Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat.

Three cDNAs encoding the antifungal protein Ag-AFP from the fungus Aspergillus giganteus, a barley class II chitinase and a barley type I RIP, all regulated by the constitutive Ubiquitin1 promoter from maize, were expressed in transgenic wheat. In 17 wheat lines, stable integration and inheritance of one of the three transgenes has been demonstrated over four generations. The formation of powdery mildew (Erysiphe graminis f. sp. tritici) or leaf rust (Puccinia recondita f. sp. tritici) colonies was significantly reduced on leaves from afp or chitinase II- but not from rip I-expressing wheat lines compared with non-transgenic controls. The increased resistance of afp and chitinase II lines was dependent on the dose of fungal spores used for inoculation. Heterologous expression of the fungal afp gene and the barley chitinase II gene in wheat demonstrated that colony formation and, thereby, spreading of two important biotrophic fungal diseases is inhibited approximately 40 to 50% at an inoculum density of 80 to 100 spores per cm2.     

A two-component regulator mediates population-density-dependent expression of the Bradyrhizobium japonicum nodulation genes

Bradyrhizobium japonicum nod gene expression was previously shown to be population density dependent. Induction of the nod genes is highest at low culture density and repressed at high population densities. This repression involves both NolA and NodD2 and is mediated by an extracellular factor found in B. japonicum conditioned medium. NolA and NodD2 expression is maximal at high population densities. We demonstrate here that a response regulator, encoded by nwsB, is required for the full expression of the B. japonicum nodYABC operon. In addition, NwsB is also required for the population-density-dependent expression of both nolA and nodD2. Expression of nolA and nodD2 in the nwsB mutant remained at a basal level, even at high culture densities. The nwsB defect could be complemented by overexpression of a second response regulator, NodW. Consistent with the fact that NolA and NodD2 repress nod gene expression, the expression of a nodY-lacZ fusion in the nwsB mutant was unaffected by culture density. In plant assays with GUS fusions, nodules infected with the wild type showed no nodY-GUS expression. In contrast, nodY-GUS expression was not repressed in nodules infected with the nwsB mutant. Nodule competition assays between the wild type and the nwsB mutant revealed that the addition of conditioned medium resulted in a competitive advantage for the nwsB mutant.     

Response regulator DegU of Listeria monocytogenes regulates the expression of flagella-specific genes

An isogenic mutant of Listeria monocytogenes EGD with a deletion of the response regulator gene degU showed a lack of motility due to the absence of flagella. In the present study, we used two-dimensional gel electrophoresis, mass-spectrometry and microarray analyses to identify the listerial genes that depend on DegU for expression. We found that the two L. monocytogenes operons encoding flagella-specific genes and the monocistronically transcribed flaA gene are positively regulated by DegU at 24 degrees C, but are not expressed at 37 degrees C.     

Motifs and cis-regulatory modules mediating the expression of genes co-expressed in presynaptic neurons

Background  

Hundreds of proteins modulate neurotransmitter release and synaptic plasticity during neuronal development and in response to synaptic activity. The expression of genes in the pre- and post-synaptic neurons is under stringent spatio-temporal control, but the mechanism underlying the neuronal expression of these genes remains largely unknown.     

Incongruent expression profiles between human and mouse orthologous genes suggest widespread neutral evolution of transcription control

Rapid rates of evolution can signify either a lack of selective constraint and the consequent accumulation of neutral alleles, or positive Darwinian selection driving the fixation of advantageous alleles. Based on a comparison of 1,350 orthologous gene pairs from human and mouse, we show that the evolution of gene expression profiles is so rapid that it is comparable to that of paralogous gene pairs or randomly paired genes. The expression divergence in the entire set of orthologous pairs neither strongly correlates with sequence divergence, nor focuses in any particular tissue. Moreover, comparing tissue expressions across the orthologous gene pairs, we observe that any human tissue is more similar to any other human tissue examined than to its corresponding mouse tissue. Collectively, these results indicate that, while some differences in expression profiles may be due to adaptive evolution, the levels of divergence are mostly compatible with a neutral mode of evolution, in which a mutation for ectopic expression may rise to fixation by random drift without significantly affecting the fitness. A disturbing corollary of these findings is that knowledge of where the gene is expressed may not carry information about its function.     

Mutated polyadenylation signals for controlling expression levels of multiple genes in mammalian cells

A set of mutated SV40 early polyadenylation signals (SV40pA) with varying strengths is generated by mutating the AATAAA sequence in the wild-type SV40pA. They are shown to control the expression level of a gene over a 10-fold range using luciferase reporter genes in transient transfection assays. The relative strength of these SV40pA variants remains similar under three commonly used mammalian promoters and in five mammalian cell lines. Application of SV40pA variants for controlling expression level of multiple genes is demonstrated in a study of monoclonal antibody (mAb) synthesis in mammalian cells. By using SV40pA variants of different strengths, the expression of light chain (LC) and heavy chain (HC) genes encoded in a single vector is independently altered which results in different ratios of LC to HC expression spanning a range from 0.24 to 16.42. The changes in gene expression are determined by measuring mRNA levels and intracellular LC and HC polypeptides. It is found that a substantial decrease of HC expression, which increases the LC/HC mRNA ratio, only slightly reduces mAb production. However, reducing the LC expression by a similar magnitude, which decreases the LC/HC mRNA ratio results in a sharp decline of mAb production to trace amounts. This set of SV40pA variants offers a new tool for accurate control of the relative expression levels of multiple genes. It will have wide-ranging applications in fields related to the study of biosynthesis of multi-subunit proteins, proteomic research on protein interactions, and multi-gene metabolic engineering.     

Long-distance signals positively regulate the expression of iron uptake genes in tobacco roots

Long-distance signals generated in shoots are thought to be associated with the regulation of iron uptake from roots; however, the signaling mechanism is still unknown. To elucidate whether the signal regulates iron uptake genes in roots positively or negatively, we analyzed the expressions of two representative iron uptake genes: NtIRT1 and NtFRO1 in tobacco (Nicotiana tabacum L.) roots, after shoots were manipulated in vitro. When iron-deficient leaves were treated with Fe(II)-EDTA, the expressions of both genes were significantly reduced; nevertheless iron concentration in the roots maintained a similar level to that in roots grown under iron-deficient conditions. Next, all leaves from tobacco plants grown under the iron-deficient condition were excised. The expression of two genes were quickly reduced below half within 2 h after the leaf excision and gradually disappeared by the end of a 24-h period. The NtIRT1 expression was compared among the plants whose leaves were cut off in various patterns. The expression increased in proportion to the dry weight of iron-deficient leaves, although no relation was observed between the gene expression and the position of excised leaves. Interestingly, the NtIRT1 expression in hairy roots increased under the iron-deficient condition, suggesting that roots also have the signaling mechanism of iron status as well as shoots. Taken together, these results indicate that the long-distance signal generated in iron-deficient tissues including roots is a major factor in positive regulation of the expression of NtIRT1 and NtFRO1 in roots, and that the strength of the signal depends on the size of plants.