scholarly journals Isolation of Developmentally Regulated Genes fromToxoplasma gondii by a Gene Trap with the Positive and Negative Selectable Marker Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase

1998 ◽  
Vol 18 (2) ◽  
pp. 807-814 ◽  
Author(s):  
Laura J. Knoll ◽  
John C. Boothroyd
Keyword(s):  
Antimicrobial Agents ◽  
Selectable Marker ◽  
Insertion Site ◽  
Gene Trap ◽  
Specific Gene ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
S1 Nuclease ◽  
Nuclease Digestion

ABSTRACT Within its intermediate host, Toxoplasma gondiiswitches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterlessHXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasmafamily of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.

scholarly journals Isolation and Characterization of Novel Phages Targeting Pathogenic Klebsiella pneumoniae

2021 ◽  
Vol 11 ◽  
Author(s):  
Na Li ◽  
Yigang Zeng ◽  
Rong Bao ◽  
Tongyu Zhu ◽  
Demeng Tan ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Bacterial Infections ◽  
Antimicrobial Agents ◽  
Multidrug Resistant ◽  
S1 Nuclease ◽  
Isolation And Characterization ◽  
Comprehensive Knowledge ◽  
Future Challenges ◽  
Lactamase Gene

Klebsiella pneumoniae is a dominant cause of community-acquired and nosocomial infections, specifically among immunocompromised individuals. The increasing occurrence of multidrug-resistant (MDR) isolates has significantly impacted the effectiveness of antimicrobial agents. As antibiotic resistance is becoming increasingly prevalent worldwide, the use of bacteriophages to treat pathogenic bacterial infections has recently gained attention. Elucidating the details of phage-bacteria interactions will provide insights into phage biology and the better development of phage therapy. In this study, a total of 22 K. pneumoniae isolates were assessed for their genetic and phenotypic relatedness by multi-locus sequence typing (MLST), endonuclease S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), and in vitro antibiotic susceptibility testing. In addition, the beta-lactamase gene (blaKPC) was characterized to determine the spread and outbreak of K. pneumoniae carbapenemase (KPC)-producing enterobacterial pathogens. Using these ST11 carbapenem-resistant K. pneumoniae isolates, three phages (NL_ZS_1, NL_ZS_2, and NL_ZS_3) from the family of Podoviridae were isolated and characterized to evaluate the application of lytic phages against the MDR K. pneumoniae isolates. In vitro inhibition assays with three phages and K. pneumoniae strain ZS15 demonstrated the strong lytic potential of the phages, however, followed by the rapid growth of phage-resistant and phage-sensitive mutants, suggesting several anti-phage mechanisms had developed in the host populations. Together, this data adds more comprehensive knowledge to known phage biology and further emphasizes their complexity and future challenges to overcome prior to using phages for controlling this important MDR bacterium.

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins, osteocalcin, and matrix Gla protein in vitro

1994 ◽  
Vol 160 (2) ◽  
pp. 255-264 ◽  
Author(s):  
Leesa M. Barone ◽  
Michael A. Aronow ◽  
Melissa S. Tassinari ◽  
Donna Conlon ◽  
Gary S. Stein ◽  
...  
Keyword(s):  
Vitamin K ◽  
Matrix Gla Protein ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Differential Effects

scholarly journals Inducible Gene Trapping with Drug-Selectable Markers and Cre/loxP To Identify Developmentally Regulated Genes

2004 ◽  
Vol 24 (22) ◽  
pp. 9930-9941 ◽  
Author(s):  
You-Tzung Chen ◽  
Pentao Liu ◽  
Allan Bradley
Keyword(s):  
Retinoic Acid ◽  
Acid Treatment ◽  
Gene Trap ◽  
In Vitro Differentiation ◽  
Retinoic Acid Treatment ◽  
Developmentally Regulated ◽  
Gene Trapping ◽  
Gene Traps ◽  
Inducible Gene

ABSTRACT Gene trapping in mouse embryonic stem cells is an important genetic approach that allows simultaneous mutation of genes and generation of corresponding mutant mice. We designed a selection scheme with drug selection markers and Cre/loxP technology which allows screening of gene trap events that responded to a signaling molecule in a 96-well format. Nine hundred twenty gene trap clones were assayed, and 258 were classified as gene traps induced by in vitro differentiation. Sixty-five of the in vitro differentiation-inducible gene traps were also responsive to retinoic acid treatment. In vivo analysis revealed that 85% of the retinoic acid-inducible gene traps trapped developmentally regulated genes, consistent with the observation that genes induced by retinoic acid treatment are likely to be developmentally regulated. Our results demonstrate that the inducible gene trapping system described here can be used to enrich in vitro for traps in genes of interest. Furthermore, we demonstrate that the cre reporter is extremely sensitive and can be used to explore chromosomal regions that are not detectable with neo as a selection cassette.

scholarly journals Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei

2020 ◽  
Vol 48 (15) ◽  
pp. 8704-8723
Author(s):  
Joseph T Smith Jr. ◽  
Eva Doleželová ◽  
Brianna Tylec ◽  
Jonathan E Bard ◽  
Runpu Chen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
High Throughput Sequencing ◽  
Environmental Changes ◽  
Developmental Regulation ◽  
Complex Life Cycle ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Life Cycle Stages

Abstract Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3′ to 5′ progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.

scholarly journals Identification of the Ω4514 Regulatory Region, a Developmental Promoter of Myxococcus xanthus That Is Transcribed In Vitro by the Major Vegetative RNA Polymerase

2002 ◽  
Vol 184 (12) ◽  
pp. 3348-3359 ◽  
Author(s):  
Tong Hao ◽  
Dvora Biran ◽  
Gregory J. Velicer ◽  
Lee Kroos
Keyword(s):  
Rna Polymerase ◽  
Myxococcus Xanthus ◽  
Insertion Site ◽  
Regulatory Region ◽  
Developmental Expression ◽  
Binding Motif ◽  
Developmentally Regulated ◽  
Coa Transferase ◽  
Negative Autoregulation

ABSTRACT Ω4514 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. DNA upstream of the insertion site was cloned, and the promoter was localized. The promoter resembles vegetative promoters in sequence, and σA RNA polymerase, the major form of RNA polymerase in growing M. xanthus, initiated transcription from this promoter in vitro. Two complete open reading frames were identified downstream of the promoter and before the Ω4514 insertion. The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators. ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation. Tn5 lac Ω4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase. An orf1 disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of orf2 nor insertion Ω4514 in orf3 caused a defect. Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases. ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an orf1 mutant. Also, multiple upstream DNA elements are necessary for full developmental expression. These results suggest that transcriptional activation also regulates the operon. Ω4514 is the first example of a developmentally regulated M. xanthus operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.

Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1737-1737
Author(s):  
Liyan Pang ◽  
Xun Wang ◽  
Yuhuan Wang ◽  
Gerd Blobel ◽  
Mortimer Poncz
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Critical Role ◽  
Regulatory Region ◽  
Maturation Stage ◽  
Specific Gene ◽  
Mrna Levels ◽  
Specific Regulation

Abstract The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

Tissue-specific gene expression of prolactin receptor in the acute-phase response induced by lipopolysaccharides

2004 ◽  
Vol 287 (4) ◽  
pp. E750-E757 ◽  
Author(s):  
Ana M. Corbacho ◽  
Giuseppe Valacchi ◽  
Lukas Kubala ◽  
Estibaliz Olano-Martín ◽  
Bettina C. Schock ◽  
...  
Keyword(s):  
Acute Phase ◽  
Acute Phase Response ◽  
Prolactin Receptor ◽  
Phase Response ◽  
Specific Gene ◽  
Mrna Levels ◽  
Tissue Specific ◽  
Specific Regulation

Acute inflammation can elicit a defense reaction known as the acute-phase response (APR) that is crucial for reestablishing homeostasis in the host. The role for prolactin (PRL) as an immunomodulatory factor maintaining homeostasis under conditions of stress has been proposed; however, its function during the APR remains unclear. Previously, it was shown that proinflammatory cytokines characteristic of the APR (TNF-α, IL-1β, and IFNγ) induced the expression of the PRL receptor (PRLR) by pulmonary fibroblasts in vitro. Here, we investigated the in vivo expression of PRLR during lipopolysaccharide (LPS)-induced APR in various tissues of the mouse. We show that PRLR mRNA and protein levels were downregulated in hepatic tissues after intraperitoneal LPS injection. Downregulation of PRLR in the liver was confirmed by immunohistochemistry. A suppressive effect on mRNA expression was also observed in prostate, seminal vesicle, kidney, heart, and lung tissues. However, PRLR mRNA levels were increased in the thymus, and no changes were observed in the spleen. The proportion of transcripts for the different receptor isoforms (long, S1, S2, and S3) in liver and thymus was not altered by LPS injection. These findings suggest a complex tissue-specific regulation of PRLR expression in the context of the APR.

Transcription of a trout protamine gene in vitro: the effects of alteration of promoters

1984 ◽  
Vol 62 (5) ◽  
pp. 291-300 ◽  
Author(s):  
Jacek M. Jankowski ◽  
Gordon H. Dixon
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Tata Box ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Promoter Sequences ◽  
Transcription System ◽  
Nuclease Digestion ◽  
Simplex Virus

An in vitro approach has been used to study trout protamine gene expression using various recombinant plasmids containing trout protamine genes as templates in the HeLa cell lysate transcription system. The specific RNA transcript which is protected against S1 nuclease digestion by hybridization to the protamine gene sequence is α-amanitin sensitive (1 μg/mL), showing that RNA polymerase II is involved. The sizes of transcripts from templates linearized with Bam HI, Rsa I, and Hpa II (all downstream from the putative TATA box) are consistent with those predicted from the known sequence of the protamine gene. Digestion at an Alu I site only 14 base pairs (bp) upstream from TATA box has no effect on the accuracy of transcription in vitro; however, cutting at an Ava II site 9 bp downstream from the TATA box (reading from the first T) abolishes transcription. Chimeric plasmids, in which a herpes simplex virus (HSV-1) thymidine kinase (tk) promoter is tandemly inserted upstream from the trout protamine DNA sequences or as a replacement of the natural protamine promoter, were constructed. Use of these plasmids allowed an examination in a single assay of eight different putative promoter sequences (TATAAAA, TATAAA, TACAAA, TATATA, TATTTAA, CATATTA, TATATTAT, and TATTTAT) that are localized in either the protamine or the tk genes. The canonical TATAAAA promoter (the natural protamine promoter) was the strongest one and, in its presence, none of the others were used significantly for transcription. However, when this promoter was removed the weaker promoters were able to promote transcription.

scholarly journals Wnt-11 Expression Promotes Invasiveness and Correlates with Survival in Human Pancreatic Ductal Adeno Carcinoma

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 921 ◽  
Author(s):  
Dart ◽  
Arisan ◽  
Owen ◽  
Hao ◽  
Jiang ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
Ductal Adenocarcinoma ◽  
Boyden Chamber ◽  
Mrna Levels ◽  
Rna Seq ◽  
Developmentally Regulated ◽  
Mesenchymal Transition ◽  
Adeno Carcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer, proving difficult to manage clinically. Wnt-11, a developmentally regulated gene producing a secreted protein, has been associated with various carcinomas but has not previously been studied in PDAC. The present study aimed to elucidate these aspects first in vitro and then in a clinical setting in vivo. Molecular analyses of Wnt-11 expression as well as other biomarkers involved qRT-PCR, RNA-seq and siRNA. Proliferation was measured by MTT; invasiveness was quantified by Boyden chamber (Matrigel) assay. Wnt-11 mRNA was present in three different human PDAC cell lines. Wnt-11 loss affected epithelial-mesenchymal transition and expression of neuronal and stemness biomarkers associated with metastasis. Indeed, silencing Wnt-11 in Panc-1 cells significantly inhibited their Matrigel invasiveness without affecting their proliferative activity. Consistently with the in vitro data, human biopsies of PDAC showed significantly higher Wnt-11 mRNA levels compared with matched adjacent tissues. Expression was significantly upregulated during PDAC progression (TNM stage I to II) and maintained (TNM stages III and IV). Wnt-11 is expressed in PDAC in vitro and in vivo and plays a significant role in the pathophysiology of the disease; this evidence leads to the conclusion that Wnt-11 could serve as a novel, functional biomarker PDAC.

scholarly journals Transcriptional Networks in the Liver: Hepatocyte Nuclear Factor 6 Function Is Largely Independent of Foxa2

2005 ◽  
Vol 25 (16) ◽  
pp. 7069-7077 ◽  
Author(s):  
Nir E. Rubins ◽  
Joshua R. Friedman ◽  
Phillip P. Le ◽  
Liping Zhang ◽  
John Brestelli ◽  
...  
Keyword(s):  
Current Model ◽  
Cdna Array ◽  
Interaction Model ◽  
Transcriptional Networks ◽  
Specific Gene ◽  
Mrna Levels ◽  
Significant Difference ◽  
And Control

ABSTRACT A complex network of hepatocyte nuclear transcription factors, including HNF6 and Foxa2, regulates the expression of liver-specific genes. The current model, based on in vitro studies, suggests that HNF6 and Foxa2 interact physically. This interaction is thought to synergistically stimulate Foxa2-dependent transcription through the recruitment of p300/CBP by HNF6 and to inhibit HNF6-mediated transcription due to the interference of Foxa2 with DNA binding by HNF6. To test this model in vivo, we utilized hepatocyte-specific gene ablation to study the binding of HNF6 to its targets in the absence of Foxa2. Chromatin immunoprecipitation using anti-HNF6 antibodies was performed on chromatin isolated from Foxa2 loxP/loxP Alfp.Cre and control mouse livers, and HNF6 binding to its target, Glut2, was determined by quantitative PCR. In contrast to the current model, we found no significant difference in HNF6 occupancy at the Glut2 promoter between Foxa2-deficient and control livers. In order to evaluate the Foxa2/HNF6 interaction model on a global scale, we performed a location analysis using a microarray with 7,000 mouse promoter fragments. Again, we found no evidence that HNF6 binding to its targets in chromatin is reduced in the presence of Foxa2. We also examined the mRNA levels of HNF6 targets in the liver using a cDNA array and found that their expression was similar in Foxa2-deficient and control mice. Overall, our studies demonstrate that HNF6 binds to and regulates its target promoters in vivo in the presence and absence of Foxa2.

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