scholarly journals Integrity of the Mod(mdg4)-67.2 BTB Domain Is Critical to Insulator Function in Drosophila melanogaster

2006 ◽  
Vol 27 (3) ◽  
pp. 963-974 ◽  
Author(s):  
Anton Golovnin ◽  
Alexander Mazur ◽  
Marina Kopantseva ◽  
Maria Kurshakova ◽  
Pavel V. Gulak ◽  
...  
Keyword(s):  
Amino Acids ◽  
Drosophila Melanogaster ◽  
Binding Sites ◽  
Protein Dimerization ◽  
Mutant Proteins ◽  
Btb Domain ◽  
Acidic Domain ◽  
Insulator Function ◽  
Domain Protein ◽  
Carboxyl Terminal

ABSTRACT The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function.

scholarly journals The BTB domain of bric à brac mediates dimerization in vitro.

1995 ◽  
Vol 15 (6) ◽  
pp. 3424-3429 ◽  
Author(s):  
W Chen ◽  
S Zollman ◽  
J L Couderc ◽  
F A Laski
Keyword(s):  
Amino Acids ◽  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Zinc Finger ◽  
Zinc Finger Proteins ◽  
Protein Dimerization ◽  
Conserved Motif ◽  
Btb Domain

The gene bric à brac (bab) is required for the proper development of the limbs and ovary in Drosophila melanogaster. bab encodes a BTB domain (also called a POZ domain), an approximately 115-amino-acid conserved motif found primarily in the N termini of zinc finger proteins. In this paper, we show that the BTB domain of bab can mediate protein dimerization in vitro. In addition, we demonstrate that the first 51 amino acids of the bab BTB domain are sufficient for dimerization, and we identify amino acids within this region that are required for binding.

scholarly journals The Carboxyl-Terminal Region of Human Cytomegalovirus IE1491aa Contains an Acidic Domain That Plays a Regulatory Role and a Chromatin-Tethering Domain That Is Dispensable during Viral Replication

2005 ◽  
Vol 79 (1) ◽  
pp. 225-233 ◽  
Author(s):  
Jens Reinhardt ◽  
Geoffrey B. Smith ◽  
Christopher T. Himmelheber ◽  
Jane Azizkhan-Clifford ◽  
Edward S. Mocarski
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Deletion Mutant ◽  
Viral Gene Expression ◽  
Mutant Virus ◽  
Serine Phosphorylation ◽  
Viral Gene ◽  
Acidic Domain ◽  
Carboxyl Terminal

ABSTRACT The human cytomegalovirus major immediate-early (α) protein IE1491aa plays an important role in controlling viral gene expression at low multiplicities of infection. With a transient complementation assay, full-length IE1491aa enhanced the growth of ie1 mutant virus CR208 20-fold better than a deletion mutant lacking 71 carboxyl-terminal amino acids (IE11-420aa). A 16-amino-acid domain between amino acids 476 and 491 was both necessary and sufficient for chromatin-tethering activity; however, this domain was completely dispensable for complementation of CR208 replication. The proximal 55-amino-acid acidic domain (amino acids 421 to 475) was found to be most important for function. A deletion mutant lacking only this domain retained chromatin-tethering activity but failed to complement mutant virus. Interestingly, serine phosphorylation (at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455) was not required for complementation. These results show that IE1491aa is composed of at least two domains that support replication, a region located between amino acids 1 and 399 that complements ie1 mutant virus replication to low levels and an acidic domain between amino acids 421 and 479 that dramatically enhances complementation.

scholarly journals Cloning and Characterization of the scute (sc) Gene of Drosophila subobscura

Genetics ◽  
1996 ◽  
Vol 144 (3) ◽  
pp. 1043-1051
Author(s):  
L M Botella ◽  
C Doñoro ◽  
L Sáchez ◽  
C Segarra ◽  
B Granadino
Keyword(s):  
Amino Acids ◽  
Drosophila Melanogaster ◽  
Binding Sites ◽  
Untranslated Region ◽  
Sequence Comparisons ◽  
Dna Binding Sites ◽  
Bhlh Proteins ◽  
Transgenic Flies ◽  
Dual Functions

Abstract The scute (sc) gene, a member of the achaete-scute complex of Drosophila melanogaster, has dual functions: sisterless (sis-b) function required for sex determination and dosage compensation and scute function, which is involved in neurogenesis. The sc homologue of D. subobscura was cloned. It lacks introns and encodes a single 1.7-kb transcript slightly larger than that of D. melanogaster (1.6 kb). The Sc protein of D. subobscura is slightly larger than that of D. melanogaster (382 vs. 345 amino acids). Sequence comparisons between both species show the Sc protein to have a highly conserved bHLH domain. Outside this domain, amino acid replacements are not randomly distributed. Two additional conserved domains, of 20 and 36 amino acids, are present near the C-terminal end. They may represent domains confering specificity upon the Sc protein with respect to other proteins of the achaete-scute complex. In its 3′ untranslated region, Sc RNA contains uridine stretches, putative Sxl protein DNA-binding sites. The D. subobscura Sc protein can cooperate with other D. melanogaster bHLH proteins because D. subobscura sc supplies sis-b function when introduced into D. melanogaster transgenic flies mutant for sc.

Conserved amino acids in metal-binding motifs of PDE3A are involved in substrate and inhibitor binding

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3380-3386 ◽  
Author(s):  
Wei Zhang ◽  
Robert W. Colman
Keyword(s):  
Amino Acids ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Divalent Cations ◽  
Binding Motif ◽  
Inhibitor Binding ◽  
Binding Motifs ◽  
Mutant Proteins ◽  
Conserved Amino Acids

Abstract The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A▵1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (kcat/Km). Mutants H752A, H756A, and E825A had kcat of less than 0.0008 s−1 to 0.0475 s−1 compared to PDE3A▵1, with 1.86 second−1, with unchanged Km. Although E866A had a kcat of 0.235 s−1, the Kmfor cyclic adenosine monophosphate (cAMP) was increased 11-fold and the Ki for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3A▵1. The Ki of H836A for cGMP was 177-fold higher than that of PDE3A▵1. The Km for E971A was 5-fold higher than PDE3A▵1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn2+, Mg2+, or Co2+. This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.

Conserved amino acids in metal-binding motifs of PDE3A are involved in substrate and inhibitor binding

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3380-3386 ◽  
Author(s):  
Wei Zhang ◽  
Robert W. Colman
Keyword(s):  
Amino Acids ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Divalent Cations ◽  
Binding Motif ◽  
Inhibitor Binding ◽  
Binding Motifs ◽  
Mutant Proteins ◽  
Conserved Amino Acids

The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A▵1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (kcat/Km). Mutants H752A, H756A, and E825A had kcat of less than 0.0008 s−1 to 0.0475 s−1 compared to PDE3A▵1, with 1.86 second−1, with unchanged Km. Although E866A had a kcat of 0.235 s−1, the Kmfor cyclic adenosine monophosphate (cAMP) was increased 11-fold and the Ki for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3A▵1. The Ki of H836A for cGMP was 177-fold higher than that of PDE3A▵1. The Km for E971A was 5-fold higher than PDE3A▵1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn2+, Mg2+, or Co2+. This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.

scholarly journals The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 926
Author(s):  
Maria C. Martins ◽  
Susana F. Fernandes ◽  
Bruno A. Salgueiro ◽  
Jéssica C. Soares ◽  
Célia V. Romão ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Reductase Activity ◽  
Conserved Residues ◽  
Mutant Proteins ◽  
E Coli ◽  
C Terminus ◽  
Bona Fide ◽  
Oxygen Reductase ◽  
Soluble Enzymes

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

scholarly journals Major Carboxyl Terminal Fragments Generated by γ-Secretase Processing of the Alzheimer Amyloid Precursor Are 50 and 51 Amino Acids Long

2013 ◽  
Vol 21 (5) ◽  
pp. 474-483 ◽  
Author(s):  
Inga Pinnix ◽  
Jorge A. Ghiso ◽  
Miguel A. Pappolla ◽  
Kumar Sambamurti
Keyword(s):  
Amino Acids ◽  
Carboxyl Terminal

scholarly journals Genetic and Molecular Characterization of sting, a Gene Involved in Crystal Formation and Meiotic Drive in the Male Germ Line of Drosophila melanogaster

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 749-760 ◽  
Author(s):  
Armin Schmidt ◽  
Gioacchino Palumbo ◽  
Maria P Bozzetti ◽  
Patrizia Tritto ◽  
Sergio Pimpinelli ◽  
...  
Keyword(s):  
Amino Acids ◽  
Drosophila Melanogaster ◽  
Null Allele ◽  
Germ Line ◽  
Meiotic Drive ◽  
P Element ◽  
Crystal Formation ◽  
Male Sterile ◽  
Male Sterile Mutation

Abstract The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Ste+ flies and star-shaped in Ste flies, once again as observed in cry– males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry– males, a 0.7-kb mRNA is produced.

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