The mistletoe lectin I--phloretamide structure reveals a new function of plant lectins.

The X-ray structure at 2.7A resolution of the complex between the European mistletoe lectin I (Viscum album, ML-I) and the plant growth hormone, 3-(p-hydroxyphenyl)-propionic acid amide (phloretamide, PA) from xylem sap has revealed the binding of PA at the so far undescribed hydrophobic cavity located between the two subunits of this ribosome-inhibiting protein. No such cavity is observed in related lectins. The binding of PA is achieved through interactions with the non-conserved residues Val228A, Leu230A, Arg388B, and the C-terminal Pro510B. It is conceivable that binding of PA to ML-I is part of a defence mechanism of the parasite against the host, whereby the parasite prevents the growth hormone of the host from interfering with its own regulatory system. The specific binding of PA to ML-I indicates that heterodimeric RIPs are multifunctional proteins whose functions in the cell have not yet been fully recognized and analyzed.

Effect of small molecules on cell reprogramming.

The essential idea of regenerative medicine is to fix or replace tissues or organs with alive and patient-specific implants. Pluripotent stem cells are able to indefinitely self-renew and differentiate into all cell types of the body which makes them a potent substantial player in regenerative medicine. The easily accessible source of induced pluripotent stem cells may allow obtaining and cultivating tissues in vitro. Reprogramming refers to regression of mature cells to its initial pluripotent state. One of the approaches affecting pluripotency is the usage of low molecular mass compounds that can modulate enzymes and receptors leading to the formation of pluripotent stem cells (iPSCs). It would be great to assess the general character of such compounds and reveal their new derivatives or modifications to increase the cell reprogramming efficiency. Many improvements in the methods of pluripotency induction have been made by various groups in order to limit the immunogenicity and tumorigenesis, increase the efficiency and accelerate the kinetics. Understanding the epigenetic changes during the cellular reprogramming process will extend the comprehension of stem cell biology and lead to potential therapeutic approaches. There are compounds which have been already proven to be or for now only putative inducers of the pluripotent state that may substitute for the classic reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc) in order to improve the time and efficiency of pluripotency induction. The effect of small molecules on gene expression is dosage-dependent and their application concentration needs to be strictly determined. In this review we analysed the role of small molecules in modulations leading to pluripotency induction, thereby contributing to our understanding of stem cell biology and uncovering the major mechanisms involved in that process.

Regulatory RNAs in mammals.

Recent years have brought a dramatic change in our understanding of the role of ribonucleic acids (RNAs) within the cell. In addition to the already well-known classes of RNAs that take part in the transmission of genetic information from DNA to proteins, a new highly heterogeneous group of RNA molecules has emerged. The regulatory nonprotein-coding RNAs (npcRNAs) have been shown to be involved in modulation of gene expression on both the transcriptional and post-transcriptional level. They participate in mechanisms of chromatin modification, regulation of transcription factor activity, and influencing mRNA stability, processing, and translation. npcRNAs are key factors in genetic imprinting, dosage compensation of X-chromosome-linked genes, and many processes of differentiation and development.

Aminoacyl-tRNA synthetases database.

Aminoacyl-tRNA synthetases (AARSs) are at the center of the question of the origin of life. They constitute a family of enzymes integrating the two levels of cellular organization: nucleic acids and proteins. AARSs arose early in evolution and are believed to be a group of ancient proteins. They are responsible for attaching amino acid residues to their cognate tRNA molecules, which is the first step in the protein synthesis. The role they play in a living cell is essential for the precise deciphering of the genetic code. The analysis of AARSs evolutionary history was not possible for a long time due to a lack of a sufficiently large number of their amino acid sequences. The emerging picture of synthetases' evolution is a result of recent achievements in genomics [Woese,C., Olsen,G.J., Ibba,M. and Söll,D. (2000) Microbiol. Mol. Biol. Rev., 64, 202-236]. In this paper we present a short introduction to the AARSs database. The updated database contains 1047 AARS primary structures from archaebacteria, eubacteria, mitochondria, chloroplasts and eukaryotic cells. It is the compilation of amino acid sequences of all AARSs known to date, which are available as separate entries via the WWW at http://biobases.ibch.poznan.pl/aars/.

The non-coding RNAs as riboregulators.

The non-coding RNAs database (http://biobases.ibch.poznan.pl/ncRNA/) contains currently available data on RNAs, which do not have long open reading frames and act as riboregulators. Non-coding RNAs are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing to control cell growth and differentiation. Some of them are connected with several well known developmental and neuro-behavioral disorders. We have divided them into four groups. This paper is a short introduction to the database and presents its latest, updated edition.

The primary structure of lupin seed 5.8 S ribosomal RNA.

The lack of colinearity between nucleotide sequence of the lupin 5.8 S rDNA gene (Rafalski, A.J., Wiewiórowski, M. and Soll, D. (1983) FEBS Lett. 152, 241-246) and 5.8 S rRNA of other plants (Erdmann, V.A. and Wolters, J. (1986) Nucleic Acids Res. 14, r1-r59.) prompted us to clarify this point by sequencing the native lupin 5.8 S rRNA. The sequence analysis was carried out using enzymatic and chemical methods. Lupin seed 5.8 S rRNA contains 164 nucleotides, including four modified ones: two residues of 2'-O-methylguanosine, one pseudouridine and one 2'-O-methyladenosine. The nucleotide sequence homology with the other plant 5.8 S rRNAs is approx. 88-96%.

The nucleotide sequence of 5S ribosomal RNA from the parasitic nematode Ascaris suum. Evolutionary relationships in nematodes.

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.

Compilation of ribosomal 5S ribonucleic acid nucleotide sequences: eukaryotic 5S rRNAs.

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 primary structure of six leucine isoacceptor tRNAs of yellow lupin seeds. The structural requirements for amber tRNA suppressor activity.

Six tRNA(Leu) isoacceptors from yellow lupin seeds were purified, sequenced, and their readthrough properties over the UAG stop codon were tested using TMV RNA as a messenger. The tested tRNAs(Leu) did not show amber suppressor activity. The partial structure of tRNA(Gln), a minor species in yellow lupin, was also determined. Comparison of the nucleotide sequence of all known isoacceptors of tRNA(Tyr), tRNA(Gln) and tRNA(Leu) from plants, mammals and ciliates enabled us to find general structural requirements for tRNA to be a UAG suppressor. From the partial sequence of lupin tRNA(Gln) we suggest that it will have readthrough properties.

Structural analysis of plant ribosomal 5S RNAs. Visualisation of novel tertiary interactions by cleavage of lupin and wheat 5SrRNAs with ribonuclease H.

A model for the tertiary structure of plant 5S rRNA, previously proposed by our laboratory (Joachimiak, A. et al. (1990) Int. J. Biol. Macromol., in press) was tested by specific cleavage of the plant 5S rRNA in the presence of synthetic oligodeoxynucleotides. The hexanucleotides used in this study were complementary to different portions of loops C, D and E, the nucleotides of which have recently been proposed to be involved in tertiary hydrogen bonds. The results obtained strongly support the interaction of loops C and D by nucleotides C34, C35, C36, A37 and G85, G86, G87, U88, respectively. Digestion pattern of loop E (domain gamma, nucleotides 66-110) suggests a possible different arrangement of this part of the plant 5S rRNA molecule, when compared with other eukaryotes.

B-Z DNA reversible conformation changes effected by high pressure.

There are numerous data showing that a DNA molecule with alternate pirymidine-purine sequence can adopt a left-handed, double-helical Z-DNA conformation. Such structural changes of DNA occur as a consequence of environmental conditions (e.g. 4 M NaCl) or chemical modification (e.g. methylation or bromination of bases). In this paper, we found for the first time that high pressure (several kilobars) can change the DNA conformation from the B to the Z form. When the pressure is reduced to an atmospheric one, DNA conformation returns back to the B-form. The Z-DNA structure formation was confirmed by circular dichroism (CD) and ultraviolet (UV) measurements. However, we found, that the values of the ratio of absorbance at the wavelengths 295 and 257 nm in the range of 0.3-0.4 is not a fully conclusive proof for the Z-DNA conformation. Although the ratio is typical for Z-DNA form, it is not obvious that the negative band in CD spectrum will be observed. On the other hand, methylated DNA does not undergo B----Z DNA transitions at the high pressure. These conformational changes of DNA molecules could be interpreted as the effect a of different hydration of various DNA forms.

Aminoacyl-tRNAs. Diversity before and unity after interaction with EF-Tu:GTP.

Evidence is presented for a new role for elongation factor EF-Tu. This involves conformational restraint or conformational selection of any aminoacyl-tRNA for channeling it to the ribosomal decoding site.

Chemical probes for tRNA tertiary structure. Comparative alkylation of tRNA with methylnitrosourea, ethylnitrosourea and dimethylsulfate.

The tertiary structure of tRNA in solution can be proved by chemical modification experiments. Three reagents, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea and dimethylsulfate which are known to alkylate nucleic acids at nucleophilic centers were compared. It is found that N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea mainly react with phosphate residues and dimethylsulfate only with the bases. With dimethylsulfate the extent of alkylation of guanosines is about one order of magnitude higher than that of the phosphates by the nitroso compounds.

Reduction in the amount of 8-hydroxy-2'-deoxyguanosine in the DNA of SV40-transformed human fibroblasts as compared with normal cells in culture.

DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age-related diseases including cancer. Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8-hydroxy-2'-deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently. Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold higher levels of oh8dG are detected in the DNA of cultured normal human skin fibroblasts as compared with SV40-transformed human fibroblasts MRC-5V2. In comparison, the levels of oh8dG were similar in the growth medium of both types of cells. Applications of this method range from studies on the genomic stability and instability of normal and cancerous cells to the clinical and laboratory testing of toxic substances and drugs.

Evidence for the presence of kinetin in DNA and cell extracts.

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.

New model of tertiary structure of plant 5S rRNA is confirmed by digestions with alpha-sarcin.

The cytotoxin alpha-sarcin was employed to test the model of secondary and tertiary structures of plant 5S rRNAs, which we recently proposed [(1990) Int. J. Biol. Macromol. (in press)]. alpha-Sarcin is a novel ribonuclease that hydrolyzes phosphodiester bonds adjacent to purines in nucleic acids. The digestion pattern obtained for lupin and wheat germ 5S rRNAs strongly suggests the existence of tertiary interactions between residues C34, C35, C36, A37 and G85, G86, G87, U88 as previously proposed. The results on the secondary structure of plant 5S rRNA are in line with a previously proposed model.

A mechanism for the in vivo formation of N6-furfuryladenine, kinetin, as a secondary oxidative damage product of DNA.

Recently, we have reported the presence of kinetin (N6-furfuryladenine) in commercially available DNA, in freshly extracted cellular DNA and in plant cell extracts. We have also found that kinetin has electrochemical properties which can be used for monitoring the level of this modified base in DNA. Here, for the first time, we propose a mechanism for the formation of kinetin in DNA in vivo, based on the analyses of its mass spectra. Since hydroxy radical oxidation at the carbon 5' of the deoxyribose residue yields furfural, we propose that this aldehyde reacts with the amino group of adenine and, after intramolecular rearrangement, kinetin is formed in vivo. Thus kinetin is the first stable secondary DNA damage product known to date with very well defined cytokinin and anti-aging properties, linked to oxidative processes in the cell. These results also indicate that N6-furfuryladenine or kinetin is an important component of a new salvage pathway of hydroxy radicals constituting a 'free radical sink'. In this way, the cells can neutralize the harmful properties of hydroxyl radical reaction products, such as furfural, and respond to oxidative stress by inducing defence mechanisms of maintenance and repair.

Dual hydrolytic role for Pb(II) ions.

RNA phosphodiester bonds can be cleaved by metal ions, of which Pb2+ is one of the most effective. It can cleave both generally and site-specifically, depending on the substrate and the conditions. In addition, metal ions are also known to cleave ester bonds between amino acid and the 3'-end of transfer RNA. Here we report that in aminoacylated transfer RNA, Pb2+ ions cleave internucleotide bonds in the 3'-end of tRNA and also cleaves the bond between tRNA and its amino-acid, attached at the 3'-end via an ester bond to the terminal ribose in aminoacyl tRNA. The two reactions proceed at different rates. The rate of deacylation is significantly faster than the rate of cleavage of phosphodiester bonds, with a pH-optimum of 7. This dual hydrolytic role is not seen for other metal ions examined, namely Zn(II), Cd(II) and Mn(II). The rate of the two kinds of hydrolyses by Pb2+ ions is compared with that of other metal-ions. The mechanism of cleavage is investigated further by modification of the 3'-end of tRNA.

The primary structure of wheat germ tRNAArg--the substrate for arginyl-tRNAArg:protein transferase.

Besides its major role in protein synthesis, wheat germ arginyl-tRNAArg can serve as an amino acid donor in an enzymatic reaction to bovine serum albumin catalysed by the enzyme arginyl-tRNAArg: protein transferase. The nucleotide sequence of the tRNAArg involved in this reaction was determined to be: pG-A-C-U-C-C-G-U-m1G-m2G-C-C-C-A-A-D-Gm-G-A-X-A-A-G-G-C-m2(2) G-C-U-G-G-U-Cm-U-I-C-G-m2A-A-A-C-C-A-G-A-G-A-D-U-m5C-U-G-G-G-T-psi -C-G-m1 A-U-C-C-C-C-A-G-C-G-G-A-G-U-C-G-C-C-AOH. We suggest that the decapentanucleotide 5'-G-U-Pu-m2G-C-N-C-A-A-D-Gm-G-A-X-A-3', localized in the D-region, interacts specifically with the protein transferase.

Conservation of plant cytoplasmic tRNA structure. Nucleotide sequence of rape tRNAPhe.

The primary structure of rape seeds tRNAPhe has been determined. It is identical to that of wheat germ, pea and barley tRNAsPhe, and of the minor tRNAPhe species of yellow lupin seeds.