Biomedical subjects
Publications and source records attributed to J Barciszewski.
5S Ribosomal RNA is the smallest RNA component of the ribosomes. Due to relatively simple isolation and sequencing procedures as well as a potential use of the sequence data in evolutionary analyses, the amount of known nucleotide sequences on both RNA and DNA levels was rapidly growing. In this paper we present the updated (March 1996) compilation of eukaryotic 5D rRNA and 5S rDNA sequences.
The compilation of 5S rRNA and 5S rRNA gene nucleotide sequences as of 30 September 1996, contains a total of 1661 primary structures of 5S rRNAs or their genes, which is an increase of 928 new sequence entries over the last compilation. It covers sequences from 54 archaea, 449 eubacteria, 34 plastids, nine mitochondria and 430 eukaryotes. The databank uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information. The taxonomic classification of organisms was totally updated. Now the database is also available via anonymous FTP or WWW.
We synthesized and used hammerhead ribozymes designed for the GUC and AUC target sequences located in the terminal loop C of animal and plant 5S rRNAs to verify its involvement in a higher order structure formation of ribosomal RNA. The activity of both GUC and AUC specific ribozymes were proven to be active against synthetic analogs of the stem III and loop C in the two types of 5S rRNAs. In contrast we did not observe any ribozyme-catalysed hydrolysis within the loop C of the native 5S rRNAs. We considered two possible explanations of the results: an involvement of the loop C in long range interactions and lack of a structural flexibility to form proper tertiary structure of the complex with the ribozyme. From our data it seems that the single stranded nucleotides within the loop C can not form correct RNA duplex structure required for ribozyme activity. It is clear that hammerhead ribozymes can be used for inhibition of different steps of expression of genetic information only if the target sequence occurs in appropriate context, that allows to form correct secondary and tertiary structure of the ribozyme-substrate complex.
New data are presented on the interaction of model synthetic peptides containing an arginine-rich region of human immunodeficiency virus (HIV-Tat), with native RNA molecules: tRNA(Phe) of Saccharomyces cerevisiae and 5S rRNA from Lupinus luteus. Both RNA species form complexes with the Tat1 (GRKKRRQRRRA) and Tat2 (GRKKRRQRRRAPQDSQTHQASLSKQPA) peptides, as shown by electrophoretic gel shift and RNase footprint assays, and CD measurements. The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe as well as in the loop D of 5S rRNA is specifically protected against RNases. Our data indicate direct interactions of guanine of RNA moieties with arginine residues. These interactions seem similar to those observed in DNA-protein complexes, but different from those previously observed in the TAR RNA-Tat complexes.
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Recently several new evidences have appeared on biological role of native short peptides. This is an overview on two of them occurring in plants: systemin and ENOD 40.
In contrast to the current view that kinetin (N6-furfuryladenine) is an unnatural and synthetic compound, we have detected it in commercially available DNA, in freshly extracted cellular DNA from human cells and in plant cell extracts by two independent methods. First, we discovered that N6-furfuryladenine has electrochemical properties that can be applied for monitoring this modified base by a HPLC/UV/EC method. Second, we have confirmed electrochemical assignments by mass-spectrometric analysis. A pathway of kinetin formation is proposed in which the formation of furfural by oxidative damage of the deoxyribose moiety of DNA is followed by its reaction with adenine residues to form N6-furfuryladenine. Since this modification can lead to mutations, the odd DNA base has to be removed by repair enzymes.
We have determined the nucleotide sequence of 5S ribosomal RNA from the parasitic nematode Ascaris suum. The analysis of all Nematoda 5S rRNAs and their genes shows that this group must have diverged from other Metazoa at early stages of evolution. This conclusion is supported by the sequence variability in single-stranded regions which are strongly conserved in animal 5S rRNAs.
The amino acid sequences of a number of closely related proteins ("napin") isolated from Brassica napus were determined by mass spectrometry without prior separation into individual components. Some of these proteins correspond to those previously deduced (napA, BngNAP1, and gNa), chiefly from DNA sequences. Others were found to differ to a varying extent (BngNAP1', BngNAP1A, BngNAP1B, BngNAP1C, gNa', and gNaA). The short chains of gNa and gNa' and of BngNAP1 and BngNAP1' differ by the replacement of N-terminal proline by pyroglutamic acid; the long chains of gNaA and BngNAP1B contain a six amino acid stretch, MQGQQM, which is present in gNa (according to its DNA sequence) but absent from BngNAP1 and BngNAP1C. These alternations of sequences between napin isoforms are most likely due to homologous recombination of the genetic material, but some of the changes may also be due to RNA editing. The amino acids that follow the untruncated C termini of those napin chains for which the DNA sequences are known (napA, BngNAP1, and gNa) are aromatic amino acids. This suggests that the processing of the proprotein leading to the C termini of the two chains is due to the action of a protease that specifically cleaves a G/S-F/Y/W bond.
Four different chemically synthesized single stranded complementary oligonucleotides: DNA I, d(GCGCGCATATAT); RNA I, r(AUAUAUGCGCGC): RNA II, r(GGCCGGUUAAUU); and RNA III, r(AAUUAACCGGCC) were studied in order that the effects of high pressure on heteroduplex and homoduplex structures could be understood. The oligonucleotides were subjected to a high pressure at low and/or high salt buffer and analyzed by circular dichroism spectroscopy. In these conditions, both DNA-RNA and RNA-RNA duplexes with different purine-pyrimidine sequences change their conformation. The heteroduplex DNA I-RNA I with the complementary alternating purine-pyrimidine sequence, does not change its conformation of A type at high salt alone or at high salt and high pressure applied together. The homoduplex RNA II-RNA III with purine-purine-pyrimidine-pyrimidine sequence does not change strongly its. A-RNA conformation either. However, a structure of the homoduplex is affected by high pressure alone or with high salt as concluded from shifting the maximum of the CD spectrum to around 265 nm and inducing higher Cotton effect. These observations clearly suggest some conformational changes of the homoduplex. A single stranded oligonucleotide (RNA I) and oligodeoxynucleotide (DNA I) alone showed up a different conformation. The CD spectrum of RNA I is similar to that of A-RNA structure, out that of DNA I shows a very small Cotton effect and has not an ordered structure.
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Comparative analysis of the nucleotide sequences of plant 5S rRNA genes shows numerous discrepancies with the mature 5S rRNAs. Detailed inspection of these differences revealed that they are mainly due to single nucleotide substitutions, insertions and deletions. In several cases also longer insertions were found, two of which are particularly interesting. Their location in loops resembles tRNA introns. One of these insertions is 79 nucleotides long and can potentially form a tRNA-like structure, with 50% homology to plant tRNAGly. This observation led us to propose that, in the maturation of some 5S rDNA transcripts, editing and splicing mechanisms may be involved.
We have cloned and sequenced glutamate-tRNA synthetase (GluRS) and glutaminyl-tRNA synthetase (GlnRS) from Arabidopsis thaliana. They have conservative motifs found in all known GlxRS genes. For Lupinus luteus we found only one gene of GlxRS. At the moment we do not know exactly, whether it corresponds to GlnRS or GluRS.
Eukaryotic transcription factor IIIA (TF IIIA) is know to activate specifically transcription of 5S rRNA gene. It also interacts with mature 5S rRNA. The best known TF IIIA has been purified from Xenopus laevis. Up to know this protein has not been isolated from plants. In this paper we show for the first time purification and properties of TF IIIA like protein from tulip (Tulipa whittalli). It shows 5S rRNA and 5S rRNA gene binding activity.
We studied interactions between various domains of transcriptional factor IIIA (TFIIIA) from Xenopus laevis containing variable number of zinc finger with 5S ribosomal RNA (5S rRNA) from plants. The results clearly show that the complexes of peptide-RNA are formed in addition they strongly support that the tertiary structure of 5S rRNA is well conserved among eukaryotes. We optimalized conditions of complex formation reaction in order to study topography of these complexes.
In the presence and absence of methionine initiator tRNA (tRNA(iMet)) theophylline applied for 1 h starting 6 h after induction increased significantly (2-fold) interferon (IFN) yield. Theophylline did not change the kinetics of IFN production in the presence of tRNA(iMet). The shut-off stage was present and the production was prolonged to 24 h after IFN induction. In comparison with the values obtained in untreated cells, poly I:C plus DEAE dextran (inducing complex) caused an about 2-2.5 fold increase of cyclic adenosine 3'5'-monophosphate (cAMP) level. The similar results were observed in the cells treated with inducing complex plus tRNA(iMet). About 2-fold higher cAMP level occurred in the cells under the influence of the inducing complex plus theophylline in comparison to the level obtained without theophylline. The addition of tRNA(iMet) did not influence the above presented data.
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