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.

Characterization and expression analysis of the yellow lupin (Lupinus luteus L.) gene coding for nodule specific proline-rich protein.

The LlPRP2 gene coding for a proline-rich protein shows a high level of similarity to, as well as significant differences from the family of ENOD2 nodule-specific genes. Several sequence motifs with putative regulatory function were identified in the 5' and 3' noncoding regions of the LlPRP2 gene. Northern blot analysis revealed that the expression of the LlPRP2 gene begins 9 days after inoculation of yellow lupin roots with Bradyrhizobium sp. (Lupinus); the expression is restricted to symbiotic nodules and is not detected in other tissues or organs. Detailed hybridization analysis showed that, when expression is activated, the LlPRP2 transcript is modified so as to produce at least three bands and a continuous distribution of decay intermediates. The modification of the LlPRP2 transcript probably involves degradation from the 5'- and/or 3'-ends of the RNA molecules. Southern blot analysis indicates that only one gene is present in the yellow lupin genome. The presence of genes homologous to the LlPRP2 gene was confirmed for three cultivars of yellow lupin and for Lupinus angustifolius. However, LlPRP2 homologues were not detected in Lupinus albus cv. Bac, indicating that this plant may lack the ENOD2 sequence.

Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues.

The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the "Class II" hemoglobins.

A plant-derived edible vaccine against hepatitis B virus.

The infectious hepatitis B virus represents 42 nm spherical double-shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein.

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.

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.

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.

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.

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.

In situ hybridization of two nodule-specific gene products from yellow lupin.

We have applied in situ hybridization technique to examine the expression of two tissue-specific plant genes represented by cDNA clones: pLN-13 and pLN-50 during the symbiosis of Lupinus luteus with Bradyrhizobium lupini. Both genes are expressed in root nodules and their transcripts are restricted to the infected zone of the nodule as revealed with 35S-or 3H-labelled antisense RNA probes. The number of grains over bacteroid-containing cells after hybridization with 3H-labelled probes differed during three tested phases of nodule development: at the 14th, 21st and 28th day after the infection with B. lupini. As control, apical root meristems and segments of root hair zones of uninfected plants were used for incubation with RNA of either sense or antisense orientation preceded by RNAse A digestion. The data obtained confirm that both genes are organ-specific and reveal their developmentally regulated expression pattern during the course of root nodule morphogenesis.

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.

Autoradiographic study of 3H-uridine incorporation into nucleoli and DNA/3H-5S DNA in situ hybridization in root nodules and uninfected Lupinus luteus L. tissues.

The autoradiographic method was used to compare the 3H-uridine incorporation and the number of hybridization sites with 3H-5S DNA in yellow lupin root apical meristem, root hair of uninfected roots as well as in root nodule cortex and bacteriod-containing tissue. It has been shown that the number of hybridization sites is proportional to the ploidy level, but not to rRNA synthesis, which is most intense in root apical meristem and young bacteriod-containing cells.

Localization of 5S DNA by in situ hybridization in metaphase chromosomes of Vicia faba L.

The tritium-labelled cloned 5S DNA from Lupinus luteus was used for localization of 5S RNA genes in Vicia faba subsp. minor metaphase chromosomes. In situ hybridization sites were found to be localized in chromosomes I and VI. In chromosome I the probe hybridized to the region adjoining NOR whereas in chromosome VI silver grains were found in the median part of the long arm. After prolonged exposure the autoradiographic grains expanded in the proximal part of that chromosome arm.

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.