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G L Igloi

Publications and source records attributed to G L Igloi.

At least 19 recordsLinked to original sources
PubMed1992

Affinity electrophoretic detection of primary amino groups in nucleic acids: application to modified bases of tRNA and to aminoacylation.

Thiolation of primary amino groups in tRNA with the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate gives rise to species which are retarded during electrophoresis in organomercury-containing polyacrylamide gels. Since such amino groups occur, as far as is known, only as part of the modified bases 3-(3-amino-3-carboxypropyl)uridine and N-2-(5-amino-5-carboxypentyl)cytidine or as the alpha-amino group of aminoacylated tRNAs, this extension of the principle of affinity electrophoresis can be used for the detection and analysis of a specific functional group in both single tRNA species and in a mixed population. The strength of the interaction may be quantified and provides information on the chemical environment/conformation of the derivatized bases.

Amines↗
PubMed1992

Structure of a gene encoding heat-shock protein HSP70 from the unicellular alga Chlamydomonas reinhardtii.

The structure of a gene encoding a 70-kDa heat-shock protein (HSP70) from the unicellular alga, Chlamydomonas reinhardtii, is described. This gene shows a remarkable expression pattern, because it is inducible by light as well as by elevated temperature [von Gromoff et al., Mol. Cell. Biol. 9 (1989) 3911-3918]. As a first step in the investigation of trans-acting factors involved in environmentally controlled expression of this hsp70 gene, the nucleotide sequence of the entire gene, including its 5'- and 3'-flanking regions was determined. Although the deduced amino acid sequence exhibits a high degree of conservation to the HSP70 from higher plants, the C. reinhardtii gene has a unique structure among the members of the hsp70 gene family. While most hsp70 genes have only one or no intron, the coding region of the C. reinhardtii gene is interrupted by six introns. Besides putative TATA and CCAAT boxes, two heat-shock elements (HSE) were found in the promoter region, and a third HSE motif was located within the fourth intron. A computer search for regulatory cis-acting elements revealed a noted similarity of a 5'-upstream sequence motif to the G-box motif conserved in higher plants. A polyadenylation recognition sequence canonical for nuclear genes of C. reinhardtii is located downstream from the coding sequence.

Amino Acid Sequence↗
PubMed1992

RNA editing in tobacco chloroplasts leads to the formation of a translatable psbL mRNA by a C to U substitution within the initiation codon.

The psbL gene which codes for a 38 amino acid peptide of photosystem II, together with the photosynthetic genes psbE and psbF, is contained in a conserved position of many species of higher plant plastomes. The alignment of the psbL nucleotide sequences from ten species shows strong conservation, which is indicative of a functional gene. The tobacco and spinach psbL genes have, however, an ACG codon instead of the initiator ATG codon observed in the homologous position of the other eight species. Evidence is presented that in tobacco chloroplasts a translatable psbL mRNA containing an AUG initiator codon is formed by a C to U editing of the ACG codon. This observation, following the previously reported editing of an rpl2 gene in maize chloroplasts, underlines a more widespread occurrence of this type of posttranscriptional mRNA modification and demonstrates its presence in a dicotyledon plant.

Base Sequence↗
PubMed1992

Evolutionary analysis of the plastid-encoded gene for the alpha subunit of the DNA-dependent RNA polymerase of Pyrenomonas salina (Cryptophyceae).

The nucleotide sequence of the gene coding for the plastid-encoded alpha subunit of DNA-dependent RNA polymerase from the cryptomonad alga Pyrenomonas salina was determined. The deduced amino-acid sequence, corresponding to a 35.2 kDa polypeptide, was compared to homologues from other organisms. Evolutionary relationships were analyzed in detail by the parsimony method together with bootstrap analysis. The deduced phylogenetic tree shows that the cryptomonad gene is the most ancient type of known plastid-encoded RNA polymerase.

Amino Acid Sequence↗
PubMed1991

Editing of a chloroplast mRNA by creation of an initiation codon.

Primary mRNA transcripts in several systems are edited by single base substitutions, small deletions or insertions to yield functional messenger RNA species. Mitochondrial mRNAs in particular, including those from plants, seem to be the subject of extensive editing, unlike mRNAs encoded by chloroplast DNA, for which the prediction of amino-acid sequence from the corresponding gene sequence is generally unambiguous. Occasionally, however, an ACG codon appears at the 5' terminus of chloroplast genes, where the initiation codon ATG would be expected. Here we present evidence for a C----U editing that is responsible for the conversion of the ACG codon to an AUG initiation codon in the mRNA transcript from the rpl2 gene of the maize plastome, showing that mRNA editing can also occur in chloroplasts.

Amino Acid Sequence↗
PubMed1991

Identification of in vivo processing intermediates and of splice junctions of tRNAs from maize chloroplasts by amplification with the polymerase chain reaction.

Total RNA from chloroplasts of maize seedlings was used for polymerase chain reaction (PCR) mediated amplification of tRNA precursors and of mature tRNAs encoded by the two split tRNA genes of the ribosomal spacer (tRNA(lle)GAU and tRNA(Ala)UGC) and the single intron-containing tRNA(Gly)UCC gene. Sequence analysis of DNAs amplified from the mature tRNAs by combinations of exon specific primers allows unambiguous identification of the respective splice junctions. Primer combinations in which 5'- or 3'-flanking precursor tRNA sequences are included, leads to the amplification of processing intermediates in which 5'-terminal extensions are still present, whereas no PCR products corresponding to 3'-terminal extensions could be detected. From this it is concluded that in chloroplasts the 5'-terminal endonucleolytic cleavage by RNase P occurs as one of the final steps in the tRNA processing pathway of which the endonucleolytic cleavage at the 3' side probably occurs prior to the splicing of the intron sequences.

Base Sequence↗
PubMed1991

Demonstration of nucleomorph-encoded eukaryotic small subunit ribosomal RNA in cryptomonads.

In cryptomonads, unicellular phototrophic flagellates, the plastid(s) is (are) located in a special narrow compartment which is bordered by two membranes; it harbours neither mitochondria nor Golgi dictyosomes but comprises eukaryotic ribosomes and starch grains together with a small organelle called the nucleomorph. The nucleomorph contains DNA and is surrounded by a double membrane with pores. It is thought to be the vestigial nucleus of a phototrophic eukaryotic endosymbiont. Cryptomonads are therefore supposed to represent an intermediate state in the evolution of complex plastids from endosymbionts. We have succeeded in isolating pure nucleomorph fractions, and can thus provide, using pulsed field gel electrophoresis, polymerase chain reaction and sequence analysis, definitive proof for the eukaryotic nature of the symbiont and its phylogenetic origin.

Animals↗
PubMed1990

Nucleotide sequence of the maize chloroplast rpo B/C1/C2 operon: comparison between the derived protein primary structures from various organisms with respect to functional domains.

The genes (rpo B/C1/C2) coding for the beta, beta', beta" subunits of maize (Zea mays) chloroplast RNA polymerase have been located on the plastome and their nucleotide sequences established. The operon is part of a large inversion with respect to the tobacco and spinach chloroplast genomes and is flanked by the genes trnC and rps2. Notable features of the nucleotide sequence are the loss of an intron in rpoC1 and an insertion of approximately 450 bp in rpoC2 compared to the dicotyledons tobacco, spinach and liverwort. The derived amino acid sequence of this additional monocotyledon specific sequence is characterized by acidic heptameric repeat units containing stretches of glutamic acid, tyrosines and leucines with regular spacing. Other structural motifs, such as a nucleotide binding domain in the beta subunit and a zinc finger in the beta' subunit, are compared at the amino acid level throughout the RNA polymerase subunits with the enzymes from other organisms in order to identify functionally important conserved regions.

Amino Acid Sequence↗
PubMed1988

Interaction of tRNAs and of phosphorothioate-substituted nucleic acids with an organomercurial. Probing the chemical environment of thiolated residues by affinity electrophoresis.

The interactions of 4-thiouridine and 5-[(methylamino)methyl]-2-thiouridine in tRNA and of phosphorothioate esters in nucleic acids with an organomercurial have been investigated. For this purpose, an affinity electrophoretic system has been developed in which the mercury derivative has been covalently immobilized in a standard polyacrylamide gel. The retardation of thiolated macromolecules was found to be sensitive to the chemical environment of the sulfur atom, giving characteristic interaction constants dependent on the nature of the modification and its accessibility to binding. The interaction could, in the case of tRNA, be abolished by conventional specific chemical modification of the thiolated bases, as well as by irradiation with 32P-derived beta-emission. Not only has the fractionation of sulfur-modified from unmodified species been attained but a quantitative application of the technique has made it possible to study the binding of mercury and, by competition, that of magnesium in terms of the conformation of tRNA.

Autoradiography↗
PubMed1987

The use of permanganate as a sequencing reagent for identification of 5-methylcytosine residues in DNA.

The use of permanganate as a reagent for DNA sequencing by chemical degradation has been studied with respect to its specificity for 5-methylcytosine residues. At weakly acidic pH and room temperature, 0.2 mM potassium permanganate reacts preferentially with thymine, 5-methylcytosine, and to a lesser extent with purine residues, while cytosine remains essentially intact. Permanganate oxidation is, therefore, a suitable DNA sequencing reaction for positive discrimination between 5-methylcytosine and unmethylated cytosine.

5-Methylcytosine↗
PubMed1987

Functional in vivo verification in E. coli of promoter activities from the rDNA/tDNA(Val)(GAC) leader region of Zea mays chloroplasts.

Restriction fragments containing upstream sequences of the rRNA operon from Zea mays chloroplasts were tested for promoter activity in vivo by insertion into an E. coli promoter-probe vector. The expression of this vector's reporter gene, which codes for alkaline phosphatase, was stimulated more than 1,500-fold upon linkage with the chloroplast rRNA promoter. Site specific mutagenesis of the invariant T of the -10 sequence of this promoter reduced the expression of the reporter gene to 2% of the wild type. This indicates that the chloroplast rRNA promoter, which directs transcriptional initiation 117 bp upstream of the 16S rRNA gene, is also active in the bacterial system. A restriction fragment further upstream containing the gene for tRNA(Val) (GAC) also showed strong promoter activity (29% as compared with the rRNA promoter). This promoter activity probably reflects the chloroplast promoter directing the synthesis of the tRNA(Val) (GAC) primary transcript. Surprisingly, this restriction fragment also displayed promoter activity (13% compared with the rRNA promoter) in reverse orientation.

Base Sequence↗
PubMed1985

Affinity electrophoresis for monitoring terminal phosphorylation and the presence of queuosine in RNA. Application of polyacrylamide containing a covalently bound boronic acid.

An affinity electrophoretic method has been developed to study the state of terminal phosphorylation of RNAs and the presence of the hypermodified base Q in tRNA. It is based on the copolymerization of acryloylaminophenylboronic acid into standard polyacrylamide gels and the interaction of this derivative with free cis-diol groups present in the RNA. In the case of terminal phosphorylation, free ribose groups are present either as such, or may be introduced by enzymatic reactions specific for a particular phosphorylation pattern (e.g. using T4 RNA ligase or guanylyltransferase). Additionally, tRNA species containing the Q base may be resolved from Q-lacking tRNAs by boronate affinity electrophoresis. The introduction of a non-destructive, one-step electrophoretic procedure not only offers an alternative to classical analytical methods, but also provides a means of isolating such populations of RNAs for which other methods are unavailable or are less convenient.

Acrylamide↗
PubMed1978

Aminoacyl-tRNA synthetases from yeast: generality of chemical proofreading in the prevention of misaminoacylation of tRNA.

The specificity of valyl-, phenylalanyl-, and tyrosyl-tRNA synthetases from yeast has been examined by a series of stringent tests designed to eliminate the possibility of artefactual interference. Valyl-tRNA synthetase, as well as activating a number of amino acid analogues, will accept alanine, cysteine, isoleucine, and serine in addition to threonine as substrates for both ATP-PPi exchange and transfer to some tRNAVal species. The transfer is not observed if atempts are made to isolate the appropriate aminoacyl-tRNAVal-C-C-A but its role in the overall aminoacylation can be suspected from both the formation of a stable aminoacyl-tRNAVal-C-C-A(3'NH2) compound and from the stoichiometry of ATP hydrolysis during the aminoacylation of the native tRNA. Similar tests with phenylalanyl-tRNA synthetase indicate that this enzyme will also activate and transfer other naturally occurring amino acids, namely, leucine, methionine, and tyrosine. The tyrosine enzyme, which lacks the hydrolytic capacity of the other two enzymes (von der Haar, F., & Cramer, F (1976) Biochemistry 15, 4131--4138) is probably absolutely specific for tyrosine. It is concluded that chemical proofreading, in terms of an enzymatic hydrolysis of a misacylated tRNA, plays an important part in maintaining the specificity in the overall reaction and that this activity may be more widespread than has so far been suspected.

Amino Acids↗
PubMed1978

Threonyl-tRNA synthetase from yeast: aminoacylation of tRNA on its non-accepting 3'-terminal hydroxyl group and its behaviour in enzyme-catalyzed deacylation.

Methods have been developed by which tRNA Thr may be aminoacylated at the normally non-accepting 3'-terminal ribose OH. Two of the methods utilize the mischarging ability of the synthetases under special conditions of low salt concentration and presence of organic solvents. The third method demonstrates for the first time that for some synthetases the 2',3' specificity may be manipulated by use of similar special conditions. In the case of threonyl-tRNA synthetase, Thr-tRNAThr-C-C-A(3'd) has been synthesised by this method. The behaviour of threonyl esters of tRNAThr-C-C-A, tRNAThr-C-C-A(2'd) and tRNA Thr-C-C-A-(3'd) in the free enzyme-catalyzed deacylation has been studied and the results indicate that the cis diol functional group is necessary for this hydrolysis. The position on the terminal ribose from which the amino acid is removed in this reaction remains to be identified.

Adenosine Monophosphate↗