Transcription and Translation of the Sex Message in the Smut Fungus Ustilago Violacea

1974 ◽  
Vol 16 (1) ◽  
pp. 49-62
Author(s):  
J. E. CUMMINS ◽  
A. W. DAY
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Information Exchange ◽  
Plasma Membranes ◽  
Cellular Protein ◽  
Mating Activity ◽  
Ustilago Violacea ◽  
Protein Levels ◽  
Amino Acid Turnover ◽  
Mating Conditions

In Ustilago violacea conjugation involves a period of courtship followed by the cooperative assembly of a conjugation tube between paired sporidia of opposite mating type. During courtship information is exchanged between the paired sporidia directing transcription and translation of a ‘sex message’. This information exchange is completed even though the plasma membranes and walls of the mating sporidia remain intact until after the sex message has been translated. Once translation is completed the copulatory organelle is assembled. Inhibitor studies show that translation of specific mRNA species (sex message) is essential during the 3-4 h period of courtship prior to assembly and that transcription of this sex message is completed 15-30 min prior to the completion of translation. Amino acid incorporation studies show that even though the cellular protein levels remain constant during courtship there is extensive amino acid turnover as the sporidia adjust to mating conditions. However, an enhancement of amino acid turnover by mating, in contrast to unmixed sporidia, is not detectable. Prototrophic strains continue to synthesize stable RNA during courtship while amino acid auxotrophs discontinue stable RNA synthesis immediately on transfer to mating conditions. Both prototrophs and auxotrophs show extensive RNA turnover during courtship but a specific enhancement of turnover due to mating activity is not detectable from the labelling pattern alone. A round of DNA replication may be completed during courtship but mating activity does not influence the gross pattern of DNA replication in either of the mating types.

scholarly journals Functional interaction of a novel cellular protein with the papillomavirus E2 transactivation domain.

1997 ◽  
Vol 17 (12) ◽  
pp. 7208-7219 ◽  
Author(s):  
D E Breiding ◽  
F Sverdrup ◽  
M J Grossel ◽  
N Moscufo ◽  
W Boonchai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Cellular Protein ◽  
Bovine Papillomavirus ◽  
Transactivation Domain

The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.

scholarly journals PROTEIN SYNTHESIS AND AMINO ACID TURNOVER IN TISSUE CULTURE

1952 ◽  
Vol 196 (1) ◽  
pp. 51-68 ◽  
Author(s):  
H.W. Gerarde ◽  
Marion. Jones ◽  
Theodore. Winnick
Keyword(s):  
Tissue Culture ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Turnover

scholarly journals Genetic Relationships in the Toxin-Producing Fungal Endophyte, Alternaria oxytropis Using Polyketide Synthase and Non-Ribosomal Peptide Synthase Genes

2021 ◽  
Vol 7 (7) ◽  
pp. 538
Author(s):  
Rebecca Creamer ◽  
Deana Baucom Hille ◽  
Marwa Neyaz ◽  
Tesneem Nusayr ◽  
Christopher L. Schardl ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyketide Synthase ◽  
Genetic Relationships ◽  
Protein Sequences ◽  
Fungal Endophyte ◽  
Melanin Synthesis ◽  
Melanin Biosynthesis ◽  
Protein Levels ◽  
Oxytropis Sericea ◽  
And Function

The legume Oxytropis sericea hosts a fungal endophyte, Alternaria oxytropis, which produces secondary metabolites (SM), including the toxin swainsonine. Polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) enzymes are associated with biosynthesis of fungal SM. To better understand the origins of the SM, an unannotated genome of A. oxytropis was assessed for protein sequences similar to known PKS and NRPS enzymes of fungi. Contigs exhibiting identity with known genes were analyzed at nucleotide and protein levels using available databases. Software were used to identify PKS and NRPS domains and predict identity and function. Confirmation of sequence for selected gene sequences was accomplished using PCR. Thirteen PKS, 5 NRPS, and 4 PKS-NRPS hybrids were identified and characterized with functions including swainsonine and melanin biosynthesis. Phylogenetic relationships among closest amino acid matches with Alternaria spp. were identified for seven highly conserved PKS and NRPS, including melanin synthesis. Three PKS and NRPS were most closely related to other fungi within the Pleosporaceae family, while five PKS and PKS-NRPS were closely related to fungi in the Pleosporales order. However, seven PKS and PKS-NRPS showed no identity with fungi in the Pleosporales or the class Dothideomycetes, suggesting a different evolutionary origin for those genes.

scholarly journals Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker.

1994 ◽  
Vol 127 (6) ◽  
pp. 1799-1813 ◽  
Author(s):  
E de Beus ◽  
J S Brockenbrough ◽  
B Hong ◽  
J P Aris
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Protein ◽  
Nucleolar Protein ◽  
Mrna Levels ◽  
Multicopy Plasmid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Levels ◽  
Significant Amino Acid Sequence ◽  
Ribosomal Protein L3

We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM1, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM1 mRNA. The consequences of overexpressing NOP2 from the GAL10 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure.

Integration of in vitro neurotoxicity data with biokinetic modelling for the estimation of in vivo neurotoxicity

2007 ◽  
Vol 26 (4) ◽  
pp. 333-338 ◽  
Author(s):  
Anna Forsby ◽  
Bas Blaauboer
Keyword(s):  
Exposure Period ◽  
Cellular Protein ◽  
In Vitro Toxicity ◽  
Target Tissue ◽  
Receptor Function ◽  
Human Neuroblastoma ◽  
Protein Levels ◽  
Effective Doses

Risk assessment of neurotoxicity is mainly based on in vivo exposure, followed by tests on behaviour, physiology and pathology. In this study, an attempt to estimate lowest observed neurotoxic doses after single or repeated dose exposure was performed. Differentiated human neuroblastoma SH-SY5Y cells were exposed to acrylamide, lindane, parathion, paraoxon, phenytoin, diazepam or caffeine for 72 hours. The effects on protein synthesis and intracellular free Ca2+concentration were studied as physiological endpoints. Voltage operated Ca2 +channel function, acetylcholine receptor function and neurite degenerative effects were investigated as neurospecific endpoints for excitability, cholinergic signal transduction and axonopathy, respectively. The general cytotoxicity, determined as the total cellular protein levels after the 72 hours exposure period, was used for comparison to the specific endpoints and for estimation of acute lethality. The lowest concentration that induced 20% effect (EC 20) obtained for each compound, was used as a surrogate for the lowest neurotoxic level (LOEL) at the target site in vivo. The LOELs were integrated with data on adsorption, distribution, metabolism and excretion of the compounds in physiologically-based biokinetic (PBBK) models of the rat and the lowest observed effective doses (LOEDs) were estimated for the test compounds. A good correlation was observed between the estimated LOEDs and experimental LOEDs found in literature for rat for all test compounds, except for diazepam. However, when using in vitro data from the literature on diazepam's effect on gamma-amino butyric acid (GABA)A receptor function for the estimation of LOED, the correlation between the estimated and experimental LOEDs was improved from a 10 000-fold to a 10-fold difference. Our results indicate that it is possible to estimate LOEDs by integrating in vitro toxicity data as surrogates for lowest observed target tissue levels with PBBK models, provided that some knowledge about toxic mechanisms is known. Human & Experimental Toxicology (2007) 26, 333—338

scholarly journals The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

2009 ◽  
Vol 29 (18) ◽  
pp. 5008-5019 ◽  
Author(s):  
Makiko Komata ◽  
Masashige Bando ◽  
Hiroyuki Araki ◽  
Katsuhiko Shirahige
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Terminal Region ◽  
Methyl Methanesulfonate ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Hydroxyurea Treatment ◽  
Dna Replication Checkpoint ◽  
Direct Binding

ABSTRACT Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

scholarly journals Bub1 in Complex with LANA Recruits PCNA To Regulate Kaposi's Sarcoma-Associated Herpesvirus Latent Replication and DNA Translesion Synthesis

2015 ◽  
Vol 89 (20) ◽  
pp. 10206-10218 ◽  
Author(s):  
Zhiguo Sun ◽  
Hem Chandra Jha ◽  
Erle S. Robertson
Keyword(s):  
Dna Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Kinase Domain ◽  
Cellular Protein ◽  
Nuclear Antigen ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Molecular Bridge

ABSTRACTLatent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requirestrans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells.IMPORTANCEDuring latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.

Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein

1985 ◽  
Vol 5 (6) ◽  
pp. 1442-1448
Author(s):  
G A Adams ◽  
J K Rose
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
G Protein ◽  
Abnormal Behavior ◽  
Animal Cells ◽  
Viral Glycoprotein ◽  
Protein Levels ◽  
Membrane Spanning ◽  
Membrane Anchoring ◽  
Isoleucine Residue

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed.

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