scholarly journals Deletion of the 389 N-Terminal Residues of the Transcriptional Activator AREA Does Not Result in Nitrogen Metabolite Derepression in Aspergillus nidulans

1998 ◽  
Vol 180 (21) ◽  
pp. 5762-5764 ◽  
Author(s):  
Mark X. Caddick ◽  
Herbert N. Arst
Keyword(s):  
Amino Acids ◽  
Aspergillus Nidulans ◽  
Deletion Mutant ◽  
Transcriptional Activator ◽  
Gene Replacement ◽  
Untranslated Regions ◽  
Homologous Gene ◽  
Heterologous Promoter ◽  
Native Promoter ◽  
N Terminus

ABSTRACT Utilizing a homologous gene replacement in order to retain the native promoter and 5′ and 3′ untranslated messenger regions (and thereby ensure physiological validity), we have shown that deletion of the N-terminal 389 amino acids of the transcriptional activator AREA does not result in nitrogen metabolite derepression inAspergillus nidulans. Our results provide no evidence for a modulating interaction involving the N terminus of AREA and contrast with those of H. K. Lamb, A. L. Dodds, D. R. Swatman, E. Cairns, and A. R. Hawkins (J. Bacteriol. 179:6649–6656, 1997), who used nontargeted ectopic copies of a construct containing a heterologous promoter and untranslated regions. Results obtained with this deletion mutant, nevertheless, provide further evidence for the dispensability of large portions of AREA.

scholarly journals The relA/spoT-Homologous Gene inStreptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

1998 ◽  
Vol 180 (16) ◽  
pp. 4123-4132 ◽  
Author(s):  
Oscar H. Martínez-Costa ◽  
Miguel A. Fernández-Moreno ◽  
Francisco Malpartida
Keyword(s):  
Amino Acids ◽  
Deletion Mutant ◽  
Streptomyces Coelicolor ◽  
Stringent Response ◽  
Morphological Differentiation ◽  
Antibiotic Biosynthesis ◽  
Homologous Gene ◽  
E Coli

ABSTRACT Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined.S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence thatS. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of anS. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. colitruncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, arel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition,E. coli relA in an S. coelicolor reldeletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-typerel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.

scholarly journals Mating Type Protein Mat1-2 from Asexual Aspergillus fumigatus Drives Sexual Reproduction in Fertile Aspergillus nidulans

2008 ◽  
Vol 7 (6) ◽  
pp. 1029-1040 ◽  
Author(s):  
Wioletta Pyrzak ◽  
Karen Y. Miller ◽  
Bruce L. Miller
Keyword(s):  
Aspergillus Fumigatus ◽  
Aspergillus Nidulans ◽  
Mating Type ◽  
Sexual Development ◽  
Genetic System ◽  
Gene Replacement ◽  
Sexual Cycle ◽  
Homologous Gene ◽  
Gene Exchange ◽  
First Report

ABSTRACT The lack of an experimentally amenable sexual genetic system in Aspergillus fumigatus is a major limitation in the study of the organism's pathogenesis. A recent comparative genome analysis revealed evidence for potential sexuality in A. fumigatus. Homologs of mating type genes as well as other genes of the “sexual machinery” have been identified in anamorphic A. fumigatus. The mat1-2 gene encodes a homolog of MatA, an HMG box mating transcriptional factor (MatHMG) that regulates sexual development in fertile Aspergillus nidulans. In this study, the functionalities of A. fumigatus mat1-2 and the Mat1-2 protein were determined by interspecies gene exchange between sterile A. fumigatus and fertile A. nidulans. Ectopically integrated A. fumigatus mat1-2 (driven by its own promoter) was not functional in a sterile A. nidulans ΔmatA strain, and no sexual development was observed. In contrast, the A. fumigatus mat1-2 open reading frame driven by the A. nidulans matA promoter and integrated by homologous gene replacement at the matA locus was functional and conferred full fertility. This is the first report showing that cross species mating type gene exchange between closely related Ascomycetes did not function in sexual development. This is also the first report demonstrating that a MatHMG protein from an asexual species is fully functional, with viable ascospore differentiation, in a fertile homothallic species. The expression of mat1-2 was assessed in A. fumigatus and A. nidulans. Our data suggest that mat1-2 may not be properly regulated to allow sexuality in A. fumigatus. This study provides new insights about A. fumigatus asexuality and also suggests the possibility for the development of an experimentally amenable sexual cycle.

Post-proline endopeptidase, further characterization of the enzyme from pig kidneys

1984 ◽  
Vol 49 (8) ◽  
pp. 1846-1853 ◽  
Author(s):  
Karel Hauzer ◽  
Tomislav Barth ◽  
Linda Servítová ◽  
Karel Jošt
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Relative Molecular Mass ◽  
Enzyme Molecule ◽  
Thiol Compounds ◽  
Modified Method ◽  
N Terminus

A post-proline endopeptidase (EC 3.4.21.26) was isolated from pig kidneys using a modified method described earlier. The enzyme was further purified by ion exchange chromatography on DEAE-Sephacel. The final product contained about 95% of post-proline endopeptidase. The enzyme molecule consisted of one peptide chain with a relative molecular mass of 65 600 to 70 000, containing a large proportion of acidic and alifatic amino acids (glutamic acid, aspartic acid and leucine) and the N-terminus was formed by aspartic acid or asparagine. In order to prevent losses of enzyme activity, thiol compounds has to be added.

scholarly journals Generation of Antibodies against Foot-and-Mouth-Disease Virus Capsid Protein VP4 Using Hepatitis B Core VLPs as a Scaffold

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 338
Author(s):  
Jessica Swanson ◽  
Rennos Fragkoudis ◽  
Philippa C. Hawes ◽  
Joseph Newman ◽  
Alison Burman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis B ◽  
Capsid Protein ◽  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Specific Antibodies ◽  
Mouth Disease Virus ◽  
N Terminus ◽  
Foot And Mouth

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the economically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neutralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.

scholarly journals HbxB Is a Key Regulator for Stress Response and β-Glucan Biogenesis in Aspergillus nidulans

2021 ◽  
Vol 9 (1) ◽  
pp. 144
Author(s):  
Sung-Hun Son ◽  
Mi-Kyung Lee ◽  
Ye-Eun Son ◽  
Hee-Soo Park
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Aspergillus Nidulans ◽  
Deletion Mutant ◽  
Phenotypic Analysis ◽  
Spore Viability ◽  
Fungal Development ◽  
Expression Of Genes ◽  
Trehalose Biosynthesis

Homeobox transcription factors are conserved in eukaryotes and act as multi-functional transcription factors in filamentous fungi. Previously, it was demonstrated that HbxB governs fungal development and spore viability in Aspergillus nidulans. Here, the role of HbxB in A. nidulans was further characterized. RNA-sequencing revealed that HbxB affects the transcriptomic levels of genes associated with trehalose biosynthesis and response to thermal, oxidative, and radiation stresses in asexual spores called conidia. A phenotypic analysis found that hbxB deletion mutant conidia were more sensitive to ultraviolet stress. The loss of hbxB increased the mRNA expression of genes associated with β-glucan degradation and decreased the amount of β-glucan in conidia. In addition, hbxB deletion affected the expression of the sterigmatocystin gene cluster and the amount of sterigmatocystin. Overall, these results indicated that HbxB is a key transcription factor regulating trehalose biosynthesis, stress tolerance, β-glucan degradation, and sterigmatocystin production in A.nidulans conidia.

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

scholarly journals A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

2001 ◽  
Vol 75 (14) ◽  
pp. 6329-6336 ◽  
Author(s):  
T. Arazi ◽  
Y. M. Shiboleth ◽  
A. Gal-On
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Foot And Mouth Disease ◽  
Systemic Infection ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Immunogenic Epitope ◽  
Mouth Disease Virus ◽  
N Terminus

ABSTRACT Systematic deletion and peptide tagging of the amino-terminal domain (NT, ∼43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5′ end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.

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