glsA, a Volvox gene required for asymmetric division and germ cell specification, encodes a chaperone-like protein

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 649-658 ◽  
Author(s):  
S.M. Miller ◽  
D.L. Kirk
Keyword(s):  
Mitotic Spindle ◽  
Site Directed Mutagenesis ◽  
Cell Specification ◽  
Division Plane ◽  
Binding Domains ◽  
Acid Protein ◽  
Germ Cell Specification ◽  
Asymmetric Divisions ◽  
Dividing Cells ◽  
Amino Acid Protein

The gls genes of Volvox are required for the asymmetric divisions that set apart cells of the germ and somatic lineages during embryogenesis. Here we used transposon tagging to clone glsA, and then showed that it is expressed maximally in asymmetrically dividing embryos, and that it encodes a 748-amino acid protein with two potential protein-binding domains. Site-directed mutagenesis of one of these, the J domain (by which Hsp40-class chaperones bind to and activate specific Hsp70 partners) abolishes the capacity of glsA to rescue mutants. Based on this and other considerations, including the fact that the GlsA protein is associated with the mitotic spindle, we discuss how it might function, in conjunction with an Hsp70-type partner, to shift the division plane in asymmetrically dividing cells.

scholarly journals Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

1998 ◽  
Vol 64 (5) ◽  
pp. 1650-1656 ◽  
Author(s):  
Peter W. Coschigano ◽  
Thomas S. Wehrman ◽  
L. Y. Young
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Molecular Mass ◽  
Cysteine Residue ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Pyruvate Formate Lyase ◽  
Calculated Molecular Mass ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

scholarly journals Genetic Localization and Molecular Characterization of Two Key Genes (mitAB) Required for Biosynthesis of the Antitumor Antibiotic Mitomycin C

1999 ◽  
Vol 181 (7) ◽  
pp. 2199-2208 ◽  
Author(s):  
Yingqing Mao ◽  
Mustafa Varoglu ◽  
David H. Sherman
Keyword(s):  
Amino Acid ◽  
Mitomycin C ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequence Analysis ◽  
Resistance Locus ◽  
Antitumor Antibiotic ◽  
Carbamoyl Phosphate ◽  
Acid Protein ◽  
Key Genes ◽  
Amino Acid Protein

ABSTRACT Mitomycin C (MC) is an antitumor antibiotic derived biosynthetically from 3-amino-5-hydroxybenzoic acid (AHBA),d-glucosamine, and carbamoyl phosphate. A gene (mitA) involved in synthesis of AHBA has been identified and found to be linked to the MC resistance locus, mrd, inStreptomyces lavendulae. Nucleotide sequence analysis showed that mitA encodes a 388-amino-acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase fromAmycolatopsis mediterranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of theS. lavendulae chromosomal copy of mitAcompletely blocked the production of MC. The function ofmitA was confirmed by complementation of an S. lavendulae strain containing a K191A mutation in MitA with AHBA. A second gene (mitB) encoding a 272-amino-acid protein (related to a group of glycosyltransferases) was identified immediately downstream of mitA that upon disruption resulted in abrogation of MC synthesis. This work has localized a cluster of key genes that mediate assembly of the unique mitosane class of natural products.

scholarly journals Genetic and Biochemical Characterization of Salmonella enterica Serovar Typhi Deoxyribokinase

2000 ◽  
Vol 182 (4) ◽  
pp. 869-873 ◽  
Author(s):  
Lise Tourneux ◽  
Nadia Bucurenci ◽  
Cosmin Saveanu ◽  
Pierre Alexandre Kaminski ◽  
Madeleine Bouzon ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Gene Encoding ◽  
E Coli ◽  
Salmonella Enterica Serovar Typhi ◽  
Acid Protein ◽  
Serovar Typhimurium ◽  
Amino Acid Protein

ABSTRACT We identified in the genome of Salmonella entericaserovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between theuhpA and ilvN genes, is absent inEscherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. colicorrespond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coliribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased theV max of deoxyribokinase by a factor of 2.5 and increased the K m for deoxyribose by a factor of 70, compared to the parent enzyme.

scholarly journals Structure of the N-terminal fragment ofEscherichia coliLon protease

2010 ◽  
Vol 66 (8) ◽  
pp. 865-873 ◽  
Author(s):  
Mi Li ◽  
Alla Gustchina ◽  
Fatima S. Rasulova ◽  
Edward E. Melnikov ◽  
Michael R. Maurizi ◽  
...  
Keyword(s):  
Binding Sites ◽  
Spatial Relationship ◽  
Anomalous Scattering ◽  
Lon Protease ◽  
Binding Domains ◽  
Acid Protein ◽  
Α Helix ◽  
Almost All ◽  
Relationship Of ◽  
Amino Acid Protein

The structure of a recombinant construct consisting of residues 1–245 ofEscherichia coliLon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function fromBordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.

scholarly journals Not All H3K4 Methylations Are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification

2017 ◽  
Vol 65 (3) ◽  
pp. 460-475.e6 ◽  
Author(s):  
Deqing Hu ◽  
Xin Gao ◽  
Kaixiang Cao ◽  
Marc A. Morgan ◽  
Gloria Mas ◽  
...  
Keyword(s):  
Germ Cell ◽  
Primordial Germ Cell ◽  
Cell Specification ◽  
Germ Cell Specification

scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

scholarly journals LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

2006 ◽  
Vol 398 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Yukiko Mizutani ◽  
Akio Kihara ◽  
Yasuyuki Igarashi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Family Members ◽  
Fatty Acyl ◽  
Ceramide Synthase ◽  
Broad Substrate Specificity ◽  
Acid Protein ◽  
Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

Germ Cell Specification in Mice

Science ◽  
2007 ◽  
Vol 316 (5823) ◽  
pp. 394-396 ◽  
Author(s):  
K. Hayashi ◽  
S. M. C. de Sousa Lopes ◽  
M. A. Surani
Keyword(s):  
Germ Cell ◽  
Cell Specification ◽  
Germ Cell Specification

scholarly journals Synergistic Action of Prolactin (PRL) and Androgen on PRL-Inducible Protein Gene Expression in Human Breast Cancer Cells: A Unique Model for Functional Cooperation between Signal Transducer and Activator of Transcription-5 and Androgen Receptor

2002 ◽  
Vol 16 (7) ◽  
pp. 1696-1710 ◽  
Author(s):  
Jean-Louis Carsol ◽  
Sébastien Gingras ◽  
Jacques Simard
Keyword(s):  
Androgen Receptor ◽  
Reporter Gene ◽  
Potential Interaction ◽  
Site Directed Mutagenesis ◽  
Signal Transducer ◽  
Human Breast ◽  
Maximal Increase ◽  
Binding Domains ◽  
Inducible Protein ◽  
Half Site

Abstract The signal transducer and activator of transcription 5 (Stat5) has been shown to cooperate with some nuclear receptors. However, an interaction has never been demonstrated with the androgen receptor (AR). Given that the PRL-inducible protein/gross cystic disease fluid-15 (PIP/GCDFP-15) is both a PRL-controlled and an androgen-controlled protein, we used its promoter region to investigate the potential interaction between Stat5 and androgen receptor. Dihydrotestosterone or PRL alone slightly modulated or did not modulate the luciferase activity of all reporter gene constructs. In contrast, a maximal increase was observed using the −1477+42 reporter gene construct after exposure to both dihydrotestosterone and PRL. The requirement of half-site androgen-responsive elements and two consensus Stat5-binding elements, Stat5#1 and Stat5#2, was determined by site-directed mutagenesis. Activated Stat5B binds with a higher affinity to Stat5#2 than to Stat5#1. Stat5AΔ749 and Stat5BΔ754 mutants demonstrated that the Stat5 trans-activation domain is involved in the hormonal cooperation. The cooperation depends on the PRL-induced phosphorylation on Tyr694 in Stat5A and Tyr699 in Stat5B, as demonstrated using the Stat5AY694F and Stat5BY699F proteins. The use of AR Q798E, C619Y, and C784Y mutants showed that trans-activation, DNA-binding, and ligand-binding domains of AR are essential. Our study thus suggests a functional cooperation between AR and Stat5.

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