Transgene silencing may be mediated by aberrant sense promoter sequence transcripts generated from cryptic promoters.

To investigate whether silencing of a T-DNA-carried nptII gene in five single-copy transgenic Arabidopsis lines might be due to position effects, genomic DNA flanking the insertions was analysed for gene density, GC content, presence of short repeats and transposable elements, i.e. factors suggested to promote silencing. No single, common factor could explain the observed silencing. However, in two lines, a transcript covering the nos promoter driving the nptII gene was detected. In sibling sublines with approximately 100% silencing, the nos promoter was heavily methylated. In silico analysis suggested the presence of cryptic core promoters upstream of the nos promoter, in one case in the plant DNA and in the other in a short inverted TDNA region. These fragments were able to drive reporter gene expression in transgenic Arabidopsis plants. Our results indicate that methylation and silencing of transgenic promoters may be mediated by aberrant RNA transcribed from cryptic promoters at the transgene insertion site.

The Arabidopsis thaliana genome contains at least 29 active genes encoding SET domain proteins that can be assigned to four evolutionarily conserved classes.

SET domains are conserved amino acid motifs present in chromosomal proteins that function in epigenetic control of gene expression. These proteins can be divided into four classes as typified by their Drosophila members E(Z), TRX, ASH1 and SU(VAR)3-9. Homologs of all four classes have been identified in yeast and mammals, but not in plants. A BLASTP screening of the Arabidopsis genome identified 37 genes: three E(z) homologs, five trx homologs, four ash1 homologs and 15 genes similar to Su(var)3-9. Seven genes were assigned as trx-related and three as ash1-related. Only four genes have been described previously. Our classification is based on the characteristics of the SET domains, cysteine-rich regions and additional conserved domains, including a novel YGD domain. RT-PCR analysis, cDNA cloning and matching ESTs show that at least 29 of the genes are active in diverse tissues. The high number of SET domain genes, possibly involved in epigenetic control of gene activity during plant development, can partly be explained by extensive genome duplication in Arabidopsis. Additionally, the lack of introns in the coding region of eight SU(VAR)3-9 class genes indicates evolution of new genes by retrotransposition. The identification of putative nuclear localization signals and AT-hooks in many of the proteins supports an anticipated nuclear localization, which was demonstrated for selected proteins.

Stability of barley aleurone transcripts: Dependence on protein synthesis, influence of the starchy endosperm and destabilization by GA3.

We have studied the stability of Barley aleurone and embryo expressed (Balem) transcripts in aleurone layers. The Per1, Ole1 and Ole2 transcripts are abundant during desiccation and in dry resting seeds, while B12D and B22E transcripts are expressed mainly during seed maturation and germination. From 21 to 40 days post anthesis (DPA) incubation of aleurone layers resulted in a substantial, but differential reduction in the levels of these transcripts. In contrast, Balem transcript levels in aleurone layers of incubated embryoless grains were (except for B22E) similar to those of freshly dissected layers. Cycloheximide lowered transcript levels significantly. This indicates that a protein-synthesis-dependent mRNA-stabilizing mechanism is active in the aleurone cells when attached to the starchy endosperm. At the onset of seed desiccation (40 DPA), half-lives of transcripts to be stored in the dry seed were up to several days longer than the half-life of B22E, which decreases during seed maturation. While the Per1, Ole1 and Ole2 transcript levels decline rapidly in the aleurone layers of mature, germinating seeds, the genes are actively transcribed and their transcripts highly stable in the aleurone of incubated embryoless seeds. The expression of Ole1 and Ole2, as well as Per1, can be repressed 100-1 000-fold by gibberellic acid (GA3) in a dose-dependent manner. Abscisic acid can counteract the GA3 repression. Incubations with transcriptional and translational inhibitors indicate that GA3 inhibits the transcription of these genes and at the same time induces a protein-synthesis-dependent mechanism destabilizing their mRNA molecules present.

The frequency of silencing in Arabidopsis thaliana varies highly between progeny of siblings and can be influenced by environmental factors.

In a collection of 111 transgenic Arabidopsis thaliana lines, silencing of the nptII gene was observed in 62 (56%) of the lines and three distinct nptII-silencing phenotypes were identified. Two T-DNA constructs were used, which differed in distance and orientation of the marker gene relative to the border sequences. Comparison of the sets of lines generated with each vector, indicate that the T-DNA construct configuration influence the incidence of lines displaying silencing, as well as the distribution of silencing phenotypes. Twenty lines were investigated more thoroughly. The frequency of silencing varied between siblings in 19 lines, including three lines containing a single T-DNA copy. The last line showed 100% silencing. The gus gene present in both constructs could be expressed in the presence of a silenced nptII gene. Investigation of methylation at a single site in the pnos promoter revealed partial methylation in multi-copy lines, but no methylation in single-copy lines. For 16 lines, the overall frequencies of silencing differed significantly between control plants and plants exposed to temperature stress; in 11 of these lines at the 0.1% level. In several cases, the frequency of silencing in progeny of stress-treated plants was higher than for the control group, while other lines showed higher frequencies of kanamycin-resistant progeny for the stress-treated sibling plants.

The dormancy-related peroxiredoxin anti-oxidant, PER1, is localized to the nucleus of barley embryo and aleurone cells.

Protection against desiccation-induced injury, including damage by reactive oxygen species (ROS), is a necessary component of the genetic programmes active during late seed development. Likewise, protection against ROS respiration by-products is required during seed imbibition and germination. Late embryogenesis abundant (LEA) proteins are proposed to protect seed tissues against desiccation-induced damage. Specifically, the atypical Lea gene Per1 in barley (Hordeum vulgare L.) has been proposed to play a protective role in embryo and aleurone cells against free-radical damage during late seed development and early imbibition. PER1 represents a subgroup of the peroxiredoxin family of thiol-requiring anti-oxidants with one conserved cysteine residue (1-Cys), and displays in vitro anti-oxidant activity. In this work, we use antiserum generated against PER1 to study protein accumulation patterns as well as localization at the tissue, cellular and subcellular level. While previous studies have shown the Per1 transcript to be dormancy-related, we show here that the protein level is maintained in imbibed dormant seeds, but not in non-dormant seeds. Our data identify the location of this seed-specific peroxiredoxin as the nucleus of immature embryos and aleurone layers. Highest levels of protein are detected in nucleoli. In contrast, in mature imbibed dormant seeds, cytosolic levels are comparable to that of the nucleus. A putative nuclear localization signal (NLS) of bipartite nature was identified in the C-terminal end of the PER1 sequence. Protective roles for PER1 in seeds are discussed.

Identification of sequence homology between the internal hydrophilic repeated motifs of group 1 late-embryogenesis-abundant proteins in plants and hydrophilic repeats of the general stress protein GsiB of Bacillus subtilis.

Late embryogenesis abundant (LEA) proteins are speculated to protect against water stress in plants. Group 1 LEA proteins are hydrophilic and vary mainly in the numbers of an extremely hydrophilic internal 20-amino-acid motif. This motif is present up to four times in Arabidopsis thaliana and Hordeum vulgare Group 1 proteins and has been described in numerous plant species. However, no similarity has yet been described between Group 1 genes or gene products and those from non-plant species. We report here the striking similarity between the repeated internal motif of Group 1 LEA proteins and a repeated hydrophilic motif present in a stress-related protein (GsiB) from Bacillus subtilis.

The expression of a peroxiredoxin antioxidant gene, AtPer1, in Arabidopsis thaliana is seed-specific and related to dormancy.

We have isolated a gene, AtPer1, from the dicotyledon Arabidopsis thaliana, which shows similarity to the 1-cysteine (1-Cys) peroxiredoxin family of antioxidants. In higher plants, members of this group of antioxidants have previously only been isolated from monocotyledons. It has been suggested that seed peroxiredoxins protect tissues from reactive oxygen species during desiccation and early imbibition and/or are involved in the maintenance of/protection during dormancy. AtPer1 expression is restricted to seeds. Despite differences in seed development between monocots and dicots, AtPer1 shows an expression pattern during seed development and germination similar to the dormancy-related transcript Per1 in barley. In situ hybridization identifies AtPer1 as the first aleurone-expressed transcript characterized in developing Arabidopsis seeds. The transcript is also expressed in the embryo. AtPer1 expression in seeds is unaltered in an ABA-deficient mutant (aba-1) during seed development, while expression in seeds of an ABA-insensitive mutant (abi3-1) is reduced. The transcript is not induced in vegetative tissue in response to stress by ABA or drought. AtPer1 transcript levels are correlated to germination frequencies of wildtype seeds, but AtPer1 transcript abundance is not sufficient for expression of dormancy in non-dormant mutants. Hypotheses on peroxiredoxin function are discussed in view of the results presented here.

A peroxiredoxin antioxidant is encoded by a dormancy-related gene, Per1, expressed during late development in the aleurone and embryo of barley grains.

Antioxidants can remove damaging reactive oxygen species produced as by-products of desiccation and respiration during late embryogenesis, imbibition of dormant seeds and germination. We have expressed a protein, PER1, encoded by the Balem (barley aleurone and embryo) transcript previously called B15C, and show it to reduce oxidative damage in vitro. PER1 shares high similarity to a novel group of thiol-requiring antioxidants, named peroxiredoxins, and represents a subgroup with only one conserved cysteine residue (1-Cys). PER1 is the first antioxidant belonging to the 1-Cys subgroup shown to be functionally active, and the first peroxiredoxin of any kind to be functionally described in plants. The steady state level of the transcript, Per1, homologous to a dormancy-related transcript (pBS128) from bromegrass (Bromus secalinus), increases considerably in imbibed embryos from dormant barley (Hordeum vulgare L.) grains. Our investigations also indicate that Per1 transcript levels are dormancy-related in the aleurone layer of whole grains. In contrast to most seed-expressed antioxidants Per1 disappears in germinating embryos, and in the mature aleurone the transcript is down-regulated by the germinating embryo or by gibberellic acid (GA). Our data show that the barley seed peroxiredoxin is encoded by a single Per1 gene. Possible roles of the PER1 peroxiredoxin in barley grains during desiccation, dormancy and imbibition are discussed.

The transcripts encoding two oleosin isoforms are both present in the aleurone and in the embryo of barley (Hordeum vulgare L.) seeds.

Two transcripts (Ole-1 and Ole-2) encoding two oleosin isoforms homologous to the 18 and 16 kDa oleosins of maize, respectively, have been isolated from developing barley embryos and aleurone layers where lipid bodies are highly abundant organelles. For each of the isoforms the aleurone and embryo transcripts are identical, indicating that the same genes are expressed in both tissues. The temporal accumulation of the two transcripts during seed development is similar. At a low frequency, lipid bodies are found also in starchy endosperm cells of barley. Accordingly, a low transcript level is observed for both oleosins during starchy endosperm development.

Transcripts encoding an oleosin and a dormancy-related protein are present in both the aleurone layer and the embryo of developing barley (Hordeum vulgare L.) seeds.

In cereal seeds, the aleurone layer and the embryo share several characteristics, including synthesis and accumulation of lipid bodies, desiccation tolerance and dormancy. A number of Balem transcripts present in both the barley aleurone layer and the embryo have been cloned by differential screening of a cDNA library from aleurone layers of immature barley grains. The Balem clones constitute two subgroups, one for which the transcripts are detectable in aleurone layers and embryos of developing seeds only (B23D and B15C), and another for which transcripts are present also in germinating embryos and in maternal tissues (B12D, B14E and B31E). Sequence analysis identified B23D and B15C as the barley homologues of the 18 kDa oleosin of maize embryos (72% amino acid identity) and the dormancy-associated transcript pBS128 from Bromus secalinus (95% identity), respectively. In situ hybridization experiments demonstrate that in the embryo, the B23D transcript is mainly present in the scutellum, whereas the B15C transcript is predominantly present in shoot and root apices. Using anther-derived embryos and embryogenic cell suspensions, it is demonstrated that the B23D and B15C transcripts can be used as molecular markers for somatic embryogenesis. The functions of the transcripts in the second Balem subgroup remain unknown. Further studies on the Balem transcripts may shed light on the molecular basis for the extensive similarities between the embryo and the aleurone layer of the endosperm in the grass family.

Primary structure of a novel barley gene differentially expressed in immature aleurone layers.

As a direct approach to elucidate the molecular biology of barley aleurone cell development, we differentially screened an aleurone cDNA library made from poly(A)+ RNA of immature grains for clones representing transcripts present in the aleurone but not in the starchy endosperm. For one of these clones, B22E, which hybridies to a 0.7 kb transcript, Northern and in situ hybridization revealed that expression is under complex spatial, temporal and hormonal control in barley grains. cDNAs corresponding to B22E transcripts were isolated from aleurone/pericarp and embryo of developing grains, and from germinating scutella. Among these were the nearly full-length aleurone/pericarp clone pB22E.a16 (541 bp). cDNAs matching the sequence of this clone (type 1 transcript) were found for all tissues investigated. In addition, cDNAs with an extra 12 bp insertion (type 2 transcript) were obtained from germinating scutella. The two different transcripts can encode novel barley proteins of 115 and 119 amino acids, respectively. A gene designated B22EL8 was isolated and sequenced; it encodes the type 1 B22E transcript and contains two introns of 145 and 125 bp. Particle bombardment of barley aleurone with a B22EL8 promoter-GUS (beta-glucuronidase) construct demonstrates that the promoter (3 kb) is active in developing barley grains. The promoter is not, however, active in the seeds of tobacco plants transgenic for the B22EL8 gene, indicating the existence of sequences specific for monocots. A comparison of 1.4 kb of upstream sequence of B22E with the maize c1 promoter reveals a number of short, identical sequences which may be responsible for aleurone cell-specific gene transcription.

Cell-autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assay for transposon excision in transgenic plants.

We describe a genetic switch based on the Ac transposable element of maize and the rolC gene of Agrobacterium rhizogenes, a dominant gene, which has pleiotropic effects on plant growth and morphology. Moreover, rolC gene expression under the control of the 35S cauliflower mosaic virus promoter decreases chlorophyll content in transgenic tobacco plants. Chlorophyll is a visible cell-autonomous marker, and it is shown here that the reduction in chlorophyll content caused by the rolC gene product allows us to monitor, in palisade or spongy mesophyll cells, Ac excision events resulting in rolC gene expression as pale-green sectors and spots. Our results indicate that the rolC gene product behaves in a cell-autonomous manner during leaf development, at least as far as chlorophyll accumulation is concerned. In addition, the rolC gene can be useful to evaluate visually if and when a transposable element is active. Most important, we propose the use of a transposable element as a tool to activate expression of morphogenetic genes in a clonal population of cells. This could be particularly useful when studying genes affecting growth and development whose constitutive expression can severely impair regeneration of transgenic plants.

Subcellular localization of proteins encoded by the phenotypically cryptic plasmid of Neisseria gonorrhoeae: biological evidence for outer membrane association of the cppB gene product.

Almost all clinical isolates of Neisseria gonorrhoeae harbour a plasmid of 4.2 kb with no known function. A genetic model based on the DNA sequence of the plasmid, with ten open reading frames, has been proposed by Korch et al., (1985). To address the question of the function of the encoded proteins, some of which are expressed when the plasmid is harboured by Escherichia coli, the subcellular locations of such proteins were investigated in minicells of Escherichia coli DS410. The protein CppB, earlier proposed to be a membrane-spanning polypeptide, was found associated with the outer membrane. Up to five other cryptic plasmid proteins were found to be localized in the periplasm.

Barley aleurone cell development: molecular cloning of aleurone-specific cDNAs from immature grains.

The cloning of 11 different homology groups of cDNAs representing genes expressed in aleurone, but not in starchy endosperm of 20-day-old barley grains is described. Among the cDNAs, four are aleurone-specific, while the remaining are also expressed in the embryo, but not in any other part of the plant.Sequence analysis of one of the aleurone-specific clones, B11E, reveals an open reading frame coding for an unidentified 10.4 kDa protein with a putative signal sequence and a possible metal-binding finger. The B11E gene has a high GC content in the 5' leader sequence (63%), as well as in the coding region (70%) compared to known cDNAs from the barley starchy endosperm. Northern analysis of B11E indicates maximum mRNA abundance around mid-phase of grain development.When isolated immature aleurone/pericarp is incubated in tissue culture medium (MS) the B11E message disappears, indicating a requirement for a diffusible factor from the intact grain for its continued presence.

Molecular characterization and comparison of plasmid content in seven different strains of Neisseria gonorrhoeae.

The plasmid content of one penicillin sensitive and six penicillin resistant strains of Neisseria gonorrhoeae has been examined. All strains harbour a small, phenotypically cryptic plasmid of 4.1 kilo base pairs (kb). Four of the penicillin resistant strains carry a beta-lactamase-producing plasmid of 7.3 kb. One of these also carries a large plasmid of about 40 kb. The two remaining penicillin resistant strains harbour a smaller beta-lactamase-producing plasmid of 5.5 kb. The plasmids have been subjected to digestion with a number of restriction endonucleases, and their restriction maps have been compared. Judging by the maps, the cryptic (C-) plasmids show great similarities. Except that two of them have 54 additional base-pairs (bp), and two have a HpaII site instead of a DdeI site, no differences were found. The larger beta-lactamase-producing (B-) plasmids have identical maps. The smaller seem to be homologous with the larger, except for a deletion of 1.8 kb. There is no correlation between the variant of C-plasmid and type of B-plasmid harboured in the penicillin resistant strains. The evolutionary implications suggested by this finding are discussed.

Polypeptides encoded by cryptic plasmids from Neisseria gonorrhoeae.

Almost all clinical isolates of Neisseria gonorrhoeae harbor a small, phenotypically cryptic plasmid of approximately 4.1 kb. In this study several polypeptides encoded by two variants of such plasmids, one (pSB01C) having a deletion of approximately 50 bp as compared to the other (p31788C), have been identified, and the position of the genes for two of the proteins determined. The cryptic plasmids were cloned into the HindIII site of the vectors pBR322 and pACYC184. The resulting recombinant plasmids were transformed into the Escherichia coli minicell producing strain DS410 (minA, minB) and the plasmid-encoded proteins analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pSB01C derivatives express two distinct proteins of 22 and 16 kDa and p31788C two other proteins of 24 and 18.5 kDa. Additionally, both plasmids express common proteins of 32.5, 9, and 7.5 kDa. The genes coding for the 24- and the 7.5 kDa proteins have been mapped by restriction enzyme analysis of Tn5 insertions suppressing the expression. The additional 50 bp in p31788C are localized to the coding region of the 24-kDa protein, and the 22-kDa protein of pSB01C is possibly a shortened form of the former due to the lacking 50 bp.