scholarly journals Functional analysis of the Antirrhinum floral homeotic DEFICIENS gene in vivo and in vitro by using a temperature-sensitive mutant

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2861-2875 ◽  
Author(s):  
S. Zachgo ◽  
E.d. Silva ◽  
P. Motte ◽  
W. Trobner ◽  
H. Saedler ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Flower Development ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Organ Development ◽  
Mads Box ◽  
Binding Studies

Flowers of the temperature-sensitive DEFICIENS (DEF) mutant, def-101, display sepaloid petals and carpelloid stamens when grown at 26 degrees C, the non-permissive temperature. In contrast, when cultivated under permissive conditions at 15 degrees C, the morphology of def-101 flowers resembles that of the wild type. Temperature shift experiments during early and late phases of flower development revealed that second and third whorl organ development is differentially sensitive to changes in DEF expression. In addition, early DEF expression seems to control the spatially correct initiation of fourth whorl organ development. Reduction of the def-101 gene dosage differentially affects organogenesis in adjacent whorls: at the lower temperature development of petals in the second whorl and initiation of carpels in the centre of the flower is not affected while third whorl organogenesis follows the mutant (carpelloid) pattern. The possible contribution of accessory factors to organ-specific DEF functions is discussed. In situ analyses of mRNA and protein expression patterns during def-101 flower development at 15 degrees C and at 26 degrees C support previously proposed combinatorial regulatory interactions between the MADS-box proteins DEF and GLOBOSA (GLO), and provide evidence that the autoregulatory control of DEF and GLO expression by the DEF/GLO heterodimer starts after initiation of all organ primordia. Immunolocalisation revealed that both proteins are located in the nucleus. Interestingly, higher growth temperature affects the stability of both the DEF-101 and GLO proteins in vivo. In vitro DNA binding studies suggest that the temperature sensitivity of the def-101 mutant is due to an altered heterodimerisation/DNA-binding capability of the DEF-101 protein, conditioned by the deletion of one amino acid within the K-box, a protein region thought to be involved in protein-protein interaction. In addition, we introduce a mutant allele of GLO, glo-confusa, where insertion of one amino acid impairs the hydrophobic carboxy-terminal region of the MADS-box, but which confers no strong phenotypic changes to the flower. The strong mutant phenotype of flowers of def-101/glo-conf double mutants when grown in the cold represents genetic evidence for heterodimerisation between DEF and GLO in vivo. The potential to dissect structural and functional domains of MADS-box transcription factors is discussed.

scholarly journals Deprivation of protein or amino acid induces C/EBPβ synthesis and binding to amino acid response elements, but its action is not an absolute requirement for enhanced transcription

2008 ◽  
Vol 410 (3) ◽  
pp. 473-484 ◽  
Author(s):  
Michelle M. Thiaville ◽  
Elizabeth E. Dudenhausen ◽  
Can Zhong ◽  
Yuan-Xiang Pan ◽  
Michael S. Kilberg
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Activating Transcription Factor 3 ◽  
Binding Studies ◽  
Human Hepatoma Cells ◽  
Amino Acid Response

A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPβ (CCAAT/enhancer-binding protein β) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPβ association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPβ protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPβ was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPβ DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPβ binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBPβ protein was phosphorylated on Thr235 and the phospho-C/EBPβ did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPβ-deficient MEFs (mouse embryonic fibroblasts) or C/EBPβ siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPβ-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPβ is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Control of Thioredoxin Reductase Gene (trxB) Transcription by SarA in Staphylococcus aureus

2009 ◽  
Vol 192 (1) ◽  
pp. 336-345 ◽  
Author(s):  
Anand Ballal ◽  
Adhar C. Manna
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Dna Binding ◽  
Target Genes ◽  
Thioredoxin Reductase ◽  
Negative Regulator ◽  
Binding Studies ◽  
Oxidizing Agents

ABSTRACT Thioredoxin reductase (encoded by trxB) protects Staphylococcus aureus against oxygen or disulfide stress and is indispensable for growth. Among the different sarA family mutants analyzed, transcription of trxB was markedly elevated in the sarA mutant under conditions of aerobic as well as microaerophilic growth, indicating that SarA acts as a negative regulator of trxB expression. Gel shift analysis showed that purified SarA protein binds directly to the trxB promoter region DNA in vitro. DNA binding of SarA was essential for repression of trxB transcription in vivo in S. aureus. Northern blot analysis and DNA binding studies of the purified wild-type SarA and the mutant SarAC9G with oxidizing agents indicated that oxidation of Cys-9 reduced the binding of SarA to the trxB promoter DNA. Oxidizing agents, in particular diamide, could further enhance transcription of the trxB gene in the sarA mutant, suggesting the presence of a SarA-independent mode of trxB induction. Analysis of two oxidative stress-responsive sarA regulatory target genes, trxB and sodM, with various mutant sarA constructs showed a differential ability of the SarA to regulate expression of the two above-mentioned genes in vivo. The overall data demonstrate the important role played by SarA in modulating expression of genes involved in oxidative stress resistance in S. aureus.

scholarly journals Yeast 18S rRNA Dimethylase Dim1p: a Quality Control Mechanism in Ribosome Synthesis?

1998 ◽  
Vol 18 (4) ◽  
pp. 2360-2370 ◽  
Author(s):  
Denis L. J. Lafontaine ◽  
Thomas Preiss ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Small Subunit ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rrna Processing ◽  
Small Subunit Rrna ◽  
Enzymatic Function ◽  
Processing Steps

ABSTRACT One of the few rRNA modifications conserved between bacteria and eukaryotes is the base dimethylation present at the 3′ end of the small subunit rRNA. In the yeast Saccharomyces cerevisiae, this modification is carried out by Dim1p. We previously reported that genetic depletion of Dim1p not only blocked this modification but also strongly inhibited the pre-rRNA processing steps that lead to the synthesis of 18S rRNA. This prevented the formation of mature but unmodified 18S rRNA. The processing steps inhibited were nucleolar, and consistent with this, Dim1p was shown to localize mostly to this cellular compartment. dim1-2 was isolated from a library of conditionally lethal alleles of DIM1. In dim1-2strains, pre-rRNA processing was not affected at the permissive temperature for growth, but dimethylation was blocked, leading to strong accumulation of nondimethylated 18S rRNA. This demonstrates that the enzymatic function of Dim1p in dimethylation can be separated from its involvement in pre-rRNA processing. The growth rate ofdim1-2 strains was not affected, showing the dimethylation to be dispensable in vivo. Extracts of dim1-2 strains, however, were incompetent for translation in vitro. This suggests that dimethylation is required under the suboptimal in vitro conditions but only fine-tunes ribosomal function in vivo. Unexpectedly, when transcription of pre-rRNA was driven by a polymerase II PGKpromoter, its processing became insensitive to temperature-sensitive mutations in DIM1 or to depletion of Dim1p. This observation, which demonstrates that Dim1p is not directly required for pre-rRNA processing reactions, is consistent with the inhibition of pre-rRNA processing by an active repression system in the absence of Dim1p.

The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP

1992 ◽  
Vol 12 (6) ◽  
pp. 2662-2672
Author(s):  
Z Kozmik ◽  
S Wang ◽  
P Dörfler ◽  
B Adams ◽  
M Busslinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Lymphoid Cells ◽  
Binding Studies ◽  
Specific Transcription Factor ◽  
High Affinity ◽  
B Lymphoid

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.

Properties of an in vitro system for studying temperature-sensitive DNA synthesis in ts A1S9 mouse L-cells

1978 ◽  
Vol 56 (6) ◽  
pp. 444-451 ◽  
Author(s):  
Jerome Humbert ◽  
Rose Sheinin
Keyword(s):  
Dna Synthesis ◽  
Buoyant Density ◽  
Velocity Sedimentation ◽  
Synthetic Activity ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Activity Restriction ◽  
L Cells

The in vitro DNA synthesis has been observed in whole cell lysates and in cytosol and nuclear fractions of wild-type (WT-4) mouse L-cells and ts A1S9 cells which exhibit temperature-sensitive (ts) DNA replication in vivo. The product, labelled with substrate 3H-labelled TTP, is resistant to alkali and has the buoyant density (1.709 g/cm3) expected for normal mouse DNA. Pulse-chase studies, in which newly made, single-stranded DNA was analyzed by velocity sedimentation in alkaline sucrose density gradients, revealed that in vitro DNA synthesis proceeds by a discontinuous mechanism. Approximately half of the DNA made in a 30-s pulse sedimented at 3–8S; the rest was very heterogeneous with S values between [Formula: see text] and 30S. After incubation for up to 300 s, a majority of the newly made DNA (>85%) sedimented as the larger, heterogeneous material, with some cosedimenting with chromosomal size DNA.The ts DNA synthesis phenotype of ts A1S9 cells is expressed in vitro. Thus, the activity of extracts of ts cells incubated at the nonpermissive (38.5 °C) temperature was commensurate with the in vivo activity. Restriction of the ts phenotype to DNA synthesis is evident in vitro since the RNA synthetic activity of lysates of temperature-inactivated ts A1S9 cells was equivalent to that of extracts obtained from cells grown at the permissive temperature (33.5 °C). The DNA synthetic activity of nuclei from WT-4 or ts A1S9 cells grown at 33.5 °C plus homologous cytosol is equivalent to that of the whole lysate. In contrast, such cytosol preparations give little, if any, enhancement of the activity of nuclei from ts A1S9 cells incubated at 38.5 °C for 16 h. The cytosol of such temperature-inactivated cells, which are almost fully effective with nuclei of control cells, produce little or no enhancement of DNA synthesis by homologous nuclei.

scholarly journals Scanning Mutagenesis of Mcm1: Residues Required for DNA Binding, DNA Bending, and Transcriptional Activation by a MADS-Box Protein

2000 ◽  
Vol 20 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Thomas B. Acton ◽  
Janet Mead ◽  
Andrew M. Steiner ◽  
Andrew K. Vershon
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Essential Gene ◽  
Dna Bending ◽  
Mads Box ◽  
Dna Interactions ◽  
Protein Dna Interactions

ABSTRACT MCM1 is an essential gene in the yeastSaccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending. Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro. These positions have almost identical interactions with DNA in both the serum response factor-DNA and α2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA. Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding. These results suggest a general model of how MADS-box proteins recognize and bind DNA. We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell.

scholarly journals The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP.

1992 ◽  
Vol 12 (6) ◽  
pp. 2662-2672 ◽  
Author(s):  
Z Kozmik ◽  
S Wang ◽  
P Dörfler ◽  
B Adams ◽  
M Busslinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Lymphoid Cells ◽  
Binding Studies ◽  
Specific Transcription Factor ◽  
High Affinity ◽  
B Lymphoid

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.

Sign in / Sign up

Export Citation Format

Share Document