scholarly journals CZK3, a MAP Kinase Kinase Kinase Homolog in Cercospora zeae-maydis, Regulates Cercosporin Biosynthesis, Fungal Development, and Pathogenesis

2003 ◽  
Vol 16 (9) ◽  
pp. 760-768 ◽  
Author(s):  
Won-Bo Shim ◽  
Larry D. Dunkle
Keyword(s):  
Map Kinase ◽  
Map Kinases ◽  
Gray Leaf Spot ◽  
Open Reading Frame ◽  
Wild Type ◽  
Toxic Activity ◽  
Reading Frame ◽  
Cercospora Zeae Maydis ◽  
Map Kinase Kinase Kinase ◽  
Map Kinase Kinase

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2

1993 ◽  
Vol 13 (8) ◽  
pp. 4539-4548
Author(s):  
J Wu ◽  
J K Harrison ◽  
P Dent ◽  
K R Lynch ◽  
M J Weber ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Kinases ◽  
Map Kinases ◽  
Sodium Dodecyl ◽  
Rat Tissues ◽  
Tyrosine Residues ◽  
Mitogen Activated Protein ◽  
Map Kinase Kinase

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.

scholarly journals Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

1993 ◽  
Vol 13 (10) ◽  
pp. 6241-6252 ◽  
Author(s):  
M L Samuels ◽  
M J Weber ◽  
J M Bishop ◽  
M McMahon
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Map Kinases ◽  
Growth Media ◽  
Hormone Binding ◽  
Map Kinase Cascade ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Hormone Binding Domain ◽  
Map Kinase Kinase

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1

1991 ◽  
Vol 11 (5) ◽  
pp. 2593-2608 ◽  
Author(s):  
D X Tishkoff ◽  
A W Johnson ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Open Reading Frame ◽  
Strand Exchange ◽  
Wild Type ◽  
Homologous Pairing ◽  
Gene Encoding ◽  
Reading Frame ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Exchange Protein

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.

scholarly journals Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1.

1993 ◽  
Vol 13 (8) ◽  
pp. 4679-4690 ◽  
Author(s):  
D L Charest ◽  
G Mordret ◽  
K W Harder ◽  
F Jirik ◽  
S L Pelech
Keyword(s):  
Myelin Basic Protein ◽  
Map Kinase ◽  
Map Kinases ◽  
Basic Protein ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Protein Tyrosine ◽  
Mitogen Activated Protein ◽  
Erk1 Protein ◽  
Map Kinase Kinase

p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.

scholarly journals Silencing PsKPP4 , a MAP kinase kinase kinase gene, reduces pathogenicity of the stripe rust fungus

2018 ◽  
Vol 19 (12) ◽  
pp. 2590-2602 ◽  
Author(s):  
Xiaoguo Zhu ◽  
Jia Guo ◽  
Fuxin He ◽  
Yang Zhang ◽  
Chenglong Tan ◽  
...  
Keyword(s):  
Map Kinase ◽  
Stripe Rust ◽  
Rust Fungus ◽  
Kinase Gene ◽  
Stripe Rust Fungus ◽  
Map Kinase Kinase Kinase ◽  
Map Kinase Kinase

Cloning and Characterization of a Prolinase Gene (pepR) from Lactobacillus rhamnosus

1998 ◽  
Vol 64 (5) ◽  
pp. 1831-1836 ◽  
Author(s):  
Pekka Varmanen ◽  
Terhi Rantanen ◽  
Airi Palva ◽  
Soile Tynkkynen
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Gene Replacement ◽  
Lactobacillus Helveticus ◽  
Open Reading Frame ◽  
Transcriptional Unit ◽  
Wild Type ◽  
Reading Frame ◽  
Gene Expressing ◽  
Produce Acid

ABSTRACT A peptidase gene expressingl-proline-β-naphthylamide-hydrolyzing activity was cloned from a gene library of Lactobacillus rhamnosus 1/6 isolated from cheese. Peptidase-expressing activity was localized in a 1.5-kbSacI fragment. A sequence analysis of the SacI fragment revealed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long. The ORF1-encoded 34.2-kDa protein exhibited 68% identity with the PepR protein from Lactobacillus helveticus. Additional sequencing revealed the presence of another open reading frame (ORF2) following pepR; this open reading frame was 459 bp long. Northern (RNA) and primer extension analyses indicated that pepR is expressed both as a monocistronic transcriptional unit and as a dicistronic transcriptional unit with ORF2. Gene replacement was used to construct a PepR-negative strain of L. rhamnosus. PepR was shown to be the primary enzyme capable of hydrolyzing Pro-Leu in L. rhamnosus. However, the PepR-negative mutant did not differ from the wild type in its ability to grow and produce acid in milk. The clonedpepR expressed activity against dipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, and Leu-Gly-Gly and the chromogenic substrates l-leucine-β-naphthylamide andl-phenylalanine-β-naphthylamide were hydrolyzed by the PepR of L. rhamnosus.

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