Biomedical subjects

C Gatz

Publications and source records attributed to C Gatz.

33 records · Page 2Linked to original sources

PubMed1994

A dominant negative mutant of PG13 suppresses transcription from a cauliflower mosaic virus 35S truncated promoter in transgenic tobacco plants.

TGA1a and PG13 constitute a family of tobacco basic leucine zipper (bZIP) proteins that bind to activating sequence-1 (as-1), which is one of the multiple regulatory cis elements of the cauliflower mosaic virus (CaMV) 35S promoter. After truncation of the CaMV 35S promoter down to position -90 (CaMV 35S [-90] promoter), transcription stringently depends on the presence of as-1, which is recognized by nuclear DNA binding proteins called ASF-1. The role of the TGA1a/PG13 bZIP family in the formation of ASF-1 and in transcriptional activation of the CaMV 35S (-90) promoter has not yet been demonstrated in vivo. We constructed transgenic tobacco plants expressing a mutant of potato PG13, which lacks its wild-type DNA binding domain. This mutant acts as a trans-dominant inhibitor of ASF-1 formation and of expression from the CaMV 35S (-90) promoter, showing that PG13 can specifically interact with proteins necessary for these processes. Although we did not observe any other obvious phenotypic changes, these transgenic plants are a potentially valuable tool in identifying whether TGA1a and PG13 are involved in controlling promoters encoded in the plant genome.

Bacterial Proteins↗

PubMed1992

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome.

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.

Amino Acid Sequence↗

PubMed1992

Isolation and characterization of a cDNA-clone coding for potato type B phytochrome.

We have isolated and sequenced overlapping genomic and cDNA clones encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 77% identity to the Arabidopsis phyB and 50% identity to the potato phyA open reading frame, we suggest that these clones encode phyB phytochrome. However, the size of the deduced open reading frame of 1133 amino acids is smaller than the size of the other two phyB open reading frames characterized so far in higher plants, which contain 1171 or 1187 amino acids. The intron/exon structure within the coding region is conserved in phyA and phyB genes of various species. Southern blot analysis indicates that potato phyB is a single-copy gene. PhyB mRNA levels do not differ among different organs or different light regimes. Transcription initiation starts from two different start points which are 63 bp apart.

Amino Acid Sequence↗

PubMed1992

The Tn10-encoded Tet repressor blocks early but not late steps of assembly of the RNA polymerase II initiation complex in vivo.

We have studied the effect of the Tn10-encoded Tet repressor on expression from 13 cauliflower mosaic virus (CaMV) 35S promoter derivatives that contain a tet operator sequence in various positions downstream of the TATAbox. When the operator sequence was inserted less than 33 bp away from the TATAbox (position +9 with respect to the transcription start site), the repressor interfered with transcription, whereas increasing the distance to 35 bp (position +11) abolished repression. This result indicates that initiation of transcription from the CaMV 35S promoter occurs in at least two different steps: (1) binding of transcription factors, involving sequences extending to position +9; this step can be inhibited by binding of the Tet repressor protein; and (2) initiation of transcription from this complex, which is not affected by the repressor protein. We suggest that the Tet repressor can be used to investigate whether transcription conditions in vitro truly reflect the in vivo situation.

DNA Transposable Elements↗

PubMed1992

Construction of a tetracycline-inducible promoter in Schizosaccharomyces pombe.

We have developed a tightly repressed Schizosaccharomyces pombe promoter which can be efficiently induced by Tetracycline. This promoter is a derivative of the plant viral cauliflower mosaic virus 35S promoter which normally functions as a strong constitutive promoter in S. pombe. Location of three binding sites for the Tn10-encoded Tet repressor in the vicinity of the TATA-box of the CaMV 35S promoter led to a tight repression of promoter activity in the presence of the Tet repressor protein. Up to a 400-fold induction was observed after addition of the inducer Tetracycline, which inactivates the operator-binding capacity of the repressor.

Base Sequence↗

PubMed1992

Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants.

A cauliflower mosaic virus (CaMV) 35S promoter derivative, which is tightly repressed by the Tn 10 encoded Tet repressor in a transient expression system as well as in transgenic plants has been constructed. After treatment of transgenic plants with tetracycline (Tc) the activity of the reporter enzyme beta-glucuronidase (GUS) increased up to 500-fold in tissue culture as well as under greenhouse conditions. Efficient de-repression was achieved by Tc uptake through the roots as well as by Tc treatment of leaves of intact plants. As Tc is not very stable in the plants, this system can also be used for a transient expression of a transgene. This system provides a unique tool for regenerating transgenic plants carrying a repressed transgene and for efficiently de-repressing its activity by a specific inducer at any time point of further development.

Base Sequence↗

PubMed1991

Characterization of the interaction of plant transcription factors using a bacterial repressor protein.

Transcription initiation from a eukaryotic polymerase II promoter requires a functional interaction of regulatory transcriptional activators with at least one of the basal transcription factors binding in the vicinity of the TATA box. To characterize this type of interaction in vivo, we have inserted the bacterial Tet repressor-operator complex in nine different positions between an enhancer element (as-1) and the TATA box of the cauliflower mosaic virus (CaMV) 35S RNA promoter. A direct contact between the transcriptional activator ASF-1, which binds to as-1, and the transcriptional machinery should be affected by a repressor protein bound between them, as the spacing of only 34 base pairs (bp) between as-1 and the TATA box is too short to allow looping of the DNA around the repressor. In each construct, the distance of 34 bp was kept constant, while the position of the 19-bp tet operator relative to the TATA box differed by 2 bp. Thus, the position of the Tet repressor relative to the plant transcription factors was consecutively changed by 72 degrees, which allowed us to investigate whether repression depended on the stereospecific alignment of the repressor with the transcription factors. Binding of the Tet repressor to the operator blocked transcription only when the operator was inserted less tha 5 bp from the TATA box. In all other promoter derivatives, no inhibitory effect of the repressor was observed, which suggests that ASF-1 does not directly interact with the general transcription machinery.

Bacterial Proteins↗

PubMed1991

Repression of the CaMV 35S promoter by the octopine synthase enhancer element.

A 16 base-pair palindrome upstream of the Agrobacterium tumefaciens octopine synthase (ocs) gene functions as a positive regulatory element in plant cells (Ellis et al. (1987) EMBO J. 6, 3203-3208; Fromm et al. (1989) Plant Cell 1, 977-984). We have converted it into a negative element by locating two copies flanking the TATA-box of the constitutively expressed CaMV 35S promoter. The reduced promoter activity is very likely due to sterical hindrance of the ocs binding protein (OCSTF) x ocs complex with the transcription initiation complex. We propose that this type of constructs can be used for the identification of recognition sites for DNA-binding proteins which are labile in vitro as well as for determining the DNA-binding activity of a trans-acting factor in vivo.

Amino Acid Oxidoreductases↗

PubMed1991

Regulation of a modified CaMV 35S promoter by the Tn10-encoded Tet repressor in transgenic tobacco.

We have investigated the use of the Tn10-encoded tet repressor-operator system to regulate the expression of a suitably engineered cauliflower mosaic virus (CaMV) 35S promoter in transgenic tobacco plants. First, a transgenic plant was generated which constitutively synthesizes 600,000 Tet repressor monomers per cell. In a second transformation step, the beta-glucuronidase (gus) gene under the control of a modified CaMV 35S promoter, containing two tet operators, was stably integrated into the plant genome of a tetR+ plant. Expression of the gus gene is repressed 5-fold, if the operators are located flanking the TATA box, and 50- to 80-fold when both operators are positioned downstream of the TATA box. This indicates that Tet repressor-operator complexes can form on plant chromosomes and interfere with transcription. Maximal induction is achieved after 0.5 h upon application of only 0.1 mg/l tetracycline. This fast and efficient induction makes the system useful for specifically inducing expression of transferred genes at different stages of plant development.

Base Sequence↗

PubMed1988

Tn10-encoded tet repressor can regulate an operator-containing plant promoter.

The Tn10-encoded tet repressor-operator system was used to regulate transcription from the cauliflower mosaic virus (CaMV) 35S promoter. Expression was monitored in a transient assay system by using electric field-mediated gene transfer ("electroporation") into tobacco protoplasts. The tet repressor, being expressed in the plant cells under the control of eukaryotic transcription signals, blocks transcription of a CaMV 35S promoter chloramphenicol acetyltransferase (cat) fusion gene when the two tet operators flank the "TATA" box. In the presence of the inducer tetracycline, expression is restored to full activity. Location of the operators 21 base pairs downstream of the transcription start site does not significantly affect transcription in the presence of the repressor. These experiments show that a prokaryotic regulatory protein can function in plant cells. The tet repressor-operator complex may be useful for specifically inducing transferred genes at different stages of plant development.

DNA Transposable Elements↗

PubMed1986

Acinetobacter calcoaceticus encoded mutarotase: nucleotide sequence analysis of the gene and characterization of its secretion in Escherichia coli.

The nucleotide sequence of the mutarotase gene from Acinetobacter calcoaceticus has been determined. It reveals an open reading frame of 381 amino acids. The codon usage of A. calcoaceticus for this gene is similar to E. coli except for the amino acids Leu, Ala, Glu, and Arg where major differences exist. This did not interfere drastically with high level expression in E. coli. The regulatory sequences for the initiation of translation are similar to the ones described for E. coli. The N-terminal 20 amino acids, which are not found in the mature enzyme, show homology to signal sequences of exported proteins. In A. calcoaceticus and E. coli mutarotase is specifically secreted into the periplasmic space. Processing of the signal sequence occurs at identical sites in both organisms. The mature mutarotase consists of 361 amino acids and has a calculated molecular weight of 38457 Da. Expression of mutarotase at a high level in a recombinant E. coli destabilizes the outer membrane. This results in coordinated leakage of mutarotase and beta-lactamase into the culture broth.

Acinetobacter↗

PubMed1986

Cloning and expression of the Acinetobacter calcoaceticus mutarotase gene in Escherichia coli.

This article describes the cloning of the mutarotase gene from Acinetobacter calcoaceticus and its expression in Escherichia coli. Purification of mutarotase (EC 5.1.3.3) led to a single polypeptide of 40 kilodaltons. The sequences of 27 N-terminal and 76 C-terminal amino acids were determined. From six amino acids of the N-terminal and seven amino acids of the C-terminal portion of the protein, the sequences of two oligonucleotides were deduced. These were synthesized and used as gene probes. Completely restricted chromosomal DNA from A. calcoaceticus was size fractioned, and only fractions hybridizing with the gene probes were used to construct gene banks enriched for the mutarotase determinant. With the N-terminal gene probe, a bank of 6- to 7-kilobase-pair BclI fragments in pBR327 was obtained. A total of 1,200 candidates were screened by colony hybridization followed by dot-blot analysis of purified plasmids from positive candidates and subsequent Southern blot analysis of the respective restricted plasmids, and 500 base pairs (bp) from the 5' end of the mutarotase gene were isolated by this procedure. The 3' portion of the gene was isolated from a gene bank containing 1,500-bp-long HindIII fragments inserted in M13mp11. This bank was screened by dot-blot analysis of single-stranded phage DNA with the C-terminal gene probe. The isolated gene fragments were fused at a common restriction site in their overlapping region to yield the complete mutarotase gene. High-level expression of mutarotase in E. coli was achieved when the gene was placed under transcriptional control of the phage lambda promoter pL. More than 90% of mutarotase activity was found in the culture medium. The E. coli-derived mutarotase was purified and shown to be identical to the A. calcoaceticus-derived product with respect to the molecular weight and N-terminal amino acid sequence. The expression of mutarotase in E. coli was increased 200-fold in comparison to that the wild-type A. calcoaceticus.

Acinetobacter↗

PubMed1985

Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants.

Homologous and heterologous repressor-operator interactions among four different classes of tetracycline resistance determinants have been compared. These are represented by RP1/Tn1721 (class A), R222/Tn10 (class B), pSC101/pBR322 (class C), and RA1 (class D). By the use of the purified repressor proteins of class A (TetRA) and class B (TetRB), operator sequences of all four classes are recognized by both with an identical stoichiometry of four repressor subunits per control sequence, but with different affinities. In vitro transcription has been used to demonstrate regulatory activities of TetRA and TetRB upon all four classes of tet genes. Tetracycline acted as an inducer. A functional relationship among the tet regulatory systems was also shown in vivo by complementation of a class A tetR'-galK fusion mutant with the tetR genes of classes A, B, and C. Repression of tetRA-linked galactokinase was ca. 80% in the presence of tetRA or tetRC, and ca. 50% in the presence of tetRB. Taken together, these results demonstrate heterologous repressor-operator interaction, suggesting close relationships among the four classes of Tcr determinants.

Base Sequence↗

PubMed1984

Control of expression of the Tn10-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the tet operon regulatory region.

The promoter and operator sequences of the Tn10-encoded tetracycline resistance operon are determined in vitro by transcription studies of purified DNA restriction fragments, protection of guanosine from methylation by dimethylsulphate, and DNase I footprinting employing the purified TET repressor protein. In vitro transcription reveals three promoters with overlapping consensus sequences. Two of them, designated PR1 and PR2, are directed towards the tet repressor gene and the third, called PA, initiates transcription of the tet resistance gene. All three promoters are regulated simultaneously by the TET repressor protein, as demonstrated by in vitro transcription. Tetracycline functions as an inducer in these experiments. Two palindromic operator sequences in the tet operon control region, called O1 and O2, are occupied simultaneously by the TET repressor. Four guanosine residues in symmetric positions close to the centre of the palindromic operator sequences are protected from methylation in the repressor-operator complex. However, only one guanosine residue exhibits an enhanced reaction with dimethylsulphate under these conditions. Footprinting experiments reveal protection of phosphodiester bonds against DNase I slightly further than the palindromic sequence arrangement. Several phosphodiester bonds between the two operators are accessible for cleavage by DNase I in the repressor-operator complex. Two phosphodiester bonds within each operator sequence are cleaved by DNase I. This feature shows a clear assymmetry with the two inside cleavage positions of O1 and O2 being much less accessible for DNase I as compared to the two outside positions. A molecular mechanism of regulation of the Tn10-encoded tetracycline resistance operon is presented based on these and previous results.

Base Sequence↗

PubMed1983

Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions.

The transposon Tn10-encoded TET repressor controls the expression of tetracycline resistance as well as its own synthesis. The antibiotic tetracycline functions as an inducer for both genes, which are transcribed in divergent directions from a common start area. The interaction of the TET repressor with the regulatory sequence of the tetracycline resistance operon is investigated by equilibrium and kinetic methods. The wild-type control sequence contains two nearly identical operators separated by only ten base-pairs. A deletion mutant lacking one of the operators is constructed by controlled digestion with exonuclease Bal31. It serves to prove that the two TET operators are each occupied by a TET repressor dimer in the wild-type tet operon regulatory sequence. The association constants are approximately identical for both operators between 10(12) and 10(13) M-1 as derived from kinetic data. The half-life of the TET repressor--tet operator complex is 12 minutes when competed with tet operator DNA and two minutes when competed with the inducer tetracycline. The dissociation of the repressor--operator complex has no apparent activation enthalpy but has an activation entropy of -320 J/mol K, indicating the involvement of solvent or counterion condensation. The dissociation rate constant of the tetracycline--TET repressor complex depends strongly on temperature. The activation enthalpy is 160 kJ/mol, indicating extremely strong binding of the drug. This result is discussed with respect to the necessary sensitivity of a regulated resistance gene. The native structure of the TET repressor is a dimer, as demonstrated by molecular exclusion chromatography. The elution behavior of the TET repressor--tetracycline complex indicates clearly that the repressor--inducer complex remains a dimer. The results are discussed with respect to the regulatory functions of the components.

Base Sequence↗