Regulation of nodulin gene expression

The expression of plant genes specifically induced during rhizobial infection and the early stages of nodule ontogeny (early nodulin genes) and those induced in the mature, nitrogen-fixing nodule (late nodulin genes) is differentially regulated and tissue/cell specific. We have been interested in the signal transduction pathway responsible for symbiotic, temporal and spatial control of expression of an early (Enod2) and a late (Leghemoglobin;lb) nodulin gene from the stem-nodulated legumeSesbania rostrata, and in identifying thecis-acting elements andtrans-acting factors involved in this process (De Bruijn and Schell, 1992). By introducing chimericS. rostrata lb promoter-gus reporter gene fusions into transgenicLotus corniculatus plants, we have been able to show that thelb promoter directs an infected-cell-specific expression pattern inLotus nodules. We have been able to delimit thecis-acting element responsible for nodule-infected-cell-expression to a 78 pb region of thelb promoter (NICE Element) and have analyzed this element in detail by site-specific mutagenesis. We have studied the interaction of the NICE element, and further upstreamcis-acting elements, withtrans-acting factors of both plant- and rhizobial origin. We have obtained evidence for the involvement of rhizobial proteins in infected-cell-specific plant gene expression (Welters et al., 1993). We have purified one of the bacterial binding proteins from theS. rostrata symbiontAzorhizobium caulinodans (AcBBP1), and cloned and mutated the corresponding gene, in order to examine its symbiotic phenotype. We have also found that theS. rostrata Enod2 gene is rapidly induced by physiologically significant concentrations of cytokinins, suggesting the role of cytokinin as a potential secondary signal involved in nodulation (Dehio and De Bruijn, 1992). We are examining whether the observed cytokinin induction, as well as the nodule-specific expression pattern, are modulated by theSrEnod2 promoter.     

Primary structure and promoter analysis of leghemoglobin genes of the stem-nodulated tropical legume Sesbania rostrata: conserved coding sequences, cis-elements and trans-acting factors

Summary The primary structure of a leghemoglobin (lb) gene from the stem-nodulated, tropical legume Sesbania ostrata and two lb gene promoter regions was analysed. The S. rostrata lb gene structure and Lb amino acid composition were found to be highly conserved with previously described lb genes and Lb proteins. Distinct DNA elements were identified in the S. rostrata lb promoter regions, which share a high degree of homology with cis-active regulatory elements found in the soybean (Glycine max) lbc3 promoter. One conserved DNA element was found to interact specifically with an apparently universal, trans-acting factor present in nuclear extracts of nodules. These results suggest a conserved mechanism for nodule specific induction of lb genes in leguminous plants.     

Differential expression of the Sesbania rostrata leghemoglobin glb3 gene promoter in transgenic legume and non-legume plants

The involvement of the Sesbania rostrata glb3 gene promoter NICE (nodule-infected cell expression) element in root-enhanced expression of 5-Srglb3-uidA-3nos chimeric gene was investigated in transgenic Nicotiana tabacum plants. The full-length wild-type Srglb3 promoter directed root meristem-enhanced expression in transgenic tobacco plants. The expression pattern of nine selected Srglb3 promoter mutations in the NICE element was examined in transgenic tobacco plants and compared with the pattern observed in nodules of transgenic Lotus corniculatus plants. The results suggest that the highly conserved motifs in the NICE element play an important role in expression in roots of non-legume plants.     

Effects of steroid hormones on age-related expression and modulation of the lysozyme gene of the oviduct of Japanese quail

The expression of lysozyme gene in the oviduct of Japanese quail is age-dependent. Here we show that the expression of the gene is altered by three steroid hormones: 17-estradiol (E), progesterone (P) and glucocorticoid (dexamethasone, G), and their combinations E+P, E+G and P+G. We also show that the levels/affinities of trans-acting factors that bind to specific cis-acting elements in the promoter region of the gene change with age and after steroid administration. These factors are sequence-specific, age and steroid-dependent. It is proposed that administration of appropriate doses of steroid hormones after adulthood may extend the reproductive function and egg laying period in birds.     

Replication of ColE2 and ColE3 plasmids: The regions sufficient for autonomous replication

Summary We have localized the regions sufficient for autonomous replication on the genomes of the colicin E2 (ColE2) and colicin E3 (ColE3) plasmids and analyzed the replication functions carried by these regions. A 1.3 kb segment of each plasmid is sufficient for autonomous replication. Plasmids carrying this segment retain the replication properties of the original plasmid. The 1.3 kb segment consists of three functional portions. Firstly, a 0.9 kb region which specifies at least one trans-acting factor required for replication of each plasmid. Secondly, a 0.4 kb region located adjacent to one end of the 0.9 kb region, which is required for expression of the trans-acting factor(s) and probably contains the promoter. The region across the border of these two portions of ColE2 is involved in copy number control of the plasmid. The third portion is a 50 bp region adjacent to the other end of the 0.9 kb region, which contains a cis-acting site (origin) where replication initiates in the presence of the trans-acting factor(s). The action of the trans-acting factor(s) on the origin is plasmid specific. The 50 bp regions functioning as the origins of replication of ColE2 and ColE3 are the smallest among those in prokaryotic replicons so far identified and analyzed.     

Age- and Sex-dependent Differential Interaction of Nuclear <Emphasis Type="Italic">trans</Emphasis>-acting Factors with <Emphasis Type="Italic">Fmr-1</Emphasis> Promoter in Mice Brain

We have investigated relation between interaction of the trans-acting factors with Fmr-1 promoter and expression of FMRP isoforms in intact mouse brain as a function of age and sex. Our EMSA data reveal that among the three complexes formed with 136 bp Fmr-1 promoter fragment, the level of complex C1 significantly increases in adult brain but decreases in old brain in comparison to that in young. The level of total FMRP significantly decreases from young to old in the brain of both the sexes, however, among the three isoforms, expression of the 80-kDa isoform significantly decreases in the brain of both the sexes where as the level of 70 kDa isoform decreases in females during aging. The present finding on relation between age- and sex-dependent interaction of trans-acting factors and expression of FMRP isoforms is novel and may be relevant for regulation of Fmr-1 gene in brain function during aging.     

Minimal enhancer elements of the leghemoglobinlba andlbc3 gene promoters fromGlycine max L. have different properties

The characteristics of the soybean leghemoglobinlba gene promoter were analyzed and important promoter elements from thelba andlbc3 promoters were compared using transgenicLotus corniculatus plants. A 5 deletion analysis of thelba promoter delimited twocis-acting elements controlling expression: a distal positive element (–1254, –884) required for expression and a proximal element (–285, –60) essential for full-level activity. In contrast to the corresponding region of thelbc3 promoter, thelba proximal element is unable to control expression from the heterologous CaMV 35S enhancer. The upstream positive element of thelba gene contains a position- and orientation-independent enhancer between positions (–1091, –788). The sequence of this enhancer region is conserved in thelbc3 gene upstream (–1333, –1132) of the previously assigned strong positive element (SPE; –1090, –947). The present analysis revealed some of the properties of this extendedlbc3 SPE element. The extended element (–1364, –947) functions in both orientations from 5 locations whereas the SPE2 subcomponent (–1364, –1154) containing the conserved sequence is only active in the correct orientation. Removal of the SPE2 by internal deletion demonstrates that the SPE2 subcomponent is indispensable for the activity of thelbc3 upstream positive element. These results indicate that the upstream positive elements of thelba andlbc3 genes possess different properties although their conserved minimal enhancer sequence has similar function. This may reflect the differential expression of the twolb genes ofGlycine max L.     

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.     

Replication of ColE2 and ColE3 plasmids: Two ColE2 incompatibility functions

Summary We have identified and localized two incompatibility determinants (IncA and IncB) within a 1.3 kb segment of ColE2 sufficient for autonomous replication. The IncA determinant is localized in a region shorter than 250 bp and expresses incompatibility against both ColE2 and ColE3. The region which determines sensitivity to the IncA determinant seems to overlap with the region specifying the IncA determinant. The expression of the trans-acting factor(s) specifically required for replication of ColE2 interferes with expression of the IncA determinant against ColE2 but not against ColE3. The IncA determinant might be at least partly responsible for the copy number control of the plasmid. The IncB determinant is localized in a 50 bp region (origin) which is sufficient for initiation of replication in the presence of the trans-acting factor(s). The IncB determinant is specific for ColE2 and seems to be due to titration of the trans-acting essential replication factor(s) by binding.     

Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

Abscisic acid-inducible nuclear proteins bind to bipartite promoter elements required for ABA response and embryo-regulated expression of the carrot<Emphasis Type="Italic"> Dc3</Emphasis> gene

The carrot (Daucus carota L.) lea-class gene Dc3 is expressed in developing seeds and in vegetative tissues subject to drought and treatment with exogenous abscisic acid (ABA). Cis regulatory elements involved in seed-specific expression and in response to ABA were identified in transgenic tobacco (Nicotiana tabacum L.) using -glucuronidase (GUS) reporter gene constructs containing a series of deletion and orientation mutants of the Dc3 promoter. These experiments demonstrated that the Dc3 promoter is comprised of a proximal promoter region (PPR) and a distal promoter region (DPR). TCGTGT motifs in the DPR in combination with the PPR comprise a novel, bipartite ABA module in the Dc3 gene promoter. The PPR contains cis-acting elements responsible for the developmental regulation of Dc3 expression in seeds. Five similar sequence motifs with the consensus ACACgtGCa were identified in the PPR. Both DPR and PPR interact with common nuclear proteins that are present in embryos and are inducible by ABA in vegetative tissues.     

A mutation affecting amdS expression in Aspergillus nidulans contains a triplication of a cis-acting regulatory sequence

Summary In Aspergillus nidulans expression of the acetamidase structural gene, amdS, is under the control of at least four regulatory genes including the trans-acting amdA regulatory gene. A cis-acting mutation (amdI66) consisting of an 18 by duplication in the 5 region of the amdS gene results in very high levels of acetamidase activity but only in strains carrying semi-dominant mutations in the amdA gene. In selecting for increased amdS expression in an amdI66 amdA ^– strain, an A. nidulans strain with a mutation in the 5 region of the amdS gene was isolated. The nucleotide sequence was determined of the region containing the mutation, designated amdI666. The mutant strain carries three tandem copies of the 18 by sequence that is duplicated in the amdI66 mutation. Thus, from a strain carrying a duplication of an apparent regulatory protein binding site with little effect on gene expression, a strain has been derived that carries a triplication of the site with consequent major effects on regulation. The multiple copies of regulatory sites present in many genes may have been generated by a similar mechanism.     

Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis

Intron-bearing replacement histone H3 genes in Arabidopsis and other plants are highly and constitutively expressed. We demonstrate that the introns located within the 5-untranslated regions (5-UTR) of the two Arabidopsis replacement H3 genes will abolish the cell cycle dependence of an endogenous histone H4 promoter. We demonstrate that these introns, functionally combined with their endogenous promoters, could produce the high and constitutive expression of the replacement H3 genes observed in planta. They strongly increase gene expression whatever the promoter, from the strong 35S CaMV promoter to complete and resected promoters of cell cycle-dependent and replacement histone genes. Quantitative analysis of the extent of reporter gene enhancement in different parts of developing transgenic plantlets, ranging from 2-fold to 70-fold, supports the notion that trans-acting factors are responsible for this effect. Such factors appear most abundant in roots.     

Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus

Mutation of the core pentamer, CCGAC, of two putative low temperature responsive elements (LTREs) in the 5-proximal region of the winter Brassica napus cold-induced gene BN115 was carried out. Analyses of transient expression of the resultant mutated BN115 promoter-GUS fusions revealed the loss of low-temperature regulation by the promoter. This indicates that the CCGAC sequence is critical to the low-temperature response in the BN115 gene. In contrast, mutation of two G-boxes, CACGTG, staggered between the LTREs in the same region of the promoter did not alter cold-inducible gene expression. Replacement of a possible enhancer region of the BN115 promoter with the enhancer from the CaMV 35S promoter resulted in a several-fold increase in low temperature-induced GUS activity.     

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing

RNA interference (RNAi) is becoming a popular method for analyzing gene function in a variety of biological processes. We have used RNAi in cultured Drosophila cells to identify trans-acting factors that regulate the alternative splicing of endogenously transcribed pre-mRNAs. We have generated a dsRNA library comprising 70% of the Drosophila genes encoding RNA binding proteins and assessed the function of each protein in the regulation of alternative splicing. This approach not only identifies trans-acting factors regulating specific alternative splicing events, but also can provide insight into the alternative splicing regulatory networks of Drosophila. Here, we describe this RNAi approach to identify alternative splicing regulatory proteins in detail.     

Genetic analysis of the minimal replicon of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid

Using a combination of mutagenesis with the transposon and polymerase chain reaction subcloning, the essential elements of the replication region of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid have been identified. An open reading frame, coding for a protein with homology to Rep proteins from other Lactococcus plasmids, is essential. This protein is trans-acting and could not be replaced by the Rep protein from another Lactococcus plasmid. A second open reading frame immediately downstream from the first could be removed or inactivated with no apparent effect on plasmid replication. A region containing two 10 by direct repeats and three tandem repeats of a 22 by sequence, immediately upstream of the essential open reading frame, is also essential and probably includes the origin of replication. A 181-bp DNA fragment containing this region was sufficient to allow replication in Lactococcus if the trans-acting protein was provided on another replicon. Single-stranded replication intermediates could not be detected, suggesting that the citrate plasmid uses theta replication rather than rolling-circle replication.