scholarly journals Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans.

1990 ◽  
Vol 10 (6) ◽  
pp. 3194-3203 ◽  
Author(s):  
A Andrianopoulos ◽  
M J Hynes
Keyword(s):  
Dna Binding ◽  
Aspergillus Nidulans ◽  
Nuclear Localization ◽  
Regulatory Gene ◽  
Transcription Activation ◽  
Binding Motif ◽  
Wild Type ◽  
Amino Terminal ◽  
Carboxyl Terminal

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.

Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans

1990 ◽  
Vol 10 (6) ◽  
pp. 3194-3203
Author(s):  
A Andrianopoulos ◽  
M J Hynes
Keyword(s):  
Dna Binding ◽  
Aspergillus Nidulans ◽  
Nuclear Localization ◽  
Regulatory Gene ◽  
Transcription Activation ◽  
Binding Motif ◽  
Wild Type ◽  
Amino Terminal ◽  
Carboxyl Terminal

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.

scholarly journals Ty3 Integrase Is Required for Initiation of Reverse Transcription

2002 ◽  
Vol 76 (6) ◽  
pp. 2804-2816 ◽  
Author(s):  
M. Henrietta Nymark-McMahon ◽  
Nadejda S. Beliakova-Bethell ◽  
Jean-Luc Darlix ◽  
Stuart F. J. Le Grice ◽  
Suzanne B. Sandmeyer
Keyword(s):  
Nuclear Localization ◽  
Reverse Transcription ◽  
Catalytic Triad ◽  
Catalytic Site ◽  
Nuclear Localization Sequence ◽  
Binding Motif ◽  
Site Mutation ◽  
Amino Terminal ◽  
Carboxyl Terminal

ABSTRACT The integrase (IN) encoded by the Saccharomyces cerevisiae retroviruslike element Ty3 has features found in retrovirus IN proteins including the catalytic triad, an amino-terminal zinc-binding motif, and a nuclear localization sequence. Mutations in the amino- and carboxyl-terminal domains of Ty3 IN cause reduced accumulation of full-length cDNA in the viruslike particles. We show that the reduction in cDNA is accompanied by reduced amounts of early intermediates such as minus-strand, strong-stop DNA. Expression of a capsid (CA)-IN fusion protein (CA-IN) complemented catalytic site and nuclear localization mutants, but not DNA mutants. However, expression of a fusion of CA, reverse transcriptase (RT), and IN (CA-RT-IN) complemented transposition of catalytic site and nuclear localization signal mutants, increased the amount of cDNA in some of the mutants, and complemented transposition of several mutants to low frequencies. Expression of a CA-RT-IN protein with a Ty3 IN catalytic site mutation did not complement transposition of either a Ty3 catalytic site mutant or a nuclear localization mutant but did increase the amount of cDNA in several mutants and complement at least one of the cDNA mutants for transposition. These in vivo data support a model in which independent IN domains can contribute to reverse transcription and integration. We conclude that during reverse transcription, the Ty3 IN domain interacts closely with the polymerase domain and may even constitute a domain within a heterodimeric RT. These studies also suggest that during integration the IN catalytic site and at least portions of the IN carboxyl-terminal domain act in cis.

scholarly journals nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions.

1991 ◽  
Vol 11 (11) ◽  
pp. 5746-5755 ◽  
Author(s):  
G Burger ◽  
J Strauss ◽  
C Scazzocchio ◽  
B F Lang
Keyword(s):  
Amino Acids ◽  
Aspergillus Nidulans ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Regulatory Gene ◽  
Wild Type ◽  
Transcript Levels ◽  
Amino Terminal ◽  
Nitrate And Nitrite ◽  
Acidic Region

The nucleotide sequence of nirA, mediating nitrate induction in Aspergillus nidulans, has been determined. Alignment of the cDNA and the genomic DNA sequence indicates that the gene contains four introns and encodes a protein of 892 amino acids. The deduced NIRA protein displays all characteristics of a transcriptional activator. A putative double-stranded DNA-binding domain in the amino-terminal part comprises six cysteine residues, characteristic for the GAL4 family of zinc finger proteins. An amino-terminal highly acidic region and two proline-rich regions are also present. The nucleotide sequences of two mutations were determined after they were mapped by transformation with overlapping DNA fragments, amplified by the polymerase chain reaction. nirA87, a mutation conferring noninducibility by nitrate and nitrite, has a -1 frameshift at triplet 340, which eliminates 549 C-terminal amino acids from the polypeptide. Under the assumption that the truncated polypeptide is stable, it comprises the zinc finger domain and the acidic region, which seem not sufficient for transcriptional activation. nirAd-106, an allele conferring nitrogen metabolite derepression of nitrate and nitrite reductase activity, includes two transitions, changing a glutamic acid to a lysine and a valine to an alanine, situated between a basic and a proline-rich region of the protein. Northern (RNA) analysis of the wild type and of constitutive (nirAc) and derepressed (nirAd) mutants show that the nirA transcript does not vary between these strains, being in all cases constitutively expressed. On the other hand, transcript levels of structural genes (niaD and niiA) do vary, being highly inducible in the wild type but constitutively expressed in the nirAc mutant. The nirAd mutant appears phenotypically derepressed, because the niaD and niiA transcript levels are overinduced in the presence of nitrate but are still partially repressed in the presence of ammonium.

scholarly journals Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans

1992 ◽  
Vol 12 (5) ◽  
pp. 1932-1939
Author(s):  
P Kulmburg ◽  
D Sequeval ◽  
F Lenouvel ◽  
M Mathieu ◽  
B Felenbok
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Aspergillus Nidulans ◽  
Binding Protein ◽  
Transcriptional Activator ◽  
Binding Domain ◽  
Binding Motif ◽  
Affinity Binding ◽  
Coding Region

The ALCR protein is the transcriptional activator of the ethanol utilization pathway in the filamentous fungus Aspergillus nidulans. This activator belongs to a family of fungal proteins having a conserved DNA-binding domain containing six cysteines (C6 class) with some striking features. At variance with other motifs of this class, the binding domain of ALCR is strongly asymmetrical in relation to the central cysteines and moreover was predicted to adopt a helix-turn-helix structure. This domain of ALCR was synthesized in Escherichia coli and purified as a glutathione-S-transferase fusion protein. Our results show that the transcriptional activator ALCR is a DNA-binding protein. The DNA-binding motif contains zinc that is necessary for the specific DNA binding. The ALCR peptide binds upstream of the coding region of alcR to two specific targets with different affinities that are characterized by a conserved 5-nucleotide core, 5'-CCGCA-3' (or its reverse). One site, the lower-affinity binding site, is a direct repeat, and the other, the higher-affinity binding site, is a palindromic sequence with dyad symmetry. Therefore, the ALCR binding protein is able to recognize one DNA sequence in two different configurations. An alcR mutant obtained by deletion of the two specific targets in the cis-acting region of the alcR gene is unable to grow on ethanol and does not express any alcohol dehydrogenase activity. These results demonstrate that the binding sites are in vivo functional targets (UASalc) for the ALCR protein in A. nidulans. They corroborate prior evidence that alcR is autoregulated.

Sequences within an upstream activation site in the yeast enolase gene ENO2 modulate repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1

1990 ◽  
Vol 10 (9) ◽  
pp. 4863-4871
Author(s):  
J P Holland ◽  
P K Brindle ◽  
M J Holland
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Regulatory Gene ◽  
Major Protein ◽  
Null Mutation ◽  
Wild Type ◽  
Autonomously Replicating Sequence ◽  
Element Activity ◽  
Yeast Enolase

Transcription of the yeast enolase gene ENO2 is reduced 20- to 50-fold in strains carrying a null mutation in the positive regulatory gene GCR1. A small deletion mutation within one of two upstream activation sites (UAS elements) in the 5'-flanking region of ENO2 permitted wild-type levels of ENO2 gene expression in a strain carrying the gcr1 null mutation. These data show that sequences required for UAS element activity in GCR1 strains were required to repress ENO2 expression in a gcr1 strain. Protein factors that specifically bound to this UAS/repression site were identified. We show that the DNA-binding protein ABFI (autonomously replicating sequence-binding factor) is the major protein which binds the UAS/repression site. Minor DNA-binding activities that interact specifically with the UAS/repression site were also identified and may correspond to proteolytic breakdown products of ABFI. None of the observed binding activities were encoded by the GCR1 structural gene. A double-stranded oligonucleotide that included the UAS/repression site activated transcription of UAS-less ENO1 and ENO2 gene cassettes in vivo to wild-type levels in strains carrying the GCR1 allele as well as the gcr1 null mutation. These latter data show that the UAS/repression site is sufficient for transcriptional activation but is not sufficient to repress transcription of the enolase genes in a gcr1 genetic background.

scholarly journals Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

1997 ◽  
Vol 17 (10) ◽  
pp. 5679-5687 ◽  
Author(s):  
C P Chang ◽  
Y Jacobs ◽  
T Nakamura ◽  
N A Jenkins ◽  
N G Copeland ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Hox Proteins ◽  
Amino Terminal ◽  
Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

scholarly journals Sequences within an upstream activation site in the yeast enolase gene ENO2 modulate repression of ENO2 expression in strains carrying a null mutation in the positive regulatory gene GCR1.

1990 ◽  
Vol 10 (9) ◽  
pp. 4863-4871 ◽  
Author(s):  
J P Holland ◽  
P K Brindle ◽  
M J Holland
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Regulatory Gene ◽  
Major Protein ◽  
Null Mutation ◽  
Wild Type ◽  
Autonomously Replicating Sequence ◽  
Element Activity ◽  
Yeast Enolase

Transcription of the yeast enolase gene ENO2 is reduced 20- to 50-fold in strains carrying a null mutation in the positive regulatory gene GCR1. A small deletion mutation within one of two upstream activation sites (UAS elements) in the 5'-flanking region of ENO2 permitted wild-type levels of ENO2 gene expression in a strain carrying the gcr1 null mutation. These data show that sequences required for UAS element activity in GCR1 strains were required to repress ENO2 expression in a gcr1 strain. Protein factors that specifically bound to this UAS/repression site were identified. We show that the DNA-binding protein ABFI (autonomously replicating sequence-binding factor) is the major protein which binds the UAS/repression site. Minor DNA-binding activities that interact specifically with the UAS/repression site were also identified and may correspond to proteolytic breakdown products of ABFI. None of the observed binding activities were encoded by the GCR1 structural gene. A double-stranded oligonucleotide that included the UAS/repression site activated transcription of UAS-less ENO1 and ENO2 gene cassettes in vivo to wild-type levels in strains carrying the GCR1 allele as well as the gcr1 null mutation. These latter data show that the UAS/repression site is sufficient for transcriptional activation but is not sufficient to repress transcription of the enolase genes in a gcr1 genetic background.

scholarly journals Red Fluorescent Protein (DsRed) as a Reporter inSaccharomyces cerevisiae

2001 ◽  
Vol 183 (12) ◽  
pp. 3791-3794 ◽  
Author(s):  
Fernando Rodrigues ◽  
Martijn van Hemert ◽  
H. Yde Steensma ◽  
Manuela Côrte-Real ◽  
Cecı́la Leão
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Dsred Protein ◽  
Green Fluorescent

ABSTRACT We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.

scholarly journals Independent and Interchangeable Multimerization Domains of the AbrB, Abh, and SpoVT Global Regulatory Proteins

2005 ◽  
Vol 187 (18) ◽  
pp. 6354-6362 ◽  
Author(s):  
Fude Yao ◽  
Mark A. Strauch
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Chimeric Proteins ◽  
Dna Binding Domain ◽  
Global Regulators ◽  
Amino Terminal ◽  
Sequence Homologies ◽  
Carboxyl Terminal ◽  
Paralogous Proteins

ABSTRACT The global regulators AbrB, Abh, and SpoVT are paralogous proteins showing their most extensive sequence homologies in the DNA-binding amino-terminal regions (about 50 residues). The carboxyl-terminal portion of AbrB has been hypothesized to be a multimerization domain with little if any role in DNA-binding recognition or specificity. To investigate the multimerization potentials of the carboxyl-terminal portions of AbrB, Abh, and SpoVT we utilized an in vivo multimerization assay system based upon fusion of the domains to the DNA binding domain of the λ cI repressor protein. The results indicate that the N and C domains of all three paralogues are independent dimerization modules and that the intact Abh and SpoVT proteins are most probably tetramers. Chimeric proteins consisting of the AbrB N-terminal DNA-binding domain fused to the C domain of either Abh or SpoVT are indistinguishable from wild-type AbrB in their ability to regulate an AbrB target promoter in vivo.

scholarly journals Neural network modeling of differential binding between wild-type and mutant CTCF reveals putative binding preferences for zinc fingers 1-2

2021 ◽  
Author(s):  
Irene Miriam Kaplow ◽  
Abhimanyu Banerjee ◽  
Chuan-Sheng Foo
Keyword(s):  
Neural Network ◽  
Dna Binding ◽  
Zinc Fingers ◽  
Neural Network Modeling ◽  
Binding Motif ◽  
Wild Type ◽  
Core Motif ◽  
Binding Motifs ◽  
Binding Domains

Background: Many transcription factors (TFs), such as multi zinc-finger (ZF) TFs, have multiple DNA binding domains (DBDs) with multiple components, and deciphering the DNA binding motifs of individual components is a major challenge. One example of such a TF is CCCTC-binding factor (CTCF), a TF with eleven ZFs that plays a variety of roles in transcriptional regulation, most notably anchoring DNA loops. Previous studies found that CTCF zinc fingers (ZFs) 3-7 bind CTCF's core motif and ZFs 9-11 bind a specific upstream motif, but the motifs of ZFs 1-2 have yet to be identified. Results: We developed a new approach to identifying the binding motifs of individual DBDs of a TF through analyzing chromatin immunoprecipitation sequencing (ChIP-seq) experiments in which a single DBD is mutated: we train a deep convolutional neural network to predict whether wild-type TF binding sites are preserved in the mutant TF dataset and interpret the model. We applied this approach to mouse CTCF ChIP-seq data and, in addition to identifying the known binding preferences of CTCF ZFs 3-11, we identified a GAG binding motif for ZF1 and a weak ATT binding motif for ZF2. We analyzed other CTCF datasets to provide additional evidence that ZFs 1-2 interact with the motifs we identified, and we found that the presence of the motif for ZF1 is associated with Ctcf peak strength. Conclusions: Our approach can be applied to any TF for which in vivo binding data from both the wild-type and mutated versions of the TF are available, and our findings provide an unprecedently comprehensive understanding of the binding preferences of CTCF's DBDs.

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