Transcriptional control of the rat hepatic CYP2E1 gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4495-4505
Author(s):  
T Ueno ◽  
F J Gonzalez
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Transcriptional Control ◽  
Developmental Stages ◽  
Nuclear Extract ◽  
Binding Studies ◽  
Mobility Shift ◽  
Adult Rats ◽  
Cyp2e1 Gene

The rat hepatic CYP2E1 gene becomes transcriptionally activated within 1 day after birth. This activation can be mimicked by using the 5' end of the gene in a cell-free nuclear extract prepared from hepatocytes taken from rats at different developmental stages. Deletion analysis revealed that a positive element located between -127 and -89 was responsible for 90% of the in vitro transcription activity of adult liver extracts. Protein binding studies revealed that this region was operationally equivalent to the binding site for the factor HNF-1. Two other protein-binding regions were uncovered, one of which corresponded to the site for a CCAAT-binding factor NFY. The other site was a palindrome sequence unique to the CYP2E1 gene. These latter two factors did not significantly contribute to transcriptional activity in vitro and were not conserved between the rat and human CYP2E1 genes. Extracts prepared from fetal and newborn livers were transcriptionally inactive, whereas extracts from livers of 3-day-old rats were fully active toward the CYP2E1 gene. DNase I footprinting patterns indistinguishable between fetal and adult extracts were obtained for all three factors. However, gel mobility shift assays revealed a second, higher-mobility band produced by fetal and newborn liver extracts bound to the HNF-1 oligomer. UV-cross-linking studies showed that adult and fetal extracts had only a single 98-kilodalton protein that bound to this oligomer. In contrast, adult lung samples, also transcriptionally inactive toward the CYP2E1 gene, contained two proteins of slightly greater than 110 kilodaltons. These results suggest that the CYP2E1 gene is positively regulated in adult rats by HNF-1 or a protein similar in DNA-binding properties to HNF-1. The role of this factor or other protein-protein interactions in the lack of CYP2E1 transcription in fetal and newborn animals remains unclear.

scholarly journals Transcriptional control of the rat hepatic CYP2E1 gene.

1990 ◽  
Vol 10 (9) ◽  
pp. 4495-4505 ◽  
Author(s):  
T Ueno ◽  
F J Gonzalez
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Transcriptional Control ◽  
Developmental Stages ◽  
Nuclear Extract ◽  
Binding Studies ◽  
Mobility Shift ◽  
Adult Rats ◽  
Cyp2e1 Gene

The rat hepatic CYP2E1 gene becomes transcriptionally activated within 1 day after birth. This activation can be mimicked by using the 5' end of the gene in a cell-free nuclear extract prepared from hepatocytes taken from rats at different developmental stages. Deletion analysis revealed that a positive element located between -127 and -89 was responsible for 90% of the in vitro transcription activity of adult liver extracts. Protein binding studies revealed that this region was operationally equivalent to the binding site for the factor HNF-1. Two other protein-binding regions were uncovered, one of which corresponded to the site for a CCAAT-binding factor NFY. The other site was a palindrome sequence unique to the CYP2E1 gene. These latter two factors did not significantly contribute to transcriptional activity in vitro and were not conserved between the rat and human CYP2E1 genes. Extracts prepared from fetal and newborn livers were transcriptionally inactive, whereas extracts from livers of 3-day-old rats were fully active toward the CYP2E1 gene. DNase I footprinting patterns indistinguishable between fetal and adult extracts were obtained for all three factors. However, gel mobility shift assays revealed a second, higher-mobility band produced by fetal and newborn liver extracts bound to the HNF-1 oligomer. UV-cross-linking studies showed that adult and fetal extracts had only a single 98-kilodalton protein that bound to this oligomer. In contrast, adult lung samples, also transcriptionally inactive toward the CYP2E1 gene, contained two proteins of slightly greater than 110 kilodaltons. These results suggest that the CYP2E1 gene is positively regulated in adult rats by HNF-1 or a protein similar in DNA-binding properties to HNF-1. The role of this factor or other protein-protein interactions in the lack of CYP2E1 transcription in fetal and newborn animals remains unclear.

scholarly journals Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534 ◽  
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Pyrene-based prospective biomaterial: In vitro bioimaging, protein binding studies and detection of bilirubin and Fe3+

2019 ◽  
Vol 221 ◽  
pp. 117150 ◽  
Author(s):  
Venkatesan Srinivasan ◽  
Mariadoss Asha Jhonsi ◽  
Namasivayam Dhenadhayalan ◽  
King-Chuen Lin ◽  
Devanesan Arul Ananth ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Studies

scholarly journals Host Protein Interactions with the 3′ End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication

2000 ◽  
Vol 74 (11) ◽  
pp. 5053-5065 ◽  
Author(s):  
Jeannie F. Spagnolo ◽  
Brenda G. Hogue
Keyword(s):  
Protein Interactions ◽  
Helper Virus ◽  
Host Protein ◽  
Genome Replication ◽  
Mobility Shift ◽  
Bovine Coronavirus ◽  
Replication Assay ◽  
Infected Cells ◽  
Gel Mobility Shift

ABSTRACT RNA viruses have 5′ and 3′ untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5′ end and has a 3′ UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3′ UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3′ UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important cis-acting signal for coronavirus replication.

Controlling the Physical Behavior and Biological Performance of Liposome Formulations Through Use of Surface Grafted Poly(ethylene Glycol)

2002 ◽  
Vol 22 (2) ◽  
pp. 225-250 ◽  
Author(s):  
C. Allen ◽  
N. Dos Santos ◽  
R. Gallagher ◽  
G.N.C. Chiu ◽  
Y. Shu ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Protein Binding ◽  
Chemical Properties ◽  
Total Serum Protein ◽  
Total Serum ◽  
Poly Ethylene Glycol ◽  
Binding Studies ◽  
Poly Ethylene

The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended circulation lifetime that the polymer affords has been attributed to the reduction or prevention of protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces total serum protein binding. In this review we examine all aspects of PEG in order to gain a better understanding of how the polymer fulfills its biological role. The physical and chemical properties of the polymer are explored and compared to properties of other hydrophilic polymers. An evidence based assessment of several in vitro protein binding studies as well as in vivo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is considered as a possible means by which it extends circulation longevity. Also, a “dysopsonization” phenomenon where PEG actually promotes binding of certain proteins that then mask the vehicle is discussed.

scholarly journals Fourteen Residues of the U1 snRNP-Specific U1A Protein Are Required for Homodimerization, Cooperative RNA Binding, and Inhibition of Polyadenylation

2000 ◽  
Vol 20 (6) ◽  
pp. 2209-2217 ◽  
Author(s):  
Jacqueline M. T. Klein Gunnewiek ◽  
Reem I. Hussein ◽  
Yvonne van Aarssen ◽  
Daphne Palacios ◽  
Rob de Jong ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Structural Data ◽  
Protein Molecule ◽  
Cooperative Binding ◽  
Mobility Shift ◽  
Small Nuclear Ribonucleoprotein ◽  
U1a Protein

ABSTRACT It was previously shown that the human U1A protein, one of three U1 small nuclear ribonucleoprotein-specific proteins, autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. The U1A autoregulatory complex requires two molecules of U1A protein to cooperatively bind a 50-nucleotide polyadenylation-inhibitory element (PIE) RNA located in the U1A 3′ untranslated region. Based on both biochemical and nuclear magnetic resonance structural data, it was predicted that protein-protein interactions between the N-terminal regions (amino acids [aa] 1 to 115) of the two U1A proteins would form the basis for cooperative binding to PIE RNA and for inhibition of polyadenylation. In this study, we not only experimentally confirmed these predictions but discovered some unexpected features of how the U1A autoregulatory complex functions. We found that the U1A protein homodimerizes in the yeast two-hybrid system even when its ability to bind RNA is incapacitated. U1A dimerization requires two separate regions, both located in the N-terminal 115 residues. Using both coselection and gel mobility shift assays, U1A dimerization was also observed in vitro and found to depend on the same two regions that were found in vivo. Mutation of the second homodimerization region (aa 103 to 115) also resulted in loss of inhibition of polyadenylation and loss of cooperative binding of two U1A protein molecules to PIE RNA. This same mutation had no effect on the binding of one U1A protein molecule to PIE RNA. A peptide containing two copies of aa 103 to 115 is a potent inhibitor of polyadenylation. Based on these data, a model of the U1A autoregulatory complex is presented.

scholarly journals A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

1997 ◽  
Vol 17 (11) ◽  
pp. 6683-6692 ◽  
Author(s):  
A J Peterson ◽  
M Kyba ◽  
D Bornemann ◽  
K Morgan ◽  
H W Brock ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Point Mutations ◽  
Direct Interaction ◽  
Molecular Complexes ◽  
Polycomb Group ◽  
Ph Domain ◽  
Binding Studies

The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

Control of beta-myosin heavy chain expression in systemic hypertension and caloric restriction in the rat heart

1995 ◽  
Vol 269 (4) ◽  
pp. C1025-C1033 ◽  
Author(s):  
S. J. Swoap ◽  
F. Haddad ◽  
P. Bodell ◽  
K. M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Caloric Restriction ◽  
Heavy Chain ◽  
Pressure Overload ◽  
Nuclear Extract ◽  
Systemic Hypertension ◽  
Sprague Dawley ◽  
Mobility Shift ◽  
Concomitant Decrease

In the rat left ventricle, both pressure overload induced by abdominal aortic constriction (Abcon) and caloric restriction (CR) induce an increase in the steady-state level of the beta-myosin heavy chain (MHC) protein and mRNA. Both models also induce a concomitant decrease in the alpha-MHC protein and mRNA. The goals of this study were to 1) determine if the changes in MHC expression in the models are due to altered transcription and 2) identify the relative levels of some key factors interacting with the regulatory regions of these genes. Female Sprague-Dawley rats were randomly assigned to the following groups: 1) normal control (NC), 2) Abcon, and 3) CR. After 5 wk of experimental manipulations, myocardial nuclei were isolated. These nuclei were used for 1) nuclear run-on assays or 2) nuclear extract, which was prepared and used for gel mobility shift assays (GMSAs). Nuclear run-on assays demonstrated that the increase in beta-MHC mRNA and protein expression in both Abcon and CR can be at least partially attributed to increased transcription. The concomitant decrease in alpha-MHC content can similarly be attributed to a decrease in transcription of this gene. Furthermore, GMSAs demonstrate that nuclear extract from each group interact differently with certain elements known to be important for expression in vitro. CR nuclear extracts have a 25.6 +/- 7.2% decrease (P < 0.05 vs. NC) in interaction with a thyroid-responsive element, a potential repressor of beta-MHC transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Divergence in species and regulatory role of β-myosin heavy chain proximal promoter muscle-CAT elements

2002 ◽  
Vol 283 (6) ◽  
pp. C1761-C1775 ◽  
Author(s):  
Richard W. Tsika ◽  
John McCarthy ◽  
Natalia Karasseva ◽  
Yangsi Ou ◽  
Gretchen L. Tsika
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transgene Expression ◽  
Weight Bearing ◽  
Nuclear Extract ◽  
Proximal Promoter ◽  
Wild Type ◽  
Mobility Shift

We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair β-myosin heavy chain (βMyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized βMyHC proximal promoter elements contribute to NWB regulation.

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