format: Yoshiki Murakumo, Yuko Shimada, Takuya Kato, Yasutaka Sakurai. Molecular mechanism of transcriptional regulation of REV7, a DNA damage response gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5202.

Salt Sensing and Salt Response of Renal Neuropeptide FF Receptor 2

The causal role of dietary NaCl intake in the development of hypertension and salt sensitivity has long been recognized. The kidney sits at the fulcrum of maintaining an appropriate sodium balance and blood pressure homeostasis. As such, identifying new regulators of sodium balance in the kidney is crucial to understand the intricate mechanisms involved. Neuropeptide FF (NPFF) is a hormone expressed in the central nervous system that subserves nociception, hormonal modulation, and body temperature control. We now demonstrate for the first time that NPFF and its receptors, NPFF‐R1 and NPFF‐R2, are expressed in the kidney and endowed with pro‐hypertensive properties. The renal‐restricted bolus (1‐hr) or chronic infusion (7‐day) of NPFF increased blood pressure (*P<0.05, n=4/group) and decreased sodium excretion (UNaV; *P<0.05, N=4/group) in C57Bl/6 mice. This tubular effect was due solely to NPFF‐R2 since its silencing resulted in ~3‐fold increase in UNaV, unlike NPFF‐R1 silencing which did not affect UNaV. Therefore, we further explored the changes in NPFF‐R2 expression or activity in response to changes in salt. We now report that we have newly identified a “Sodium Response Element” (SRE), a homolog of “Dehydration‐Responsive Element” (“TACCGACAT”) in Arabidopsis thaliana genome, at the NPFF‐R2 promoter ~ 2.3 Kb upstream of the transcription start point. Exposing human renal proximal tubule cells (hRPTCs) from normal (145 μM NaCl) to low (90 μM NaCl) sodium concentration resulted in increased promoter activity (~2.5‐fold, *P<0.05, vs. DSRE, 3/group; via luciferase activity) and increased mRNA and protein expression of NPFFR2 (*P<0.05, 3–4/group; 0–8 hr). Exposure of hRPTCs from normal to high (175 μM NaCl) sodium concentration was accompanied by a reduction in promoter activity (−0.5‐fold, *P<0.05), and a decrease in mRNA and protein expression of NPFFR2 (*P<0.05, 0–8 hr). Moreover, there was a markedly increased co‐immunoprecipitation between pro‐hypertensive NPFF‐R2 and anti‐hypertensive dopamine D1 receptor, resulting in increased antagonism between the two receptors on cAMP response and sodium transport (*P<0.05, vs. fenoldopam or NPFF treatment [1 mM/30 min], 4/group). Lastly, exposure to high salt concentration resulted in greater co‐immunoprecipitation of NPFF‐R2 with Gαi3 and none with Gαs (“Gα switch”), which may explain its pro‐hypertensive activity. NPFF, via NPFF‐R2, may be a crucial determinant of sodium balance and blood pressure homeostasis and thus, may be an important target for new pharmacological and biological intervention.Support or Funding InformationR01DK039308, R01HL092196, and P01HL074940This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Research on the Osmotic Stress Gene Mining Model Based on the Arabidopsis Genome

In the field of the bioinformatics, during osmotic stress response genes mining processing, it is also very crucial to verify experimental data obtained in the course of complex experiments by using the computer. Aim of this paper is taking Arabidopsis thaliana as the experimental crop, designing technology roadmap, taking advantage of the skills of function and programming, then designing algorithms. After using the program to predict the transcription start point, the promoter sequence is extracted and simplified. In addition, different alignment methods are classified. Then, comparing the promoter sequence with the cis-element and using the formula for further processing. Finally, get the probability P value, which provide further help to experts and scholars on the basis of probability values to determine the correlation between the osmotic stress. The experimental data source of chromosomal sequences is received from Genbank database files, and cis-element sequence that associated with osmotic stress is collected from TRANSFAC and TRRD database. From this, the authors not only used the Arabidopsis promoter as the experimental data, but also use a variety of eukaryotic promoters include promoters GhNHX1 rice, cotton OsNHX1 promoter, as a comparison. Wherein the data obtained in the biological laboratory, which in the course of running the program, 70% have been verified. P value close to 0.8, this article will be treated as the promoter contains osmotic stress cis-elements, the expression of gene induced by osmotic stress. For thaliana, cotton and rice, programs running average time was 51s, 72s and 114s. Through the use of some commonly used bioinformatics gene mining algorithms, MEME algorithm and BioProspector algorithm for the same data have been processed, the average running time of the system is increasing with the increase of data. Running time of MEME algorithm increases from 60s to reach 198s, BioProspector algorithm increases from 45s to 150s model process used herein were 50s, 75s, 110s, 135s. At the same time, the authors can see in the three algorithms, the model algorithm used herein with respect to the first two more optimized. To ensure the accuracy rate, meanwhile has high speed and stabilization of higher.

Disruption of a GATA1-binding motif upstream of XG/PBDX abolishes Xga expression and resolves the Xg blood group system

AbstractThe Xga blood group is differentially expressed on erythrocytes from men and women. The underlying gene, PBDX, was identified in 1994, but the molecular background for Xga expression remains undefined. This gene, now designated XG, partly resides in pseudoautosomal region 1 and encodes a protein of unknown function from the X chromosome. By comparing calculated Xga allele frequencies in different populations with 2612 genetic variants in the XG region, rs311103 showed the strongest correlation to the expected distribution. The same single-nucleotide polymorphism (SNP) had the most significant impact on XG transcript levels in whole blood (P = 2.0 × 10−22). The minor allele, rs311103C, disrupts a GATA-binding motif 3.7 kb upstream of the transcription start point. This silences erythroid XG messenger RNA expression and causes the Xg(a−) phenotype, a finding corroborated by SNP genotyping in 158 blood donors. Binding of GATA1 to biotinylated oligonucleotide probes with rs311103G but not rs311103C was observed by electrophoretic mobility shift assay and proven by mass spectrometry. Finally, a luciferase reporter assay indicated this GATA motif to be active for rs311103G but not rs311103C in HEL cells. By using an integrated bioinformatic and molecular biological approach, we elucidated the underlying genetic basis for the last unresolved blood group system and made Xga genotyping possible.

A New Cellular Model System for Study of Regulatory Mechanisms of Human Ribosomal Gene Transcription

The rate of protein synthesis strictly correlates with the rRNA quantity in a cell. The rRNA synthesis is the first stage in the ribosomal synthesis process. It is actively regulated by such external factors as type of nutrition, growth factor, and cell stress. Thus, pre-rRNA transcription and maturation play a central role in processes of cell growth and proliferation. In recent years, numerous data have been obtained that make it possible to suggest that quantitative and qualitative changes in the rRNA synthesis are the most important molecular indicators of cell malignancy. The principle of suppressing activity of rRNA genes at various stages of their expression is used today in chemotherapeutic methods of treatment of oncological diseases. A number of data indicate that the adaptation of the nucleoli precedes the malignant transformation and is not the result of it. These data offer new possibilities for studying the relationship between the transcription of ribosomal DNA (rDNA) and cell proliferation. Understanding these subtle connections is necessary for the development of new approaches to the molecular characterization and subsequent therapy of neoplastic diseases. The aim of this work was to obtain a cellular model system that makes it possible to adequately estimate the activity of RNA polymerase I in vivo by measuring the expression level of the luciferase reporter gene. For this purpose, the regulatory region of the 6.7 kb rRNA gene preceding the transcriptional start point was recloned into the luciferase expression vector pGL4.77. The obtained construct was used to transfect the line HEK293, which resulted in the line pqA77.HEK293. The new model system opens wide prospects for studying transcription regulation mechanisms of human ribosomal genes.

The transcriptional regulator LysG (Rv1985c) of Mycobacterium tuberculosis activates lysE (Rv1986) in a lysine-dependent manner.

The Mycobacterium tuberculosis protein encoded by the Rv1986 gene is a target for memory T cells in patients with tuberculosis, and shows strong similarities to a lysine exporter LysE of Corynebacterium glutamicum. During infection, the pathogen Mycobacterium tuberculosis adapts its metabolism to environmental changes. In this study, we found that the expression of Rv1986 is controlled by Rv1985c. Rv1985c is located directly upstream of Rv1986 with an overlapping promoter region between both genes. Semiquantitative reverse transcription PCR using an isogenic mutant of Mycobacterium tuberculosis lacking Rv1985c showed that in the presence of lysine, Rv1985c protein positively upregulated the expression of Rv1986. RNA sequencing revealed the transcription start points for both transcripts and overlapping promoters. An inverted repeat in the center of the intergenic region was identified, and binding of Rv1985c protein to the intergenic region was confirmed by electrophoretic mobility shift assays. Whole transcriptome expression analysis and RNAsequencing showed downregulated transcription of ppsBCD in the Rv1985c-mutant compared to the wild type strain. Taken together, our findings characterize the regulatory network of Rv1985c in Mycobacterium tuberculosis. Due to their similarity of an orthologous gene pair in Corynebacterium glutamicum, we suggest to rename Rv1985c to lysG(Mt), and Rv1986 to lysE(Mt).

Growth-dependent activity of the cold shock cspA promoter\xa0+\xa05\u2032 UTR and production of the protein CspA in Staphylococcus aureus Newman

BackgroundResearch involving the cold shock gene cspA of the medically important bacterium Staphylococcus aureus is steadily increasing as the relationships between the activity of this gene at 37 °C and a spectrum of virulence factors (e.g., biofilm formation, capsule production) as well as stress-related genes (e.g., alkaline shock protein, asp-23 and the alternative sigma factor, sigB) are distinguished. Fundamental to each of these discoveries is defining the regulation of cspA and the production of its protein product CspA.ResultsIn this paper, primer extension analysis was used to identify a transcriptional start point at 112 bp upstream of the initiation codon of the cspA coding sequence from S. aureus Newman RNA collected at 37 °C. Based on the location of the putative −10 and −35 sites as well as putative cold shock protein binding sites, a 192 bp sequence containing an 80 bp promoter + a 112 bp 5′ UTR was generated by polymerase chain reaction. The activity of this 192 bp sequence was confirmed in a pLL38 promoter::xylE reporter gene construct. In addition, Western blots were used to confirm the production of CspA at 37 °C and demonstrated that production of the protein was not constitutive but showed growth-dependent production with a significant increase at the 6 h time point.ConclusionsThe results presented identify another regulatory region for the cold shock gene cspA of S. aureus and show growth-dependent activity of both this cspA regulatory sequence, presented as a 192 bp sequence of promoter + 5′ UTR and the production of the CspA protein at 37 °C. The presence of two active transcription start points, a −112 bp sequence defined in this work and a second previously defined at −514 bp upstream of the cspA initiation codon, suggests the possibility of interactions between these two regions in the regulation of cspA. The growth-dependent production of the cold shock protein CspA supports the availability of this protein to be a modulator of virulence and stress factor genes at 37 °C.

Functional redundancy and nonredundancy between two Troponin C isoforms in Drosophila adult muscles.

We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, Drosophila melanogaster: TpnC4 is predominantly expressed in the indirect flight muscles (IFMs), whereas TpnC41C is the main isoform in the tergal depressor of the trochanter muscle (TDT; jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of TpnC41C and compared its phenotype to a line lacking functional TpnC4 We found that the removal of either of these genes leads to expression of the other isoform in both muscle types. The switching between isoforms occurs at the transcriptional level and involves minimal enhancers located upstream of the transcription start points of each gene. Functionally, the two TpnC isoforms were not equal. Although ectopic TpnC4 in TDT muscles was able to maintain jumping ability, TpnC41C in IFMs could not effectively support flying. Simultaneous functional disruption of both TpnC genes resulted in jump-defective and flightless phenotypes of the survivors, as well as abnormal sarcomere organization. These results indicated that TpnC is required for myofibril assembly, and that there is functional specialization among TpnC isoforms in Drosophila.

Expression of the phosphodiesterase BifA facilitating swimming motility is partly controlled by FliA in Pseudomonas putida KT2440.

Flagella‐mediated motility is an important capability of many bacteria to survive in nutrient‐depleted and harsh environments. Decreasing the intracellular cyclic di‐GMP (c‐di‐GMP) level by overexpression of phosphodiesterase BifA promotes flagellar‐mediated motility and induces planktonic lifestyle in Pseudomonas. The mechanism that regulates expression of bifA gene was poorly studied. Here we showed that expression of BifA was partly controlled by flagellar sigma factor FliA (σ28) in Pseudomonas putida KT2440. FliA deletion led to an approximately twofold decrease in transcription of bifA. 5′ race assay revealed two transcription start points in bifA promoter region, with the putative σ70 and σ28 promoter sequences upstream, respectively. Point mutation in σ28 promoter region reduced transcriptional activity of the promoter in wild‐type KT2440, but showed no influence on that in fliA deletion mutant. FliA overexpression decreased the intracellular c‐di‐GMP level in a BifA‐dependent way, suggesting that FliA was able to modulate the intracellular c‐di‐GMP level and BifA function was required for the modulation. Besides, FliA overexpression enhanced swimming ability of wild‐type strain, while made no difference to the bifA mutant. Our results suggest that FliA acts as a negative regulator to modulate the c‐di‐GMP level via controlling transcription of bifA to facilitate swimming motility.

Impact of neoadjuvant chemotherapy with gemcitabine and S-1 on postoperative prognosis: A matched case-control study

Two chitinase(CTS)-encoding genes (cts) from Coccidioides immitis (CI), a respiratory fungal pathogen of humans, were cloned and sequenced. Both the genomic and cDNA sequences are presented. The transcription start points and poly(A)-addition sites were confirmed. The cts1 gene contains five introns and a 1281-bp ORF which translates a 427-amino-acid (aa) protein of 47.4 kDa. The cts2 gene contains two introns and a 2580-bp ORF which translates a 860-aa protein of 91.4 kDa. The deduced CTS1 protein showed highest homology to the Aphanocladium album and Trichoderma harzianum CTS (74% and 76%, respectively), while CTS2 showed highest homology to the CTS of Saccharomyces cerevisiae (Sc) and Candida albicans (47% and 51%, respectively). The putative N-terminal sequence of the mature CTS1 protein also showed 89% homology to the reported N-terminal sequence of a 48-kDa complement fixation antigen (CF-Ag) of Ci which has demonstrated chitinase activity. The CFAg is a clinically important antigen used in serodiagnosis of this fungal disease. CTS2 showed several of the conserved features of the Sc CTS, including putative catalytic and Ser/Thr-rich domains, and a C-terminal Cys-rich region. We propose that CTS1 and CTS2 of Ci are members of two distinct classes of fungal chitinases, an observation not previously reported for a single fungus.

Cloning identification and functional analysis of human IL-17A promoter.

To conduct the cloning identification and characterization of the sequence of human IL-17A promoter so as to analyze the regulatory mechanism of the gene expression of IL-17. First of all, the potential promoter region of IL-17A was found by means of the bioinformatics methods. Then, it was cloned into the reporter vector with PCR technique. Finally, the activity of the test promoter was determined by dual luciferase reporter system. Two transcriptional start points of the upper region, 600bp and 1000bp, of IL-17A were obtained by PCR clone and proved to have certain activities by dual luciferase reporter system. Also, they could be activated by IL-17A activator STAT3, which could start the expression of the reported gene. Clone established the regulatory region of human IL-17A promoter, which provided bases to the subsequent function research.

Genetic and Transcriptional Analyses of the Flagellar Gene Cluster in Actinoplanes missouriensis.

Actinoplanes missouriensis, a Gram-positive and soil-inhabiting bacterium, is a member of the rare actinomycetes. The filamentous cells produce sporangia, which contain hundreds of flagellated spores that can swim rapidly for a short period of time until they find niches for germination. These swimming cells are called zoospores, and the mechanism of this unique temporal flagellation has not been elucidated. Here, we report all of the flagellar genes in the bacterial genome and their expected function and contribution for flagellar morphogenesis. We identified a large flagellar gene cluster composed of 33 genes that encode the majority of proteins essential for assembling the functional flagella of Gram-positive bacteria. One noted exception to the cluster was the location of the fliQ gene, which was separated from the cluster. We examined the involvement of four genes in flagellar biosynthesis by gene disruption, fliQ, fliC, fliK, and lytA Furthermore, we performed a transcriptional analysis of the flagellar genes using RNA samples prepared from A. missouriensis grown on a sporangium-producing agar medium for 1, 3, 6, and 40 days. We demonstrated that the transcription of the flagellar genes was activated in conjunction with sporangium formation. Eleven transcriptional start points of the flagellar genes were determined using the rapid amplification of cDNA 5' ends (RACE) procedure, which revealed the highly conserved promoter sequence CTCA(N15-17)GCCGAA. This result suggests that a sigma factor is responsible for the transcription of all flagellar genes and that the flagellar structure assembles simultaneously. The biology of a zoospore is very interesting from the viewpoint of morphogenesis, survival strategy, and evolution. Here, we analyzed flagellar genes in A. missouriensis, which produces sporangia containing hundreds of flagellated spores each. Zoospores released from the sporangia swim for a short time before germination occurs. We identified a large flagellar gene cluster and an orphan flagellar gene (fliQ). These findings indicate that the zoospore flagellar components are typical of Gram-positive bacteria. However, the transcriptional analysis revealed that all flagellar genes are transcribed simultaneously during sporangium formation, a pattern differing from the orderly, regulated expression of flagellar genes in other bacteria, such as Salmonella and Escherichia coli These results suggest a novel regulatory mechanism for flagellar formation in A. missouriensis.

Effect of hypoxia on tissue factor pathway inhibitor expression in breast cancer

ESSENTIALS: A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF-7 and SK-BR-3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia-inducible factor 1α may be a target for the therapy of cancer-related coagulation and thrombosis. Activation of coagulation is a common finding in patients with cancer, and is associated with an increased risk of venous thrombosis. As a hypoxic microenvironment is a common feature of solid tumors, we investigated the role of hypoxia in the regulation of tissue factor (TF) pathway inhibitor (TFPI) expression in breast cancer. To explore the transcriptional regulation of TFPI by hypoxia-inducible factor (HIF)-1α in breast cancer cells and their correlation in breast cancer tissues. MCF-7 and SK-BR-3 breast cancer cells were cultured in 1% oxygen or treated with cobalt chloride (CoCl2 ) to mimic hypoxia. Time-dependent and dose-dependent downregulation of TFPI mRNA (quantitative RT-PCR) and of free TFPI protein (ELISA) were observed in hypoxia. Western blotting showed parallel increases in the levels of HIF-1α protein and TF. HIF-1α inhibitor abolished or attenuated the hypoxia-induced downregulation of TFPI. Luciferase reporter assay showed that both hypoxia and HIF-1α overexpression caused strong repression of TFPI promoter activity. Subsequent chromatin immunoprecipitation and mutagenesis analysis demonstrated a functional hypoxia response element within the TFPI promoter, located at -1065 to -1060 relative to the transcriptional start point. In breast cancer tissue samples, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and that of HIF-1α. This study demonstrates that HIF-1α is involved in the transcriptional regulation of the TFPI gene, and suggests that a hypoxic microenvironment inside a breast tumor may induce a procoagulant state in breast cancer patients.

두록 돼지의 등지방두께와 연관된 렙틴수용체 유전자의 신규 SNP 마커

돼지에게 있어서 지방 형질은 가장 중요한 경제형질 중 하나다. 돼지의 렙틴수용체 유전자(LEPR)는 염색체 상의 위치와 그 생리 활성 측면에서 돼지 6번 염색체 상의 지방형질 관련 양적형질좌위(QTL)에 대한 주요 후보유전자로 알려져 있다. 본 연구에서는 LEPR 유전자의 구조 변이와 돼지 경제형질과의 연관성을 분석하였다. 이를 위하여 돼지 LEPR 유전자를 포함하고 있는 박테리아 인공 염색체(BAC) 클론에 대한 샷건 염기서열 해독을 수행하여 114 kb 크기의 유전체 서열을 확보하였다. 그리고 전사개시 코돈으로부터 1.2 kb 상위 영역에서 여러 전사인자 결합부위를 발견하였다. 또한 LEPR 유전자 엑손 영역의 6개 SNP와 5’ 조절영역의 18개 SNP에 대해 550두의 두록 개체를 대상으로 연관성 분석을 수행하였다. 이들 SNP 중, −790C/G만이 등지방두께와 정육율 형질과 유의적으로 연관되어 있었으며, 2개의 미스센스 다형성 SNP를 포함하여 다른 SNP에서는 어떤 형질과도 연관성을 보이지 않았다. 결론적으로 −790C/G SNP는 두록 돼지에서 지방과 정육형질을 유전적으로 개량하는데 유용한 마커로 활용될 수 있을 것이다. Fatness is one of the most important economic traits in pigs. The leptin receptor (LEPR) gene may be a potential candidate for the fatness quantitative trait locus (QTL) on porcine chromosome 6, due to its position and physiological role. Thus, this study was carried out to evaluate the associations between structural variants in the LEPR gene and economic traits in pigs. We obtained an approximately 114-kb sequence containing the complete genomic DNA of the porcine LEPR gene, using shotgun sequencing of a bacterial artificial chromosome clone. We report the complete genomic structure of the porcine LEPR gene. Dozens of transcription factor-binding sites were found in the 1.2 kb upstream region from the transcription start point. An association study was performed with 550 Duroc pigs for 24 single-nucleotide polymorphisms (SNPs), including 6 SNPs within exons and 18 SNPs within the putative 5‘ regulatory region of the porcine LEPR gene. Among them, one SNP (−790C/G) was significantly associated with backfat thickness and lean meat percentage, whereas the others, including two SNPs with missense polymorphisms, had no effect on any phenotype. These results suggest that SNP −790C/G may be a useful marker for genetic improvements of fatness and leanness in Duroc pigs.

Insulin requires A1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium.

Gestational diabetes mellitus (GDM) associates with increased L-arginine transport and extracellular concentration of adenosine in human umbilical vein endothelial cells (HUVECs). In this study we aim to determine whether insulin reverses GDM-increased L-arginine transport requiring adenosine receptors expression in HUVECs. Primary cultured HUVECs from full-term normal (n = 38) and diet-treated GDM (n = 38) pregnancies were used. Insulin effect was assayed on human cationic amino acid transporter 1 (hCAT1) expression (protein, mRNA, SLC7A1 promoter activity) and activity (initial rates of L-arginine transport) in the absence or presence of adenosine receptors agonists or antagonists. A1 adenosine receptors (A1AR) and A2AAR expression (Western blot, quantitative PCR) was determined. Experiments were done in cells expressing or siRNA-suppressed expression of A1AR or A2AAR. HUVECs from GDM exhibit higher maximal transport capacity (maximal velocity (V max)/apparent Michaelis Menten constant (K m), V max/K m), which is blocked by insulin by reducing the V max to values in cells from normal pregnancies. Insulin also reversed the GDM-associated increase in hCAT-1 protein abundance and mRNA expression, and SLC7A1 promoter activity for the fragment -606bp from the transcription start point. Insulin effects required A1AR, but not A2AAR expression and activity in this cell type. In the absence of insulin, GDM-increased hCAT-1 expression and activity required A2AAR expression and activity. HUVECs from GDM pregnancies exhibit a differential requirement of A1AR or A2AAR depending on the level of insulin, a phenomenon that represent a condition where adenosine or analogues of this nucleoside could be acting as helpers of insulin biological effects in GDM.