Identification of gene products from cloned fragments of the left arm of λdapB2

1982 ◽  
Vol 60 (3) ◽  
pp. 338-346 ◽  
Author(s):  
George A. Mackie ◽  
Gay D. Parsons
Keyword(s):  
Molecular Weight ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Gene Products ◽  
Positive Identification ◽  
30S Subunit ◽  
Bacterial Genes ◽  
The Right ◽  
Coding Potential

A set of recombinant plasmids encompassing the bacterial substitution in λdapB2 (Mackie, G. A. (1980) J. Biol. Chem. 255, 8928–8935) has been characterized genetically by complementation and biochemically by an analysis of the proteins encoded by individual plasmids in vitro. In addition to a cluster of four genes which includes the dapB locus, a total of seven gene products has been identified. One of these is ribosomal protein S20, whose gene (rpsT) has been localized to a 0.56-kilobase segment of DNA bounded by HindIII and HindII sites. Positive identification of this gene on plasmids pGM9 and pGP2 has been achieved by analysis of the products encoded by these plasmids in vitro and by the ability of pGM9 to complement a strain lacking S20 among its 30S subunit proteins. A second gene is that for isoleucyl tRNA synthetase (ileS). Its presence on pGM21 has been ascertained by the latter's ability to direct the synthesis of a protein in vitro with the size anticipated for this gene product. Extracts of cells harbouring this plasmid also exhibit greater isoleucyl tRNA synthetase activity than parental extracts. A third gene is at least 1.3 kilobases distant from rpsT and encodes a protein of 24 000 molecular weight, of unknown function. This locus is in turn about 6.5 kilobases from λ gene E. The latter gene, and several others, all of which likely derive from λ DNA are carried on a 5.2-kilobase fragment inserted in the piasmid pGM11 which includes the junction between phage and bacterial sequences on the left side of λdapB2. This fragment of DNA is inserted into the vector in a manner which permits the coupled transcription and translation of λ genes D and E in vitro, despite the absence of their natural promoter. The most striking feature of the organization of the bacterial genes on λdapB2 is the clustering of genes for polypeptides in the right half of the bacterial substitution in contrast to the apparent minimal use of the coding potential in the left half of the substitution.

Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity

1989 ◽  
Vol 9 (5) ◽  
pp. 2089-2104
Author(s):  
A L Majumder ◽  
R A Akins ◽  
J G Wilkinson ◽  
R L Kelley ◽  
A J Snook ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Molecular Mass ◽  
Mitochondrial Protein ◽  
Gel Filtration ◽  
Nuclear Gene ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Group I Introns ◽  
Group I

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

scholarly journals Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity.

1989 ◽  
Vol 9 (5) ◽  
pp. 2089-2104 ◽  
Author(s):  
A L Majumder ◽  
R A Akins ◽  
J G Wilkinson ◽  
R L Kelley ◽  
A J Snook ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Molecular Mass ◽  
Mitochondrial Protein ◽  
Gel Filtration ◽  
Nuclear Gene ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Group I Introns ◽  
Group I

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.

scholarly journals Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 473 ◽  
Author(s):  
Takuya Umehara ◽  
Saori Kosono ◽  
Dieter Söll ◽  
Koji Tamura
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Lysine Acetylation ◽  
Trna Synthetases ◽  
E Coli ◽  
Domains Of Life ◽  
The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

l-Asparaginyl-Trna Synthetase and l-Asparagine Synthetase Activities of l-Asparaginase-Sensitive and -Resistant Forms of the Mouse Gardner Lymphoma 6C3HED

1979 ◽  
Vol 56 (6) ◽  
pp. 539-545 ◽  
Author(s):  
M. R. Davies ◽  
Lucy P. Lambert ◽  
R. D. Marshall
Keyword(s):  
Cell Lines ◽  
Tumour Cell ◽  
Trna Synthetase ◽  
Asparagine Synthetase ◽  
Ascites Fluid ◽  
Synthetase Activity ◽  
Early Passage ◽  
Tumour Cell Lines ◽  
Cells Cultured

1. The mouse Gardner lymphoma 6C3HED was grown in ascites fluid in a form sensitive to the action of l-asparaginase (line 1), in another form which was resistant to l-asparaginase (line 2) and in a third form with partial sensitivity to l-asparaginase (line 3). 2. The l-asparaginyl-tRNA synthetase activities of extracts of the tumour cells, cultured both in the mouse and in vitro, were determined. Two of the lines, 1 and 3, in early passage numbers, showed a derepression mechanism involving l-asparagine. Mutation occurred with these lines resulting in the l-asparaginyl-tRNA synthetase activity of all the tumour cell lines being the same. 3. Cells of line 1 had low l-asparagine synthetase activity, which was unchanged by altering the supply of l-asparagine in vitro. Cells of lines 2 and 3 exhibited l-asparagine synthetase activities, which changed with the supply of l-asparagine. 4. It is not certain that l-asparagine synthetase activity of l-asparaginase-sensitive cells is controlled by l-asparaginyl-tRNA acting as a corepressor.

scholarly journals Switching of filamin polypeptides during myogenesis in vitro.

1983 ◽  
Vol 96 (2) ◽  
pp. 321-329 ◽  
Author(s):  
R H Gomer ◽  
E Lazarides
Keyword(s):  
Molecular Weight ◽  
Peptide Mapping ◽  
Short Pulse ◽  
Actin Binding ◽  
Gene Products ◽  
Skeletal Myogenesis ◽  
New Class ◽  
Filament Bundles ◽  
Peptide Maps

During chicken skeletal myogenesis in vitro, the actin-binding protein filamin is present at first in association with actin filament bundles both in myoblasts and in myotubes early after fusion. Later in mature myotubes it is found in association with myofibril Z disks. These two associations of filamin are separated by a period of several days, during which the protein is absent from the cytoplasm of differentiating myotubes (Gomer, R., and E. Lazarides, 1981, Cell, 23:524-532). To characterize the two classes of filamin polypeptides we have compared, by two-dimensional peptide mapping, 125I-labeled filamin immunoprecipitated from myoblasts and fibroblasts to filamin immunoprecipitated from mature myotubes and adult skeletal myofibrils. Myoblast filamin is highly homologous to fibroblast and purified chicken gizzard filamins. Mature myotube and adult myofibril filamins are highly homologous but exhibit extensive peptide differences with respect to the other three classes of filamin. Comparison of peptide maps from immunoprecipitated 35S-methionine-labeled filamins also shows that fibroblast and myoblast filamins are highly homologous but show substantial peptide differences with respect to mature myotube filamin. Filamins from both mature myotubes and skeletal myofibrils exhibit a slightly higher electrophoretic mobility than gizzard, fibroblast, and myoblast filamins. Short pulse-labeling studies show that mature myotube filamin is synthesized as a lower molecular weight variant and is not derived from a higher molecular weight precursor. These results suggest that myoblast and mature myotube filamins are distinct gene products and that during skeletal myogenesis in vitro one class of filamin polypeptides is replaced by a new class of filamin polypeptides, and that the latter is maintained into adulthood.

Decreased Accumulation or Increased Isoleucyl-tRNA Synthetase Activity Confers Resistance to the Cyclic β-Amino Acid BAY 10-8888 in Candida albicans and Candida tropicalis

1998 ◽  
Vol 42 (7) ◽  
pp. 1581-1586 ◽  
Author(s):  
Karl Ziegelbauer
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Candida Tropicalis ◽  
Specific Activity ◽  
Trna Synthetase ◽  
Intracellular Concentration ◽  
Synthetase Activity ◽  
Resistant Mutants ◽  
Resistance Modeling

ABSTRACT BAY 10-8888, a cyclic β-amino acid, exerts its antifungal activity by inhibition of isoleucyl-tRNA synthetase activity after accumulation to a millimolar concentration inside the cell. We have selected and characterized BAY 10-8888-resistant Candida albicans mutants. Reduced BAY 10-8888 accumulation as well as increased isoleucyl-tRNA synthetase activity was observed in these mutants. Some of the mutants were cross-resistant to cispentacin, a structurally related β-amino acid, while sensitivities to 5-fluorocytosine and fluconazole remained unchanged in all mutants. All except two in vitro-resistant mutants were pathogenic in a murine candidiasis model, and BAY 10-8888 failed to cure the infection. Furthermore, we have characterized BAY 10-8888 transport and isoleucyl-tRNA synthetase activity in several Candida tropicalis strains which showed MICs higher than those of other Candida strains. An analysis of the C. tropicalis strains revealed that intracellular concentrations of BAY 10-8888 were in the millimolar range, comparable to those for C. albicans. However, these isolates expressed isoleucyl-tRNA synthetase activities about fourfold higher than those for C. albicans. To test the possibility of resistance modeling, we determined the correlations between the intracellular concentration of BAY 10-8888, the specific activity of isoleucyl-tRNA synthetase, the number of free, i.e., noninhibited, isoleucyl-tRNA synthetase molecules/cell, and growth, assuming a linear relation. We found significant correlations between growth and the intracellular concentration of BAY 10-8888 and between growth and the number of free isoleucyl-tRNA synthetase molecules/cell, but not between growth and the specific activity of isoleucyl-tRNA synthetase.

EFFECTS OF EXOGENOUS PROGESTERONE AND OESTRADIOL ON PROSTAGLANDIN F AND 13,14-DIHYDRO-15-OXO PROSTAGLANDIN F2α CONCENTRATIONS IN UTERI AND PLASMA OF OVARIECTOMIZED EWES

1977 ◽  
Vol 73 (3) ◽  
pp. 427-439 ◽  
Author(s):  
T. M. LOUIS ◽  
DILYS M. PARRY ◽  
J. S. ROBINSON ◽  
G. D. THORBURN ◽  
J. R. G. CHALLIS
Keyword(s):  
Vascular System ◽  
Jugular Vein ◽  
Prostaglandin F2α ◽  
Synthetase Activity ◽  
Ovarian Vein ◽  
Oestradiol Treatment ◽  
Prostaglandin F ◽  
Exogenous Progesterone ◽  
The Right

SUMMARY The control of prostaglandin (PG) production by steroid hormones has been investigated in non-pregnant bilaterally ovariectomized sheep, prepared with indwelling utero-ovarian venous catheters and treated with physiological amounts of oestradiol and progesterone. Oestradiol treatment alone (2 × 15 μg/day for 9 days) had no effect on the prostaglandin F (PGF) concentration in uterine caruncles or intercaruncular tissue, on the release of PGF or of 13,14-dihydro-15-oxo PGF (PGFM) from these tissues during incubation in vitro, or on the concentrations of PGF in the utero-ovarian vein or PGFM in the jugular vein. However, oestradiol did accumulate in the uterine tissues. Progesterone treatment alone (2 × 10 mg/day for 9 days) provoked a significant increase in the concentration of PGF in the caruncles, a significant increase in the release of PGF from the caruncles during incubation with arachidonic acid and increased mean concentrations of PGFM in the jugular vein. When oestradiol was superimposed on a progesterone-primed system, there was a further marked increase in the PGF content of the caruncles, release of PGF into the utero-ovarian vein, and increased concentrations of PGFM in the jugular vein. The caruncles always contained more PGF than the intercaruncular area, and released more PGF and PGFM during incubation in vitro. In the progesterone+oestradiol group, there was good correlation between the PGF concentrations in simultaneous samples from the right and left utero-ovarian veins, and for all groups there was a high correlation between utero-ovarian PGF and peripheral PGFM concentrations. The caruncular epithelium of the progesterone-treated animals contained more lipid droplets than those of the other groups. These data are consistent with a requirement for progesterone in activating 'prostaglandin synthetase' activity, and promoting PGF production, largely from the caruncles. After progesterone priming, the synthesis of PGF by the caruncles and PG release into the vascular system was increased further by oestradiol treatment, whereas oestradiol alone was without effect.

scholarly journals Transcriptional Regulation of xyn1, Encoding Xylanase I, in Hypocrea jecorina

2006 ◽  
Vol 5 (3) ◽  
pp. 447-456 ◽  
Author(s):  
Roman Rauscher ◽  
Elisabeth Würleitner ◽  
Christian Wacenovsky ◽  
Nina Aro ◽  
Astrid R. Stricker ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Glucose Repression ◽  
Low Molecular Weight ◽  
Transcriptional Level ◽  
Hypocrea Jecorina ◽  
In Vivo Footprinting ◽  
The Right ◽  
Ccaat Box

ABSTRACT Two major xylanases (XYN I and XYN II) of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) are simultaneously expressed during growth on xylan but respond differently to low-molecular-weight inducers. In vivo footprinting analysis of the xylanase1 (xyn1) promoter revealed three different nucleotide sequences (5′- GGCTAA ATGCGACATC TTAGCC -3′ [an inverted repeat of GGCTAA spaced by 10 bp], 5′-CCAAT-3′, and 5′- GGGGTC TA GACCCC -3′ [equivalent to a double Cre1 site]) used to bind proteins. Binding to the Cre1 site is only observed under repressed conditions, whereas binding to the two other motifs is constitutive. Applying heterologously expressed components of the H. jecorina cellulase regulators Ace1 and Ace2 and the xylanase regulator Xyr1 suggests that Ace1 and Xyr1 but not Ace2 contact both GGCTAA motifs. H. jecorina transformants containing mutated versions of the xyn1 promoter, leading to elimination of protein binding to the left or the right GGCTAA box revealed either strongly reduced or completely eliminated induction of transcription. Elimination of Cre1 binding to its target released the basal transcriptional level from glucose repression but did not influence the inducibility of xyn1 expression. Mutation of the CCAAT box prevents binding of the Hap2/3/5 complex in vitro and is partially compensating for the loss of transcription caused by the mutation of the right GGCTAA box. Finally, evidence for a competition of Ace1 and Xyr1 for the right GGCTAA box is given. These data prompted us to hypothesize that xyn1 regulation is based on the interplay of Cre1 and Ace1 as a general and specific repressor with Xyr1 as transactivator.

Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

1999 ◽  
Vol 82 (11) ◽  
pp. 1462-1468 ◽  
Author(s):  
José Fernández ◽  
Jari Petäjä ◽  
John Griffin
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Protein C ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V ◽  
Factor Va ◽  
Anticoagulant Action

SummaryUnfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

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