Heterosis associated gene expression in maize embryos 6 days after fertilization exhibits additive,dominant and overdominant pattern

Heterosis is important for conventional plant breeding and is intensively used to increase the productivity of crop plants. Genetic processes shortly after fertilization might be of particular importance with respect to heterosis, because coordination of the diverse genomes establishes a basis for future performance of the sporophyte. Here we demonstrate a strong crossbreeding advantage of hybrid maize embryos as early as 6 days after fertilization in a modern maize hybrid and provide the first embryo specific analysis of associated gene expression pattern at this early stage of development. We identified differentially expressed genes between hybrid embryos and the parental genotypes by a combined approach of suppression subtractive hybridization and differential screening by microarray hybridizations. Association of heterosis in embryos with genes related to signal transduction and other regulatory processes was implied by the enrichment of these functional classes among the identified gene set. Quantitative RT-PCR analysis validated the expression pattern of 7 of 12 genes analysed and revealed predominantly additive, but also dominant and overdominant expression patterns in hybrid embryos. These patterns indicate that gene regulatory interactions among parental alleles act at this early developmental stage and the genes identified provide entry points for the exploration of gene regulatory networks associated with the specification of the phenomenon heterosis in the plant life cycle.     

Identification of novel genes associated with conidiation in Beauveria bassiana with suppression subtractive hybridization

The conidiation of the entomopathogenic fungus Beauveria bassiana (Hyphomycete) is a complex process that involves the stage- and cell-type-specific expression of hundreds of genes. The suppression subtractive hybridization method was used to target genes involved in conidiation. Seventeen genes were cloned that potentially were involved in conidia formation. Six of them demonstrated differential expression between conidial and vegetative cultures. Sequence analysis showed three cDNA fragments had similarity to known genes involved in either cellular metabolism or cell regulatory processes. The other cDNA fragments showed low or no similarity to any genes previously described. The full-length cDNA and genomic sequence of a gene designated A43 was isolated. The A43 protein is composed of 180 amino acids and has 34% identity to a RNA-binding region-containing protein. The temporal expression pattern was consistent with the gene being involved in conidiation. The colony morphology of the A43 knock-out mutant had more floccus mycelium than the wild-type and also produced fewer conidia, indicating the A43 gene is involved in B. bassiana conidiation.     

Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis

Gene duplication plays an important role in the evolution of diversity and novel function and is especially prevalent in the nuclear genomes of flowering plants. Duplicate genes may be maintained through subfunctionalization and neofunctionalization at the level of expression or coding sequence. In order to test the hypothesis that duplicated regulatory genes will be differentially expressed in a specific manner indicative of regulatory subfunctionalization and/or neofunctionalization, we examined expression pattern shifts in duplicated regulatory genes in Arabidopsis. A two-way analysis of variance was performed on expression data for 280 phylogenetically identified paralogous pairs. Expression data were extracted from global expression profiles for wild-type root, stem, leaf, developing inflorescence, nearly mature flower buds, and seedpod. Gene, organ, and gene by organ interaction (G x O) effects were examined. Results indicate that 85% of the paralogous pairs exhibited a significant G x O effect indicative of regulatory subfunctionalization and/or neofunctionalization. A significant G x O effect was associated with complementary expression patterns in 45% of pairwise comparisons. No association was detected between a G x O effect and a relaxed evolutionary constraint as detected by the ratio of nonsynonymous to synonymous substitutions. Ancestral gene expression patterns inferred across a Type II MADS-box gene phylogeny suggest several cases of regulatory neofunctionalization and organ-specific nonfunctionalization. Complete linkage clustering of gene expression levels across organs suggests that regulatory modules for each organ are independent or ancestral genes had limited expression. We propose a new classification, regulatory hypofunctionalization, for an overall decrease in expression level in one member of a paralogous pair while still having a significant G x O effect. We conclude that expression divergence specifically indicative of subfunctionalization and/or neofunctionalization contributes to the maintenance of most if not all duplicated regulatory genes in Arabidopsis and hypothesize that this results in increasing expression diversity or specificity of regulatory genes after each round of duplication.     

Early and late periodic patterns of even skipped expression are controlled by distinct regulatory elements that respond to different spatial cues

We have identified the regulatory sequences required for the periodic expression of the Drosophila pair rule gene even skipped (eve). We find that the gradually changing pattern of periodic eve expression during early embryogenesis is directed by two distinct regulatory programs. Initially, eve expression in individual stripes is established by different regulatory elements, each of which responds to nonperiodic spatial cues provided, at least in part, by the gap genes. Later, coordinate expression of eve in all seven stripes is directed by a single regulatory region that responds to periodic cues provided by primary pair rule genes, including eve itself. As a consequence of this two-step regulatory program, eve functions both in the establishment of the periodic pattern of gene expression and in the subsequent specification of parasegmental boundaries.     

Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis.

We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spo0H, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spo0H encodes a sigma factor of RNA polymerase, sigma H, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by sigma H (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn917lac, in the presence and absence of sigma H. These genes had lower levels of expression in the absence of sigma H than in the presence of sigma H. Patterns of expression of the csh genes during growth and sporulation in wild-type and spo0H mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn917lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spo0H mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spo0H in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation.     

Characterization of genes with tissue-specific differential expression patterns in Populus

Like many plants, Populus has an evolutionary history in which several, both recent and more ancient, genome duplication events have occurred and, therefore, constitutes an excellent model system for studying the functional evolution of genes. In the present study, we have focused on the properties of genes with tissue-specific differential expression patterns in poplar. We identified the genes by analyzing digital expression profiles derived by mapping 90,000+ expressed sequence tags (ESTs) from 18 sources to the predicted genes of Populus. Our sequence analysis suggests that tissue-specific differentially expressed genes have less diverged paralogs than average, indicating that gene duplication events is an important event in the pathway leading to this type of expression pattern. The functional analysis showed that genes coding for proteins involved in processes of functional importance for the specific tissue(s) in which they are expressed and genes coding for regulatory or responsive proteins are most common among the differentially expressed genes, demonstrating that the expression differentiation process is under strong selective pressure. Thus, our data supports a model where gene duplication followed by gene specialization or expansion of the regulatory and responsive networks leads to tissue-specific differential expression patterns. We have also searched for clustering of genes with similar expression pattern into gene-expression neighborhoods within the Populus genome. However, we could not detect any major clustering among the analyzed genes with highly specific expression patterns. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

How much expression divergence after yeast gene duplication could be explained by regulatory motif evolution?

We used the yeast genome sequences of gene families, microarray profiles and regulatory motif data to test the current wisdom that there is a strong correlation between regulatory motif structure and gene expression profile. Our results suggest that duplicate genes tend to be co-expressed but the correlation between motif content and expression similarity is generally poor, only approximately 2-3% of expression variation can be explained by the motif divergence. Our observations suggest that, in addition to the cis-regulatory motif structure in the upstream region of the gene, multiple trans-acting factors in the gene network can influence the pattern of gene expression significantly.     

Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli.

We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp.     

Cis-regulatory properties of medaka synexpression groups

During embryogenesis, tissue specification is triggered by the expression of a unique combination of developmental genes and their expression in time and space is crucial for successful development. Synexpression groups are batteries of spatiotemporally co-expressed genes that act in shared biological processes through their coordinated expression. Although several synexpression groups have been described in numerous vertebrate species, the regulatory mechanisms that orchestrate their common complex expression pattern remain to be elucidated. Here we performed a pilot screen on 560 genes of the vertebrate model system medaka (Oryzias latipes) to systematically identify synexpression groups and investigate their regulatory properties by searching for common regulatory cues. We find that synexpression groups share DNA motifs that are arranged in various combinations into cis-regulatory modules that drive co-expression. In contrast to previous assumptions that these genes are located randomly in the genome, we discovered that genes belonging to the same synexpression group frequently occur in synexpression clusters in the genome. This work presents a first repertoire of synexpression group common signatures, a resource that will contribute to deciphering developmental gene regulatory networks.     

Molecular characterization of ubiquitin genes from Aspergillus nidulans: mRNA expression on different stress and growth conditions

We are interested in studying the ubiquitin (UBI) gene expression during different stress and growth conditions in the filamentous fungus Aspergillus nidulans. Here, we report the cloning of a cDNA clone that corresponds to a gene, ubi1, that encodes a carboxyl extension protein from A. nidulans. This cDNA corresponds to a gene that encodes a protein that showed high homology to other polyubiquitin and CEP-80 genes at the N- and C-terminus, respectively. We characterize the mRNA expression of the CEP and polyubiquitin genes during several growth and stress conditions. Expression of the ubi1 and ubi4 genes was correlated with cell growth in most of the carbon sources used, except maltose. Both ubi1 and ubi4 genes were induced upon heat-shock, although the levels of expression were raised quicker for ubi4 than for ubi1. The ubi1 and ubi4 genes displayed a very complex expression pattern in presence of drugs with a different mechanism of action suggesting that the regulatory processes controlling UBI gene expression discriminate between different stresses and can affect individually each UBI gene. The ubi1 gene was highly expressed in presence of hydrogen peroxide while the ubi4 mRNA level was not affected; several metals in our experimental conditions were not able to induce either ubi1 nor ubi4 genes.     

Inferring gene regulatory relationships by combining target-target pattern recognition and regulator-specific motif examination

Although microarray data have been successfully used for gene clustering and classification, the use of time series microarray data for constructing gene regulatory networks remains a particularly difficult task. The challenge lies in reliably inferring regulatory relationships from datasets that normally possess a large number of genes and a limited number of time points. In addition to the numerical challenge, the enormous complexity and dynamic properties of gene expression regulation also impede the progress of inferring gene regulatory relationships. Based on the accepted model of the relationship between regulator and target genes, we developed a new approach for inferring gene regulatory relationships by combining target-target pattern recognition and examination of regulator-specific binding sites in the promoter regions of putative target genes. Pattern recognition was accomplished in two steps: A first algorithm was used to search for the genes that share expression profile similarities with known target genes (KTGs) of each investigated regulator. The selected genes were further filtered by examining for the presence of regulator-specific binding sites in their promoter regions. As we implemented our approach to 18 yeast regulator genes and their known target genes, we discovered 267 new regulatory relationships, among which 15% are rediscovered, experimentally validated ones. Of the discovered target genes, 36.1% have the same or similar functions to a KTG of the regulator. An even larger number of inferred genes fall in the biological context and regulatory scope of their regulators. Since the regulatory relationships are inferred from pattern recognition between target-target genes, the method we present is especially suitable for inferring gene regulatory relationships in which there is a time delay between the expression of regulating and target genes.     

The 35UZ transposon of Drosophila melanogaster reveals differences in maintenance of transcriptional control between embryonic and larval stages.

The D. melanogaster transposon P[35UZ] contains a lacZ reporter gene fused to 35 kb of Ubx upstream sequences which drive a Ubx-like expression in embryos and in metathoracic imaginal discs. Transposition of P[35UZ] followed by analysis of different lines in wild-type and mutant backgrounds allowed us to analyze the interplay between Ubx regulatory elements, including the Polycomb response element (PRE), located inside the transposon and cis-acting regulatory elements, located outside. We found that all lines show a Ubx-like beta-galactosidase expression pattern in the embryo, but proximity to strong imaginal enhancers can change this pattern drastically. These data illustrate how maintenance of gene expression depends on the chromosomal environment and on dynamic interactions between all developmentally regulated enhancers located close to a promoter under PcG control.