Peptidyl transferase activity in wheat germ ribosomes. Effect of some antibiotics.

The formation of N-acetyl-leucyl-puromycin in a "fragment reaction" catalyzed by 80 S ribosomes from wheat germ was characterized. The reaction product was identified by high-voltage electrophoresis. The fragment reaction is inhibited by sparsomycin, blasticidin S, gougerotin and to a lesser degree by amicetin and tetracycline. Formation of an acLeu-pentanucleotide-ribosomes complex was strongly stimulated by sparsomycin.

Cloning and characterization of a nodule-enhanced glutamine synthetase-encoding gene from Lupinus luteus.

Glutamine synthetase (GS)-encoding genes in Lupinus luteus constitute a small family of genes showing different expression patterns [Boron et al., Acta Biochim. Polon. 36 (1989) 295-301]. One member of this family, the LlNGS1 gene, is strongly induced in root nodules close to the onset of nitrogen fixation and is referred to as a nodule-enhanced GS gene. We present here the structure of the nodule-enhanced LlNGS1 gene, the first gene of this class which has been sequenced. LlNGS1 is composed of twelve exons and shows structural similarity to the GS gene from Medicago sativa, indicating structure conservation of GS genes in legumes. Comparison of protein coding regions, as well as 5'-untranslated regions derived from LlNGS1 and a Lupinus angustifolius pGS5 GS cDNA clone [Grant et al., Plant Mol. Biol. 13 (1989) 481-490], revealed a high degree of shared identity between both genes, indicating that they are orthologous. The sequence of the LlNGS1 5'-flanking region (2.3 kb) contains several elements implicated in regulation of nodulin genes, as well as other characteristic DNA motifs. RNA blot hybridization analysis carried out using a probe corresponding to the LlNGS1 3'-untranslated region revealed that this gene is also transcribed in leaves, but at a barely detectable level.

Codon context effect in virus translational readthrough. A study in vitro of the determinants of TMV and Mo-MuLV amber suppression.

To assess the role of codon context on the efficiency of eukaryotic suppression of termination codons, we have compared, in a rabbit cell-free translation system, the readthrough efficiency related to two synthetic transcripts differing by the codon context around an amber codon. The codon contexts are derived from tobacco mosaic virus (TMV) and Moloney murine leukemia virus (Mo-MuLV) RNAs. The Mo-MuLV-like codon context does not promote suppression. Substituting TMV-derived triplets in the Mo-MuLV-like codon context shows that the two codons downstream from the TMV UAG signal are important determinants of suppression, as recently demonstrated in vivo.

The isolation of lupine cDNA clone coding for putative cyclin protein.

The complex of p34cdc2 protein kinase and cyclin is a key regulator of eukaryotic cell division and has been mostly investigated in yeast and animals. We have isolated a cDNA clone corresponding to cyclin from yellow lupine (Lupinus luteus). The cDNA clone CycBla-ll is 1692 bp in length and encodes a protein of predicted molecular mass 48.7 kDa. The lupine cyclin-like clone contains two domains: destruction and cyclin box, responsible for the function during the cell cycle. The amino acid comparison of CycBla-ll with conserved regions of other cyclins indicates that the lupine cDNA sequence represents mitotic cyclin of type B.

Isolation and characterization of root nodule proteins from lupin.

A group of root nodule-specific plant proteins (nodulins) has been isolated from yellow lupin (Lupinus luteus) by immunoaffinity chromatography. The cytoplasmic nodule protein extract was initially enriched in nodulins on a column with immobilized IgG fraction. It was then purified by chromatography on Sepharose 4B - bound IgG against uninfected root proteins and finally on Sepharose 4B - bound IgG against Rhizobium lupini proteins. Rocket immunoelectrophoresis showed that the nodulin preparation did not react with antibodies against root or bacterial proteins. SDS gel electrophoresis of lupin nodulins revealed at least 23 polypeptides ranging in Mr, from 7,000 to 70,000, probably representing protein subunits.

An efficient method of genomic DNA isolation from plant tissues.

The manuscript describes an easy method of isolation of plant genomic DNA. This method allowed us to isolate substantial amounts of good quality DNA from lupin (Lupinus luteus) tissues. The described method also appeared to be useful for genomic DNA isolation from tissues of other plants.

Control of protein synthesis in a wheat germ cell-free system.

Thermostable low molecular weight translational inhibitor was found in wheat germ cell-free extract. The inhibitor was formed during preincubation of wheat S-23 fraction with components of the energy-supplying system (ATP, GTP, phosphoenolpyruvate) in the absence of exogenous mRNA. The inhibitor effectively and irreversibly blocks protein synthesis in both wheat germ and rabbit reticulocyte systems. Our results seem to suggest that the inhibitor can activate wheat endogenous mRNA, which under the standard conditions does not reveal template activity but, once activated, can effectively compete with exogenous mRNA.

Isolationand in vitro translation of leghaemoglobin mRNA from yellow lupin root nodules.

Messenger RNA for leghaemoglobin, extracted from polysomes of yellow lupin root nodules, was purified by chromatography on oligo(dT1-cellulose followed by sucrose-gradient centrifugation. Leghaemoglobin mRNA activity was assayed in a cell-free protein synthesizing system derived from wheat germ. The purified mRNA was sedimented in surcrose gradient in the 9S region which corresponds to a molecular weight of about 225,000. The poly(A) segment was estimated to be 66 nucleotides in length, based on hybridization with [3H]poly(U). Analysis of the translation product using immunoprecipitation and polyacrylamide-gel electrophoresis in denaturing conditions revealed that the product made in vitro was identical with authentic leghaemoglobin.

Translation in vitro of ribonucleic acids from potato virus X, potato virus M and white clover mosaic virus.

Ribonucleic acids from three plant viruses: potato virus X (PVX), potato virus M (PVM) and white clover mosaic virus (WClMV) have been purified and used as messengers in a wheat germ cell-free system. It was demonstrated that each of these viruses contained heavy genomic RNA species of mol. wt. about 2 x 10(6), which appeared to be very efficient templates for protein synthesis in vitro. The synthesis of high-molecular-weight polypeptides: 180 x 10(3), 190 x 10(3) and 170 x 10(3) was directed by PVX, PVM and WClMV RNAs, respectively. None of the polypeptides directed by any of the RNAs studied corresponded to the coat proteins.

Translation and characterization of poly(A)-lacking RNA from lupin root nodules.

Total polysomal RNA from yellow lupin root nodules was fractionated by double oligo(dT)-cellulose chromatography. Poly(A)-containing and poly(A)-lacking RNA fractions showed considerable messenger activity in wheat germ and rabbit reticulocyte cell-free systems. The sizing of poly(A)-lacking RNA on sucrose-density gradient gives rise to separation of 14S mRNA from 22-24S mRNA species. A single polypeptide with molecular weight of 22,000 was coded for by 14S mRNA, while two polypeptides with an apparent mol. wt. of 90,000 and 87,000 were the main products of 22-24S mRNA fraction. High concentrations of unfractionated poly(A)-lacking RNA as well as the addition of poly(A) led to preferential synthesis of the 22,000 product. Preliminary results suggest the presence of m7GpppX cap structure at 5' terminus of the separated 14S and 22-24S mRNA species. This comes from the competition experiments with m7GMP and m7GTP as well as from the fact that the poly(A)-lacking RNA preparation was susceptible to methylation by methyl-transferase from vaccinia virus (methylated is the 2'-O-nucleotide adjacent to 7-methylguanosine). Digestion by T1 RNAase of methylated poly(A)-lacking RNA produced two short 5'-terminal oligonucleotides 10 and 17 nucleotides in length.

Two plant signalling peptides: systemin and ENOD 40.

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.

Reduction of bacterial genome size and expansion resulting from obligate intracellular lifestyle and adaptation to soil habitat.

Prokaryotic organisms are exposed in the course of evolution to various impacts, resulting often in drastic changes of their genome size. Depending on circumstances, the same lineage may diverge into species having substantially reduced genomes, or such whose genomes have undergone considerable enlargement. Genome reduction is a consequence of obligate intracellular lifestyle rendering numerous genes expendable. Another consequence of intracellular lifestyle is reduction of effective population size and limited possibility of gene acquirement via lateral transfer. This causes a state of relaxed selection resulting in accumulation of mildly deleterious mutations that can not be corrected by recombination with the wild type copy. Thus, gene loss is usually irreversible. Additionally, constant environment of the eukaryotic cell renders that some bacterial genes involved in DNA repair are expandable. The loss of these genes is a probable cause of mutational bias resulting in a high A+T content. While causes of genome reduction are rather indisputable, those resulting in genome expansion seem to be less obvious. Presumably, the genome enlargement is an indirect consequence of adaptation to changing environmental conditions and requires the acquisition and integration of numerous genes. It seems that the need for a great number of capabilities is common among soil bacteria irrespective of their phylogenetic relationship. However, this would not be possible if soil bacteria lacked indigenous abilities to exchange and accumulate genetic information. The latter are considerably facilitated when housekeeping genes are physically separated from adaptive loci which are useful only in certain circumstances.

Nodule-specific repression of yellow lupin protein R18.

It was found, using immunochemical techniques, that protein R18 presumably specifically repressed during development of lupin root nodules (Sikorski et al., 1989, Acta Biochim. Polon., 36, 63-72) is present in various tissues of this plant. Protein R18 was also detected in roots of four other legumes but was absent from the bacteroid-containing cells of root nodules. The data support our earlier view (Sikorski et al., op. cit.) that expression of protein R18 is regulated by the coupled mechanism of induction and repression of specific plant genes during development of lupin root nodule.

Reverse transcription and kinetic complexity analysis of mRNA from lupin root nodules.

Complementary DNA (cDNA) synthesis was performed using mRNA from lupin root nodules and from lupin uninfected roots as templates. Optimal conditions for the synthesis of 700 nucleotides long cDNA were established. Hybridization kinetics of mRNA-cDNA were performed in a homologous system from root nodules as well as in a heterologous system. Hybridization analysis revealed the presence of four frequency abundance classes within the root nodule mRNA population. In order to enrich the cDNA population in leghaemoglobin sequences, two synthetic pentadecanucleotide fragments complementary to the putative 3' ends of leghaemoglobin mRNAs were used as primers for the reverse transcription of nodule poly(A)-RNA. The resultant cDNA was enriched in rapidly hybridizing components which may contain leghaemoglobin sequences.

Identification by affinity chromatography of wheat germ ribosomal proteins which interact with RNA.

Wheat germ ribosomal proteins that bind to poly(U), tRNA and poly(A)-containing mRNA for leghaemoglobin were identified by affinity chromatography. The ribosomal proteins that associate with polynucleotides immobilized on Sepharose or on cellulose were identified by polyacrylamide gel electrophoresis. Nine 40S and nine 60S ribosomal proteins were bound to poly(U)-Sepharose. tRNA-Sepharose 4B interacted with five proteins of 40S subunit and with seven proteins of 60S subunit. Six proteins of 60S subunit were bound to poly(A)-containing mRNA for leghaemoglobin. The ribosomal proteins from the small subparticle were not determined because of irreversible binding to the column.

Structure of yellow lupine genes coding for mitotic cyclins.

Cell cycle progression in eukaryotes is controlled by complexes of p34 protein kinases and cyclins. For the first time in plants, we have established the sequence of four yellow lupine mitotic cyclin B1 genes. Their coding regions and expression pattern were also characterised recently. Structure of all the four lupine genes is similar: they consist of nine exons and eight introns, analogously located, except Luplu;CycB1;3 lacking 7th intron. Analysis of 5'-regulatory sequences of two of them showed that both comprise M-specific activators (MSA), common to plant genes induced in late G2 and early M. Putative repressor binding sites CDE/CHR found in animal G2-specific promoters can also be detected in lupine genes. Controlling region of Luplu;CycB1;4 gene that is highly activated by IAA, contains up to 7 auxin response elements, while insensible to IAA Luplu;CycB1;4 gene have no such motifs. Further studies should be undertaken to determine precisely the functions of putative regulatory elements in the expression of lupine mitotic cyclins.

Identification and synthesis in vitro of plant-specific proteins in yellow lupin root nodules.

Fifteen to twenty specific polypeptides of Mr ranging from 15 000 to 90 000 were detected using immunochemical techniques in the lupin root nodules and cell-free translation products of the nodule polysomal RNA. These polypeptides were characteristic for symbiotic state of the host plant and represented 9-18% of total nodule proteins. They were absent in uninfected roots and in protein extracts of Rhizobium lupini.

Cloning and identification of lupin nodule specific cDNA sequences.

Yellow lupin nodule specific sequences were selected by screening of cDNA library prepared from lupin nodule poly(A)+RNA. From about 3,000 clones containing fragments of lupin DNA 150-1,500 base pair long, 7% of clones carrying nodule specific sequences were identified. Among them the most abundant sequence species, represented by 32% clones, encodes leghemoglobin. Another abundant species designated pLN13 is represented by 13% clones. The Northern blot analysis of lupin mRNA confirmed nodule specificity of the cloned sequences. The nucleotide sequence of one clone, pLN281 of 225 bp, is presented.

Glutamine synthetase in Lupinus luteus. Identification and preliminary characterization of nodule-specific cDNA clone.

Two glutamine synthetase (GS) cDNA clones from L. luteus were identified and characterized. The nucleotide sequence analysis proved that they represent highly homologous but distinct mRNA species. Northern blot hybridization revealed that pc LINGS encodes the nodule-specific subunit of the GS while pcLIGS1 represents the nonspecific one present in nodule tissue as well as in uninfected roots.

Isolation and classification of a family of cyclin gene homologues in Lupinus luteus.

The lupine (Lupinus luteus cv. Ventus) cDNA clones encoding homologues of cyclin (CycB1;2, CycB1;3, CycB1;4) have been isolated from cDNA library prepared from roots inoculated with Bradyrhizobium lupini. Comparison of the deduced amino-acid sequences of CycB1;2, CycB1;3, CycB1;4 and previously described CycB1;1 (Deckert et al. 1996, Biochimie 78, 90-94) showed that they share 46-65% of identical amino acids. The presence of conserved residues (Renaudin et. al., in The Plant Cell Cycle, in the press; Renaudin et al., Plant Mol. Biol, in the press) along with phylogenetic analysis of known plant cyclins revealed that the four lupine sequences belong to subgroup 1 of B-like mitotic cyclins.