The S. Typhi leuO gene contains multiple functional promoters

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Marcos Fernández-Mora ◽  
Diego Sánchez-Popoca ◽  
Gloria Altamirano-Cruz ◽  
Grecia López-Méndez ◽  
Andrea Teresa Téllez-Galicia ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Environmental Cues ◽  
Rt Pcr ◽  
Multiple Promoters ◽  
Associated Proteins ◽  
Negative Effect ◽  
Steady State Levels ◽  
Fine Tune ◽  
Transcriptional Fusions ◽  
Lower Expression

The S. Typhi leuO gene, which codes for the LysR-type transcriptional regulator LeuO, contains five forward promoters named P3, P1, P2, P5 and P4, and two reverse promoters, P6 and P7. The activity of the forward promoters was revealed by primer extension using gene reporter fusions in an S. Typhi hns lrp mutant strain. Likewise, the activity of the reverse promoters was revealed in an hns background. Derepression of the transcription of the chromosomal gene was confirmed by RT-PCR in the hns lrp mutant. The leuOP1 transcriptional reporter fusion, which contained only the major P1 promoter, had a lower expression in a relA spoT mutant strain, indicating that the steady-state levels of the (p)ppGpp alarmone positively regulate it. In contrast, the leuOP3, leuOP5P4, leuOP6 and leuOP7 transcriptional fusions were derepressed in the relA spoT background, indicating that the alarmone has a negative effect on their expression. Thus, the search for genetic regulators and environmental cues that would differentially derepress leuO gene expression by antagonizing the action of the H-NS and Lrp nucleoid-associated proteins, or that would fine-tune the expression of the various promoters, will further our understanding of the significance that multiple promoters have in the control of LeuO expression.

scholarly journals Multiple Promoters Contribute to Swarming and the Coordination of Transcription with Flagellar Assembly in Salmonella

2010 ◽  
Vol 192 (18) ◽  
pp. 4752-4762 ◽  
Author(s):  
Christopher E. Wozniak ◽  
Fabienne F. V. Chevance ◽  
Kelly T. Hughes
Keyword(s):  
Transcriptional Regulation ◽  
Stationary Phase ◽  
Basal Body ◽  
Environmental Conditions ◽  
Swarming Motility ◽  
Multiple Promoters ◽  
Associated Proteins ◽  
Flagellar Assembly

ABSTRACT In Salmonella, there are three classes of promoters in the flagellar transcriptional hierarchy. This organization allows genes needed earlier in the construction of flagella to be transcribed before genes needed later. Four operons (fliAZY, flgMN, fliDST, and flgKL) are expressed from both class 2 and class 3 promoters. To investigate the purpose for expressing genes from multiple flagellar promoters, mutants were constructed for each operon that were defective in either class 2 transcription or class 3 transcription. The mutants were checked for defects in swimming through liquids, swarming over surfaces, and transcriptional regulation. The expression of the hook-associated proteins (FlgK, FlgL, and FliD) from class 3 promoters was found to be important for swarming motility. Both flgMN promoters were involved in coordinating class 3 transcription with the stage of assembly of the hook-basal body. Finally, the fliAZY class 3 promoter lowered class 3 transcription in stationary phase. These results indicate that the multiple flagellar promoters respond to specific environmental conditions and help coordinate transcription with flagellar assembly.

scholarly journals UBA1 and UBA2, Two Proteins That Interact with UBP1, a Multifunctional Effector of Pre-mRNA Maturation in Plants

2002 ◽  
Vol 22 (12) ◽  
pp. 4346-4357 ◽  
Author(s):  
Mark H. L. Lambermon ◽  
Yu Fu ◽  
Dominika A. Wieczorek Kirk ◽  
Marcel Dupasquier ◽  
Witold Filipowicz ◽  
...  
Keyword(s):  
Steady State ◽  
Rna Binding ◽  
Rna Recognition Motif ◽  
Dependent Manner ◽  
Binding Domains ◽  
Mrna Maturation ◽  
Associated Proteins ◽  
Steady State Levels ◽  
Transient Overexpression

ABSTRACT Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)+ RNA in the cell nucleus. Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N. plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones. The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3′ untranslated region (3′-UTR) and protecting the mRNA from exonucleolytic degradation (M. H. L. Lambermon, G. G. Simpson, D. A. Kirk, M. Hemmings-Mieszczak, U. Klahre, and W. Filipowicz, EMBO J. 19:1638-1649, 2000). To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N. plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays. Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A. thaliana. They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains. UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro. As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner. Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent. Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing. These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3′-UTRs and contributing to the stabilization of mRNAs in the nucleus.

mRNA encoding WAVE–Arp2/3-associated proteins is co-localized with foci of active protein synthesis at the leading edge of MRC5 fibroblasts during cell migration

2013 ◽  
Vol 452 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Mark Willett ◽  
Michele Brocard ◽  
Hilary J. Pollard ◽  
Simon J. Morley
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Structural Characteristics ◽  
Leading Edge ◽  
Cell Spreading ◽  
Active Protein ◽  
Associated Proteins ◽  
Steady State Levels ◽  
And Migration ◽  
Syndrome Protein

During cell spreading, mammalian cells migrate using lamellipodia formed from a large dense branched actin network which produces the protrusive force required for leading edge advancement. The formation of lamellipodia is a dynamic process and is dependent on a variety of protein cofactors that mediate their local regulation, structural characteristics and dynamics. In the present study, we show that mRNAs encoding some structural and regulatory components of the WAVE [WASP (Wiskott–Aldrich syndrome protein) verprolin homologous] complex are localized to the leading edge of the cell and associated with sites of active translation. Furthermore, we demonstrate that steady-state levels of ArpC2 and Rac1 proteins increase at the leading edge during cell spreading, suggesting that localized protein synthesis has a pivotal role in controlling cell spreading and migration.

Mesenchymal stem cells in infantile haemangioma

2011 ◽  
Vol 64 (3) ◽  
pp. 232-236 ◽  
Author(s):  
Tinte Itinteang ◽  
Anasuya Vishvanath ◽  
Darren J Day ◽  
Swee T Tan
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Neural Crest ◽  
Capillary Endothelium ◽  
Fatty Tissue ◽  
Rt Pcr ◽  
Functional Potential ◽  
Infantile Haemangioma ◽  
Mesenchymal Differentiation ◽  
Associated Proteins

BackgroundFibro-fatty deposition commonly occurs during involution of infantile haemangioma (IH). Mesenchymal stem cells have been identified in this tumour and have been proposed to be recruited from the bone marrow and/or adjacent niches, and then give rise to the fibro-fatty tissue. The authors have recently demonstrated that the capillary endothelium of proliferating IH co-expresses primitive mesodermal, mesenchymal and neural crest markers and proposed that this same endothelium has the ability to give rise to cells of mesenchymal lineage that constitute the fibro-fatty deposition.MethodsImmunohistochemistry and real-time RT-PCR were used to further characterise proliferating IHs and haemangioma explant-derived cells (HaemEDCs).ResultsThe authors have further confirmed expression of the mesenchymal-associated proteins including preadipocyte factor-1, a mesenchymal differentiation inhibition-associated cytokine. The HaemEDCs could be differentiated into osteoblasts and adipocytes, indicating their functional potential for terminal differentiation.DiscussionThe collective expression of neural crest, mesenchymal and mesenchymal differentiation inhibition-associated proteins on the endothelium of proliferating IH suggests that the cells in the capillary endothelium within the lesion possess the ability to undergo terminal mesenchymal differentiation during the proliferating phase, but are inhibited from doing so.

scholarly journals bdrF2 of Lyme Disease Spirochetes Is Coexpressed with a Series of Cytoplasmic Proteins and Is Produced Specifically during Early Infection

2005 ◽  
Vol 187 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Hongming Zhang ◽  
Abayami Raji ◽  
Michael Theisen ◽  
Paul R. Hansen ◽  
Richard T. Marconi
Keyword(s):  
Real Time ◽  
Dna Helicase ◽  
Early Infection ◽  
Rt Pcr ◽  
Inner Membrane ◽  
Phase Partitioning ◽  
Multiple Promoters ◽  
Cytoplasmic Proteins ◽  
Rapid Amplification ◽  
Triton X

ABSTRACT The Bdr proteins are polymorphic inner membrane proteins produced by most Borrelia species. In Borrelia burgdorferi B31MI, the18 bdr genes form three subfamilies, bdrD, bdrE, and bdrF. The production of at least one of the Bdr paralogs, BdrF2, is up-regulated in host-adapted spirochetes, suggesting a role for the protein in the mammalian environment. Here, we demonstrate using reverse transcriptase (RT) PCR that BBG29, BBG30, BBG31, and BBG32, which reside upstream of bdrF2 , are cotranscribed with bdrF2 as a five-gene operon. While the functions of most of these proteins are unknown, BBG32 encodes a putative DNA helicase. Real-time RT-PCR analyses demonstrated higher levels of bdrF2 transcript relative to other genes of the operon, suggesting that bdrF2 may also be transcribed independently from an internal promoter. Internal promoters were detected using the 5′ rapid amplification of cDNA ends system. The putative promoter associated with bdrF2 was found to be highly similar in sequence to the multiple promoters associated with the ospC gene. Real-time RT-PCR analyses, performed to assess the expression of these genes in infected mice, revealed that genes of the bdrF2 locus are expressed only during early infection, suggesting a role in the establishment of infection. To further characterize the proteins encoded by the bdrF2 locus, which have unknown functions, the cellular localizations of these proteins were determined by Triton X-114 extraction and phase partitioning. BBG29 and BBG31 were found to be cytoplasmic. To determine if these proteins elicit an antibody (Ab) response during infection, immunoblot analyses were performed. Abs to these proteins were not detected. Based on the analyses presented here, we offer the hypothesis that BdrF2 and other proteins encoded by the operon form an inner-membrane-associated protein complex that may interact with DNA and which carries out its functional role during transmission or the early stages of infection.

scholarly journals Neprilysin Is Poorly Expressed in the Prefrontal Cortex of Aged Dogs with Cognitive Dysfunction Syndrome

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Jesús Canudas ◽  
Daniel Insua ◽  
Leticia Sarasa ◽  
Ángela González-Martínez ◽  
María Luisa Suárez ◽  
...  
Keyword(s):  
Cognitive Dysfunction ◽  
Protective Factor ◽  
Cholinergic Neurons ◽  
Cognitively Impaired ◽  
Rt Pcr ◽  
Expression Levels ◽  
Neurodegenerative Process ◽  
Age Related ◽  
Lower Expression

Neprilysin (NEP) is the principal amyloidβ(Aβ) degrading peptidase; this activity may protect against Alzheimer’s disease (AD), the most important age-related neurodegenerative process. The aim of this work was to analyze NEP mRNA expression in the frontal cortex of dogs with and without canine cognitive dysfunction syndrome (CDS), which is considered a natural model for AD. Expression of canine cerebral NEP mRNA was assessed by RT-PCR followed by qPCR in young, aged-cognitively unimpaired (CU), and aged-cognitively impaired (CI) dogs. On average, aged-CI dogs showed 80% (P<0.01) lower expression levels of NEP mRNA than their aged-CU counterparts. Furthermore, the standard deviation of the qPCR measurements was more than 6 times higher in the cognitively healthy animals (young and aged-CU) than in the aged-CI group. Another interesting find is the determination of a positive correlation between NEP expression and the number of cholinergic neurons in basal telencephalon, indicating a probable connection between both events in these types of neurodegeneration processes. These results suggest that high expression levels of NEP might be a protective factor for canine CDS and, most likely, for other Aβ-associated neurodegenerative diseases, such as AD.

Expression of dlk1 in the Bone Marrow Cells of Patients with Myelodysplastic Syndrome and Its Clinical Significance

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5042-5042
Author(s):  
Zonghong Shao ◽  
Lanzhu Yue ◽  
Rong Fu ◽  
Lijuan Li ◽  
Erbao Ruan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Early Diagnosis ◽  
Myelodysplastic Syndrome ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Rt Pcr ◽  
Normal Controls ◽  
Lower Expression ◽  
Bone Marrow Blasts ◽  
Marrow Cells

Abstract Abstract 5042 Objective To investigate the expression of dlk1 gene (delta-like 1) in the bone marrow cells of patients with Myelodysplastic syndrome (MDS), and explore the molecular marker for early diagnosis of MDS. Methods The expression of dlk1 mRNA in the bone marrow cells of cases with MDS, AML and normal controls were measured by RT-PCR, aiming to search for the cytogenetic marker of MDS malignant clone. Results The expression of dlk1 mRNA in bone marrow cells of MDS patients (0.7342±0.3652) was significantly higher than that of normal controls (0.4801±0.1759) (P<0.05), and was significantly positively correlated with the proportion of bone marrow blasts(r=0.467,P<0.05). The expression of dlk1 mRNA significantly increased as the subtype of MDS advanced (P<0.05). Patients with abnormal karyotypes displayed significantly higher expression of dlk1 mRNA (0.9007±0.4334) than those with normal karyotypes (0.6411±0.2630) (P<0.05). Patients with higher expression of dlk1(≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower expression (<0.8) of dlk1 (0.1517±0.3109) (P<0.05). Conclusion dlk1 gene was highly expressed in MDS patients, which increased as the subtype of MDS advanced. The expression of dlk1 mRNA was significantly positively correlated with the proportion of bone marrow blasts. High expression of dlk1 gene suggests high malignant clone burden of MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Chorionic Gonadotropin-Dependent Up-Regulation of Genes Responsible for Estrogen Sulfoconjugation and Export in Granulosa Cells of Luteinizing Preovulatory Follicles

Endocrinology ◽  
2006 ◽  
Vol 147 (9) ◽  
pp. 4222-4233 ◽  
Author(s):  
Kristy A. Brown ◽  
Monique Doré ◽  
Jacques G. Lussier ◽  
Jean Sirois
Keyword(s):  
Chorionic Gonadotropin ◽  
Granulosa Cells ◽  
Human Chorionic Gonadotropin ◽  
Cell Layer ◽  
Rt Pcr ◽  
Expression Of Genes ◽  
Preovulatory Follicles ◽  
Steady State Levels ◽  
17Β Estradiol ◽  
Theca Interna

Estrogen sulfotransferase (EST) is responsible for the sulfoconjugation of estrogens, thereby changing their physical properties and preventing their action via the estrogen receptors. These sulfoconjugated steroids no longer diffuse freely across the lipid bilayer; instead, they are exported by members of the ATP-binding cassette family, such as ABCC1. The objective of this study was to investigate the regulation of EST and ABCC1 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The transcripts for EST and ABCC1 were cloned by RT-PCR, and the regulation of their mRNAs was studied in preovulatory follicles obtained during estrus at 0, 12, 24, 30, 33, 36, and 39 h after hCG. Results obtained from RT-PCR/Southern blot analyses showed significant changes in steady-state levels of both EST and ABCC1 mRNA after hCG treatment (P &lt; 0.05). In granulosa cells, a significant increase in EST transcript was observed 30–39 h after hCG. Similarly, ABCC1 transcript levels were induced in granulosa cells 12–39 h after hCG. In contrast, no significant changes in either EST or ABCC1 were detected in theca interna samples after hCG. The increase in EST and ABCC1 transcripts observed in granulosa cells was reflected in preparations of intact follicle walls, suggesting that the granulosa cell layer contributes the majority of EST and ABCC1 expression in preovulatory follicles. The present study demonstrates that follicular luteinization is accompanied not only by a decrease in 17β-estradiol biosynthesis but also by an increase in expression of genes responsible for estrogen inactivation and elimination from granulosa cells, such as EST and ABCC1, respectively.

Effects of bovine viral diarrhoea virus on the fertility of cows

2013 ◽  
Vol 61 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Sibel Yavru ◽  
Mehmet Kale ◽  
Mehmet Gulay ◽  
Orhan Yapici ◽  
Oya Bulut ◽  
...  
Keyword(s):  
Bovine Viral Diarrhoea Virus ◽  
Bovine Viral Diarrhoea ◽  
Rt Pcr ◽  
Blood Samples ◽  
Frozen Semen ◽  
Diarrhoea Virus ◽  
Mucous Discharge ◽  
Service Conception ◽  
Negative Effect ◽  
Polymerase Chain

The aim of the present study was to determine the possible relationship between bovine viral diarrhoea (BVD) virus infection and the appearance of cervical mucous discharge (CMD) and the reproductive performance of cows in oestrus. For this purpose, CMD from 97 Holstein cows in oestrus was evaluated visually before artificial insemination (AI). Cows in oestrus were inseminated with frozen semen free from BVD virus (BVDV). Blood samples were tested by enzyme-linked immunoassay (ELISA) for antigen (Ag) and antibodies (Ab) of BVDV. The presence of the BVDV genome in cervical mucus samples was tested by reverse transcriptase-polymerase chain reaction (RT-PCR). The presence of BVDV Ab, Ag or genome was not associated with abnormal cervical mucous discharge (A-CMD). However, the presence of BVDV Ag (but not of the BVDV Ab) in blood samples was associated with a lower first service conception rate (FSCR; 27.8 vs. 70.9%; P < 0.01), indicating that BVDV viraemia at the time of AI has a negative effect on the fertility of cows.

scholarly journals Role of the sRNA GcvB in regulation of cycA in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 106-114 ◽  
Author(s):  
Sarah C. Pulvermacher ◽  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Minimal Medium ◽  
Luria Bertani ◽  
Cell Physiology ◽  
Rt Pcr ◽  
Luria Bertani Broth ◽  
Increased Sensitivity ◽  
Transcriptional Fusions

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

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