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P Gornicki

Publications and source records attributed to P Gornicki.

At least 19 recordsLinked to original sources
PubMed2004

Complex nested promoters control tissue-specific expression of acetyl-CoA carboxylase genes in wheat.

Cis-acting regulatory elements of the wheat acetyl-CoA carboxylase (ACC) gene family were identified by comparing the promoter activity of 5' end gene fragments fused to a reporter gene in two transient expression systems: wheat protoplasts and epidermal cells of mature embryos. Expression of the plastid and the cytosolic ACC genes is each driven by two nested promoters responsible for the synthesis of two transcript types. The internal promoter is located in an intron removed from transcripts originating at the first promoter. These complex promoters, which are different for the cytosolic and plastid ACC genes, control tissue-specific expression of the enzymatic activity supplying cytosolic, plastid, and mitochondrial pools of malonyl-CoA. The activity of one such complex promoter, driving expression of one of the cytosolic ACC genes, was studied throughout development of transgenic wheat plants carrying a full-length promoter-reporter gene fusion. High activity of the promoter was detected in the coleoptile, in the upper sheath section of the leaf, on the top surface of the ovary, in some sections of the main veins in the lemma and glume, and in abaxial epidermis hair cells of the lemma, glume, and rachis. The findings are consistent with the developmental and environmental requirements for very-long-chain fatty acids and flavonoids, whose synthesis begins with the ACC reaction in the cytosol of these specific cell types.

Acetyl-CoA Carboxylase↗
PubMed2001

An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors.

cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.

Acetyl-CoA Carboxylase↗
PubMed2001

Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii.

Apicomplexan parasites such as Toxoplasma gondii contain a primitive plastid, the apicoplast, whose genome consists of a 35-kb circular DNA related to the plastid DNA of plants. Plants synthesize fatty acids in their plastids. The first committed step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC). This enzyme is encoded in the nucleus, synthesized in the cytosol, and transported into the plastid. In the present work, two genes encoding ACC from T. gondii were cloned and the gene structure was determined. Both ORFs encode multidomain proteins, each with an N-terminal extension, compared with the cytosolic ACCs from plants. The N-terminal extension of one isozyme, ACC1, was shown to target green fluorescent protein to the apicoplast of T. gondii. In addition, the apicoplast contains a biotinylated protein, consistent with the assertion that ACC1 is localized there. The second ACC in T. gondii appears to be cytosolic. T. gondii mitochondria also contain a biotinylated protein, probably pyruvate carboxylase. These results confirm the essential nature of the apicoplast and explain the inhibition of parasite growth in cultured cells by herbicides targeting ACC.

Acetyl-CoA Carboxylase↗
PubMed2001

Chromosome mapping and phylogenetic analysis of the cytosolic acetyl-CoA carboxylase loci in wheat.

The cytosolic isoform of plant acetyl-CoA carboxylase is a multidomain enzyme involved in the synthesis of very-long-chain fatty acids and in secondary metabolism. Chromosome mapping of wheat identified one locus containing cytosolic acetyl-CoA carboxylase genes (Acc-2) and a related partially processed pseudogene (Psi-Acc-2) in the distal region of the long arm of wheat homoeologous group 3 chromosomes. Multiple copies of the Acc-2 genes, whose presence was suggested by sequence analysis, are likely to be arranged in tandem repeats. At least three out of five genes cloned from hexaploid wheat map to this locus. Another locus containing Acc-2--related sequences is present in the distal region of the long arm of chromosome 5D. The identity of the hybridizing DNA present at this locus remains unknown. A system based on PCR-cloning and DNA sequence analysis of acetyl-CoA carboxylase genes was developed to address various phylogenetic and systematics questions in grasses. It was applied to reconstruct the phylogeny of the Acc-2 genes from D- and S-genome Aegilops and A-genome Triticum diploid species, AABB- and AAGG-genome tetraploid wheat, and AABBDD-genome hexaploid wheat, as well as from rye and barley. The combined cytogenetic and molecular evolution approach allowed assignment of gene sequences included in phylogenetic analysis to specific loci on homoeologous chromosomes. Recurring gene duplication followed by chromosome translocation and/or possible loss of some gene copies, as well as loss of introns, occurred in the gene family in different plant lineages. Two major Acc-2 clades appeared before the divergence of barley and rye. Nucleotide substitution rates in different parts of the Acc-2 gene were assessed. This analysis of the Acc-2 loci provides detailed information regarding evolutionary events at a low--copy-number locus containing important functional genes. These events are likely to be common and to play a significant role in shaping grass genomes.

Acetyl-CoA Carboxylase↗
PubMed1999

Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain.

A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3'-tail, was used to complement a yeast ACC1 mutation. These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins. Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5'-end, complement the yeast mutation. Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 microM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases. This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo. The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase. Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors. The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.

Acetyl-CoA Carboxylase↗
PubMed1999

Growth of Toxoplasma gondii is inhibited by aryloxyphenoxypropionate herbicides targeting acetyl-CoA carboxylase.

Aryloxyphenoxypropionates, inhibitors of the plastid acetyl-CoA carboxylase (ACC) of grasses, also inhibit Toxoplasma gondii ACC. Clodinafop, the most effective of the herbicides tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 microM in 2 days and effectively eliminates the parasite in 2-4 days at 10-100 microM. Clodinafop is not toxic to the host cell even at much higher concentrations. Parasite growth inhibition by different herbicides is correlated with their ability to inhibit ACC enzyme activity, suggesting that ACC is a target for these agents. Fragments of genes encoding the biotin carboxylase domain of multidomain ACCs of T. gondii, Plasmodium falciparum, Plasmodium knowlesi, and Cryptosporidium parvum were sequenced. One T. gondii ACC (ACC1) amino acid sequence clusters with P. falciparum ACC, P. knowlesi ACC, and the putative Cyclotella cryptica chloroplast ACC. Another sequence (ACC2) clusters with that of C. parvum ACC, probably the cytosolic form.

Acetyl-CoA Carboxylase↗
PubMed1997

Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets.

5'-End fragments of two genes encoding plastid-localized acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) of wheat (Triticum aestivum) were cloned and sequenced. The sequences of the two genes, Acc-1,1 and Acc-1,2, are 89% identical. Their exon sequences are 98% identical. The amino acid sequence of the biotin carboxylase domain encoded by Acc-1,1 and Acc-1,2 is 93% identical with the maize plastid ACCase but only 80-84% identical with the cytosolic ACCases from other plants and from wheat. Four overlapping fragments of cDNA covering the entire coding region were cloned by PCR and sequenced. The wheat plastid ACCase ORF contains 2,311 amino acids with a predicted molecular mass of 255 kDa. A putative transit peptide is present at the N terminus. Comparison of the genomic and cDNA sequences revealed introns at conserved sites found in the genes of other plant multifunctional ACCases, including two introns absent from the wheat cytosolic ACCase genes. Transcription start sites of the plastid ACCase genes were estimated from the longest cDNA clones obtained by 5'-RACE (rapid amplification of cDNA ends). The untranslated leader sequence encoded by the Acc-1 genes is at least 130-170 nucleotides long and is interrupted by an intron. Southern analysis indicates the presence of only one copy of the gene in each ancestral chromosome set. The gene maps near the telomere on the short arm of chromosomes 2A, 2B, and 2D. Identification of three different cDNAs, two corresponding to genes Acc-1,1 and Acc-1,2, indicates that all three genes are transcriptionally active.

Acetyl-CoA Carboxylase↗
PubMed1997

Wheat cytosolic acetyl-CoA carboxylase complements an ACC1 null mutation in yeast.

Spores harboring an ACC1 deletion derived from a diploid Saccharomyces cerevisiae strain, in which one copy of the entire ACC1 gene is replaced with a LEU2 cassette, fail to grow. A chimeric gene consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat cytosolic acetyl-CoA carboxylase (ACCase) cDNA, and yeast ACC1 3' tail was used to complement a yeast ACC1 mutation. The complementation demonstrates that active wheat ACCase can be produced in yeast. At low concentrations of galactose, the activity of the "wheat gene" driven by the GAL10 promoter is low and ACCase becomes limiting for growth, a condition expected to enhance transgenic yeast sensitivity to wheat ACCase-specific inhibitors. An aryloxyphenoxypropionate and two cyclohexanediones do not inhibit growth of haploid yeast strains containing the yeast ACC1 gene, but one cyclohexanedione inhibits growth of the gene-replacement strains at concentrations below 0.2 mM. In vitro, the activity of wheat cytosolic ACCase produced by the gene-replacement yeast strain is inhibited by haloxyfop and cethoxydim at concentrations above 0.02 mM. The activity of yeast ACCase is less affected. The wheat plastid ACCase in wheat germ extract is inhibited by all three herbicides at concentrations below 0.02 mM. Yeast gene-replacement strains will provide a convenient system for the study of plant ACCases.

Journal Article↗
PubMed1996

Structure of a gene encoding a cytosolic acetyl-CoA carboxylase of hexaploid wheat.

An entire gene encoding wheat (var. Hard Red Winter Tam 107) acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] has been cloned and sequenced. Comparison of the 12-kb genomic sequence with the 7.4-kb cDNA sequence reported previously revealed 29 introns. Within the coding region, the exon sequence is 98% identical to the known wheat cDNA sequence. A second ACCase gene was identified by sequencing fragments of genomic clones that include the first two exons and the first intron. Additional transcripts were detected by 5' and 3' RACE analysis (rapid amplification of cDNA ends). One set of transcripts had a 5' end sequence identical to the cDNA found previously and another set was identical to the gene reported here. The 3' RACE clones fall into four distinguishable sequence sets, bringing the number of ACCase sequences to six. None of these cDNA or genomic clones encodes a chloroplast targeting signal. Identification of six different sequences suggests that either the cytosolic ACCase genes are duplicated in the three chromosome sets in hexaploid wheat or that each of the six alleles of the cytosolic ACCase gene has a readily distinguishable DNA sequence.

Acetyl-CoA Carboxylase↗
PubMed1994

Wheat acetyl-coenzyme A carboxylase: cDNA and protein structure.

cDNA fragments encoding part of wheat (Triticum aestivum) acetyl-CoA carboxylase (ACC; EC 6.4.1.2) were cloned by PCR using primers based on the alignment of several biotin-dependent carboxylases. A set of overlapping clones encoding the entire wheat ACC was then isolated by using these fragments as probes. The cDNA sequence contains a 2257-amino acid reading frame encoding a 251-kDa polypeptide. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is 52-55% identical to ACC of yeast, rat, and diatom. Identity with the available C-terminal amino acid sequence of maize ACC is 66%. The biotin attachment site has the typical eukaryotic EVMKM sequence. The cDNA does not encode an obvious chloroplast targeting sequence. Various cDNA fragments hybridize in Northern blots to a 7.9-kb mRNA. Southern analysis with cDNA probes revealed multiple hybridizing fragments in hexaploid wheat DNA. Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants, indicating significant conservation among plant ACCs.

Acetyl-CoA Carboxylase↗
PubMed1993

Wheat acetyl-CoA carboxylase.

The acetyl-CoA carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent carboxylase present in wheat chloroplasts. Active acetyl-CoA carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of 14C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA carboxylase and haloxyfop.

Acetyl-CoA Carboxylase↗
PubMed1993

Genes for two subunits of acetyl coenzyme A carboxylase of Anabaena sp. strain PCC 7120: biotin carboxylase and biotin carboxyl carrier protein.

Genes for two subunits of acetyl-coenzyme A carboxylase, biotin carboxylase and biotin carboxyl carrier protein, have been cloned from Anabaena sp. strain PCC 7120. The two proteins are 181 and 447 amino acids long and show 40 and 57% identity to the corresponding Escherichia coli proteins, respectively. The sequence of the biotinylation site in Anabaena sp. strain PCC 7120 is MetLysLeu, not the MetLysMet found in other sequences of biotin-dependent carboxylases. The amino acid sequence of biotin carboxylase is also very similar (32 to 47% identity) to the sequence of the biotin carboxylase domain of other biotin-dependent carboxylases. Genes for these two subunits of acetyl-coenzyme A carboxylase are not linked in Anabaena sp. strain PCC 7120, contrary to the situation in E. coli, in which they are in one operon.

Acetyl-CoA Carboxylase↗
PubMed1991

Neighbourhood of the central fold of the tRNA molecule bound to the E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop.

The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA.

Affinity Labels↗
PubMed1990

The three-dimensional structure and function of Escherichia coli ribosomal RNA, as studied by cross-linking techniques.

A large number of intra-RNA and RNA-protein cross-link sites have been localized within the 23S RNA from E. coli 50 S ribosomal subunits. These sites, together with other data, are sufficient to constrain the secondary structure of the 23 S molecule into a compact three-dimensional shape. Some of the features of this structure are discussed, in particular, those relating to the orientation of tRNA on the 50 S subunit as studied by site-directed cross-linking techniques. A corresponding model for the 16S RNA within the 30 S subunit has already been described, and here a site-directed cross-linking approach is being used to determine the path followed through the subunit by messenger RNA.

Base Sequence↗
PubMed1989

Use of lead(II) to probe the structure of large RNA's. Conformation of the 3' terminal domain of E. coli 16S rRNA and its involvement in building the tRNA binding sites.

The present work shows that lead(II) can be used as a convenient structure probe to map the conformation of large RNA's and to follow discrete conformational changes at different functional states. We have investigated the conformation of the 3' domain of the E. coli 16S rRNA (nucleotides 1295-1542) in its naked form, in the 30S subunit and in the 70S ribosome. Our study clearly shows a preferential affinity of Pb(II) for interhelical and loop regions and suggests a high sensitivity for dynamic and flexible regions. Within 30S subunits, some cleavages are strongly decreased as the result of protein-induced protection, while others are enhanced suggesting local conformational adjustments. These rearrangements occur at functionally strategic regions of the RNA centered around nucleotides 1337, 1400, 1500 and near the 3' end of the RNA. The association of 30S and 50S subunits causes further protections at several nucleotides and some enhanced reactivities that can be interpreted in terms of subunits interface and allosteric transitions. The binding of E. coli tRNA-Phe to the 70S ribosome results in message-independent (positions 1337 and 1397) and message-dependent (1399-1400, 1491-1492 and 1505) protections. A third class of protection (1344-1345, 1393-1395, 1403-1409, 1412-1414, 1504, 1506-1507 and 1517-1519) is observed in message-directed 30S subunits, which are induced by both tRNA binding and 50S subunit association. This extensive reduction of reactivity most probably reflects an allosteric transition rather than a direct shielding.

Base Sequence↗
PubMed1989

Modified tRNAs for probing tRNA binding sites on the ribosome.

Unusual chemical properties of hypermodified nucleosides N6-(threoninocarbonyl)adenosine (t6 A) located at position 37 and 3-(3-amino-3-carboxypropyl)uridine (acp3U) located at position 20:1 have been utilized for the introduction of photoreactive azidonitrophenyl probes to the anticodon loop and to the dihydrouridine loop of yeast tRNA(mMet) and lupin tRNA(mMet), respectively. The very efficient and selective modification procedures involve condensat on of the carboxyl group of t6A with ethylenediamine in the presence of a water soluble carbodiimide followed by acylation of the newly introduced amino group with the respective N-hydroxysuccinimide ester, and acylation of the primary amino or up of acp3U with the respective N-hydroxysuccinimide ester. Binding and crosslinking of the modified, uncharged tRNAs to E coli ribosome have been studied in the presence and absence of poly(AUG) as a message. Both tRNAs carrying about 20 A long photoreactive probes retain their binding activity and upon irradiation with visible light crosslink to the ribosome with high yields showing their usefulness for structural studies on the tRNA-ribosome complex.

Adenosine↗
PubMed1985

Cross-linking of the anticodon of P and A site bound tRNAs to the ribosome via aromatic azides of variable length: involvement of 16S rRNA at the A site.

The topography of the ribosomal decoding site was explored by affinity labeling from the 5'-anticodon base, 5-(carboxymethoxy)uridine-34, of P or A site bound tRNA1Val. A nitrophenyl azide was attached to the carboxyl group of this nucleotide via side chains varying in length from 18 to 24 A. Binding of acetylvalyl-tRNA to the P site was codon dependent and that of valyl-tRNA to the A site was both codon and elongation factor Tu (EFTu) dependent. Cross-linking to both A and P sites was irradiation, probe, codon, and, in the case of the A site, EFTu dependent. Putative P-site cross-linked aminoacyl-tRNA was reactive with puromycin. The yield of cross-linking was little affected by placement of the tRNA at the A or P site but varied considerably with the length and structure of the probe side chain. When the distance from the pyrimidine C-5 atom to the azide group was 23 A, 42-45% cross-linking was obtained at each site, but when the distance was decreased to 18 A, only 7-12% was found. Placing an S-S bond in the center of the 23-A leash decreased the A-site yield to about half, while insertion of a CONH group decreased A-site cross-linking about 8-fold. P-site cross-linking was more sensitive to mercaptan quenching (50% at 0.5 mM) than was that at the A site (50% at greater than 2.0 mM) but both were partially shielded from solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗