scholarly journals Isolation of secreted proteins fromDrosophilaovaries and embryos throughin vivoBirA-mediated biotinylation

2019 ◽  
Author(s):  
Leslie M. Stevens ◽  
Yuan Zhang ◽  
Yuri Volnov ◽  
Geng Chen ◽  
David S. Stein
Keyword(s):  
Transcriptional Control ◽  
Ovarian Follicle ◽  
Structural Features ◽  
Secreted Proteins ◽  
Follicle Cells ◽  
Covalent Interaction ◽  
E Coli ◽  
Experimental Approaches ◽  
Female Germline

AbstractThe extraordinarily strong non-covalent interaction between biotin and avidin (kD = 10-14-10-16) has permitted this interaction to be used in a wide variety of experimental contexts. The Biotin Acceptor Peptide (BAP), a 15 amino acid motif that can be biotinylated by theE. coliBirA protein, has been fused to proteins of interest, making them substrates forin vivobiotinylation. Here we report on the construction and characterization of a modified BirA bearing signals for secretion and endoplasmic reticulum (ER) retention, for use in experimental contexts requiring biotinylation of secreted proteins. When expressed in theDrosophilafemale germline or ovarian follicle cells under Gal4-mediated transcriptional control, the modified BirA protein could be detected and shown to be enzymatically active in ovaries and progeny embryos. Surprisingly, however, it was not efficiently retained in the ER, and instead appeared to be secreted. To determine whether this secreted protein, now designated secBirA, could biotinylate secreted proteins, we generated BAP-tagged versions of two secretedDrosophilaproteins, Torsolike (Tsl) and Gastrulation Defective (GD), which are normally expressed maternally and participate in embryonic pattern formation. Both Tsl-BAP and GD-BAP were shown to exhibit normal patterning activity. Co-expression of Tsl-BAP together with secBirA in ovarian follicle cells resulted in its biotinylation, which permitted its isolation from both ovaries and progeny embryos using Avidin-coupled affinity matrix. In contrast, co-expression with secBirA in the female germline did not result in detectable biotinylation of GD-BAP, possibly because the C-terminal location of the BAP tag made it inaccessible to BirAin vivo. Our results indicate that secBirA directs biotinylation of proteins bound for secretionin vivo, providing access to powerful experimental approaches for secreted proteins of interest. However, efficient biotinylation of target proteins may vary depending upon the location of the BAP tag or other structural features of the protein.

scholarly journals Multiple layers of control govern expression of the Escherichia coli ibpAB heat-shock operon

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 66-76 ◽  
Author(s):  
Lena C. Gaubig ◽  
Torsten Waldminghaus ◽  
Franz Narberhaus
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Transcriptional Control ◽  
Rnase E ◽  
Translation Control ◽  
E Coli ◽  
Complex Process ◽  
Shock Proteins

The Escherichia coli ibpAB operon encodes two small heat-shock proteins, the inclusion-body-binding proteins IbpA and IbpB. Here, we report that expression of ibpAB is a complex process involving at least four different layers of control, namely transcriptional control, RNA processing, translation control and protein stability. As a typical member of the heat-shock regulon, transcription of the ibpAB operon is controlled by the alternative sigma factor σ 32 (RpoH). Heat-induced transcription of the bicistronic operon is followed by RNase E-mediated processing events, resulting in monocistronic ibpA and ibpB transcripts and short 3′-terminal ibpB fragments. Translation of ibpA is controlled by an RNA thermometer in its 5′ untranslated region, forming a secondary structure that blocks entry of the ribosome at low temperatures. A similar structure upstream of ibpB is functional in vitro but not in vivo, suggesting downregulation of ibpB expression in the presence of IbpA. The recently reported degradation of IbpA and IbpB by the Lon protease and differential regulation of IbpA and IbpB levels in E. coli are discussed.

scholarly journals In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation

2016 ◽  
Vol 113 (8) ◽  
pp. 2294-2299 ◽  
Author(s):  
Fernando F. Migone ◽  
Robert G. Cowan ◽  
Rebecca M. Williams ◽  
Kiersten J. Gorse ◽  
Warren R. Zipfel ◽  
...  
Keyword(s):  
Blood Flow ◽  
Ovarian Follicle ◽  
Multiphoton Microscopy ◽  
Follicle Cells ◽  
Endothelin Receptor ◽  
Wild Type ◽  
Transabdominal Ultrasonography ◽  
Follicle Rupture

Rupture of the ovarian follicle releases the oocyte at ovulation, a timed event that is critical for fertilization. It is not understood how the protease activity required for rupture is directed with precise timing and localization to the outer surface, or apex, of the follicle. We hypothesized that vasoconstriction at the apex is essential for rupture. The diameter and blood flow of individual vessels and the thickness of the apical follicle wall were examined over time to expected ovulation using intravital multiphoton microscopy. Vasoconstriction of apical vessels occurred within hours preceding follicle rupture in wild-type mice, but vasoconstriction and rupture were absent inAmhr2cre/+SmoM2mice in which follicle vessels lack the normal association with vascular smooth muscle. Vasoconstriction is not simply a response to reduced thickness of the follicle wall; vasoconstriction persisted in wild-type mice when thinning of the follicle wall was prevented by infusion of protease inhibitors into the ovarian bursa. Ovulation was inhibited by preventing the periovulatory rise in the expression of the vasoconstrictor endothelin 2 by follicle cells of wild-type mice. In these mice, infusion of vasoconstrictors (either endothelin 2 or angiotensin 2) into the bursa restored the vasoconstriction of apical vessels and ovulation. Additionally, infusion of endothelin receptor antagonists into the bursa of wild-type mice prevented vasoconstriction and follicle rupture. Processing tissue to allow imaging at increased depth through the follicle and transabdominal ultrasonography in vivo showed that decreased blood flow is restricted to the apex. These results demonstrate that vasoconstriction at the apex of the follicle is essential for ovulation.

scholarly journals Regulation of Drosophila yolk protein genes by an ovary-specific GATA factor.

1995 ◽  
Vol 15 (12) ◽  
pp. 6943-6952 ◽  
Author(s):  
M Lossky ◽  
P C Wensink
Keyword(s):  
Fat Body ◽  
Regulatory Element ◽  
Ovarian Follicle ◽  
In Vitro Transcription ◽  
Yolk Protein ◽  
Follicle Cells ◽  
Gata Factor ◽  
Yolk Protein Genes

The divergently transcribed yolk protein genes (Yp1 and Yp2) of Drosophila melanogaster are expressed only in adult females, in fat body tissue and in ovarian follicle cells. Using an in vitro transcription assay, we have identified a single 12-bp DNA element that activates transcription from the promoters of both Yp genes. In vivo, this regulatory element is tissue specific: it activates transcription of Yp1 and Yp2 reporter genes in follicle cells but has no detectable effect in fat body or other tissues. The sequence of the element consists of two recognition sites for the GATA family of transcription factors. We show that among the Drosophila genes known to encode GATA factors, only dGATAb is expressed in ovaries. The single transcript that we detect in ovaries is alternatively spliced or initiated to produce an ovary-specific isoform of the protein. Bacterially expressed dGATAb binds to the 12-bp element; a similar binding activity is also present in the Kc0 nuclear extracts used for in vitro transcription assays. These in vitro and in vivo results lead us to propose that dGATAb makes several developmentally regulated products, one of which is a follicle cell-specific protein activating transcription of Yp1 and Yp2 from a known regulatory element.

scholarly journals Structure of a novel dimeric SET domain methyltransferase that regulates cell motility

2018 ◽  
Author(s):  
Yulia Pivovarova ◽  
Jun Liu ◽  
Johannes Lesigang ◽  
Oliver Koldyka ◽  
Rene Rauschmeier ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Critical Role ◽  
Structural Features ◽  
Host Cells ◽  
Binding Motif ◽  
Cellular Motility ◽  
Apicomplexan Parasites ◽  
Dimerization Interface ◽  
Parasite Egress

AbstractLysine methyltransferases (KMTs) were initially associated with transcriptional control through their methylation of histones and other nuclear proteins, but have since been found to regulate many other cellular activities. The apical complex lysine (K) methyltransferase (AKMT) of the human parasite Toxoplasma gondii was recently shown to play a critical role in regulating cellular motility. Here we report a 2.1-Å resolution crystal structure of the conserved and functional C-terminal portion (aa289-709) of T. gondii AKMT. AKMT dimerizes via a unique intermolecular interface mediated by the C-terminal TPR (tetratricopeptide repeat)-like domain together with a specific zinc-binding motif that is absent from all other KMTs. Disruption of AKMT dimerization impaired both its enzyme activity and parasite egress from infected host cells in vivo. Structural comparisons reveal that AKMT is related to the KMTs in the SMYD family, with, however, a number of distinct structural features in addition to the unusual dimerization interface. These features are conserved among the apicomplexan parasites and their free-living relatives, but not found in any known KMTs in animals. AKMT therefore is the founding member of a new subclass of KMT that has important implications for the evolution of the apicomplexans.

scholarly journals The yefM-yoeB Toxin-Antitoxin Systems of Escherichia coli and Streptococcus pneumoniae: Functional and Structural Correlation

2006 ◽  
Vol 189 (4) ◽  
pp. 1266-1278 ◽  
Author(s):  
Concha Nieto ◽  
Izhack Cherny ◽  
Seok Kooi Khoo ◽  
Mario García de Lacoba ◽  
Wai Ting Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Structural Features ◽  
E Coli ◽  
Modeled Structure ◽  
The Family ◽  
Bona Fide ◽  
K 12

ABSTRACT Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM Spn ) and toxin (YoeB Spn ) products. We showed that overproduction of YoeB Spn is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB Spn -mediated toxicity could be reversed by the cognate antitoxin, YefM Spn , but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

scholarly journals Evidence for an Arginine Exporter Encoded by yggA (argO) That Is Regulated by the LysR-Type Transcriptional Regulator ArgP in Escherichia coli

2004 ◽  
Vol 186 (11) ◽  
pp. 3539-3546 ◽  
Author(s):  
Madhusudan R. Nandineni ◽  
J. Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Control ◽  
Transcriptional Regulator ◽  
Basic Amino Acid ◽  
Missense Mutations ◽  
Reading Frame ◽  
E Coli ◽  
Regulator Protein ◽  
Outward Transport

ABSTRACT The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP+ -dependent induction by arginine. Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP+ strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription. Dominant missense mutations in argP (argPd ) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained. The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E. coli strains with either the argPd mutation or multicopy yggA+ . The null yggA mutation abolished the increased arginine efflux from the argPd strain. Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for “arginine outward transport”). We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations.

scholarly journals Dynamic landscape of protein occupancy across the Escherichia coli chromosome

2020 ◽  
Author(s):  
Peter L. Freddolino ◽  
Thomas J. Goss ◽  
Haley M. Amemiya ◽  
Saeed Tavazoie
Keyword(s):  
Genetic Manipulation ◽  
Dynamic Condition ◽  
Bacterial Genome ◽  
Structural Features ◽  
Sequence Specificity ◽  
Bacterial Physiology ◽  
E Coli ◽  
Transcriptional Regulatory ◽  
Protein Occupancy

AbstractFree living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein-DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using a modification of in vivo protein occupancy display technology (IPOD-HR) applied to E. coli. We observe that the E. coli protein-DNA interactome organizes into two distinct prototypic features: (1) highly dynamic condition-dependent transcription factor occupancy by dedicated transcriptional regulators, and (2) condition-invariant kilobase scale occupancy by nucleoid factors, forming silencing domains analogous to eukaryotic heterochromatin. We show that occupancy dynamics across a range of conditions can rapidly reveal the global transcriptional regulatory organization of a bacterium. Beyond discovery of previously hidden regulatory logic, we show that these observations can be utilized to computationally determine sequence-specificity models for the majority of active transcription factors. Our study demonstrates that global observations of protein occupancy combined with statistical inference can rapidly and systematically reveal the transcriptional regulatory and structural features of a bacterial genome. This capacity is particularly crucial for non-model bacteria which are not amenable to routine genetic manipulation.

Differential sorting of constitutively co-secreted proteins in the ovarian follicle cells of Drosophila

2001 ◽  
Vol 80 (4) ◽  
pp. 271-284 ◽  
Author(s):  
Ioannis P. Trougakos ◽  
Issidora S. Papassideri ◽  
Gail L. Waring ◽  
Lukas H. Margaritis
Keyword(s):  
Ovarian Follicle ◽  
Secreted Proteins ◽  
Follicle Cells

THE OVARIAN FOLLICLE OF THE SHEEPINHIBITION OF OESTROGEN SECRETION BY LUTEINIZING HORMONE

1974 ◽  
Vol 61 (3) ◽  
pp. 455-463 ◽  
Author(s):  
R. M. MOOR
Keyword(s):  
Luteinizing Hormone ◽  
Organ Culture ◽  
Culture Medium ◽  
Ovarian Follicle ◽  
Corpora Lutea ◽  
In Vitro Techniques ◽  
Progesterone Secretion ◽  
Experimental Approaches

SUMMARY The object of this study was to test the hypothesis that levels of luteinizing hormone (LH), comparable to those circulating at oestrus, inhibit oestrogen secretion from Graafian follicles of sheep. Three experimental approaches were used. Follicles maintained in organ culture secreted high levels of oestrogen into the medium throughout a 7-day culture period; almost no progesterone was secreted under such conditions. By contrast, oestrogen secretion declined precipitously and progesterone secretion increased rapidly after the addition of LH (0·25 μg–10 μg NIH-LH-S 17/ml) to the culture medium. In experiments combining in-vivo and in-vitro techniques, follicles were obtained from sheep from which the corpora lutea had been removed 24 h previously. The large follicles explanted from these sheep secreted high levels of oestrogen throughout the 7 days in culture. Insignificant amounts of oestrogen were, however, secreted in culture by large follicles that had been explanted from sheep in which 1 mg LH had been infused between 18 and 24 h after removal of the corpus luteum. Experiments carried out entirely in vivo showed that intravenous infusion of 1 mg LH into sheep from which the corpora lutea had been removed 18 h previously prevented the ovaries from secreting, during the ensuing 22 h, the large amounts of oestrogen they would otherwise have produced. The results demonstrate that oestrogen secretion by large Graafian follicles is terminated both in vitro and in vivo by an amount of LH corresponding to that released at oestrus.

Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3’ Untranslated Regions

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Structural Features ◽  
Neonatal Rat ◽  
Regulatory Control ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Regulatory Effects

Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5’ and 3’ untranslated regions (UTRs) of mature mRNA. The 3’UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin’s 3’ UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3’UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin’s respective 3’UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin’s shorter 3’UTR sequence was inconclusive; however, results illustrated that titin’s longer 3’UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin’s variant 3’UTR sequences.

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