酵母基因上游与内含子可能存在的转录协同作用

酵母基因上游与内含子可能存在的转录协同作用     

Construction of a vnfH::lacZ fusion and study of expression from the vnfH promoter of the vanadium-dependent nitrogen fixation pathway in Azotobacter vinelandii

Abstract A lac fusion of the vnfH promoter of Azotobacter vinelandii has been constructed. An upstream sequence seems to be necessary for the activity of the promoter. The repression of expression of this promoter by fixed nitrogen is several-fold lower compared to that of the nifH promoter. Vanadium has no role in the expression of the nifH or the vnfH promoter. Molybdenum is essential for the nifH promoter and represses the vnfH promoter. Tungsten can substitute molybdenum in its regulatory role. ntrA and vnfA are essential for the expression of vnfH , but ntrC has no role.     

Efficiently finding regulatory elements using correlation with gene expression

We present an efficient algorithm for detecting putative regulatory elements in the upstream DNA sequences of genes, using gene expression information obtained from microarray experiments. Based on a generalized suffix tree, our algorithm looks for motif patterns whose appearance in the upstream region is most correlated with the expression levels of the genes. We are able to find the optimal pattern, in time linear in the total length of the upstream sequences. We implement and apply our algorithm to publicly available microarray gene expression data, and show that our method is able to discover biologically significant motifs, including various motifs which have been reported previously using the same data set. We further discuss applications for which the efficiency of the method is essential, as well as possible extensions to our algorithm.     

The MIK region rather than the C-terminal domain of AP3-like class B floral homeotic proteins determines functional specificity in the development and evolution of petals

In core eudicots, euAP3-type MADS-box genes encode a PISTILLATA (PI)-derived motif, as well as a C-terminal euAP3 motif that originated from a paleoAP3 motif of an ancestral APETALA3 (AP3)-like protein through a translational frameshift mutation. To determine the functional and evolutionary relevance of these motifs, a series of point mutation and domain-swap constructs were generated, involving CsAP3, a paleoAP3-type gene from the basal angiosperm Chloranthus spicatus encoding a truncated paleoAP3 motif, and AtAP3, a euAP3-type gene from the core eudicot Arabidopsis thaliana. The chimeric constructs were expressed in A. thaliana under the control of the AP3 promoter or the CaMV 35S promoter in an ap3 mutant or wild-type background, respectively. Significant recovery of AP3 function was obtained in both complementation and ectopic expression experiments whenever the region upstream of the C-terminal motifs (MIK region) from A. thaliana was taken, even when the PI-derived motif and the truncated paleoAP3 motif of CsAP3 substituted for the corresponding sequences from AtAP3. However, no or very weak complementation or gain-of-function was seen when the MIK region was from CsAP3. Our data suggest that changes in the MIK region rather than mutations in the C-terminal domain were of crucial importance for the evolution of the functional specificity of euAP3-type proteins in stamen and petal development.     

Characterization of the promoter of the Rhizobium etli recA gene.

The promoter of the Rhizobium etli recA gene has been identified by primer extension and by making deletions affecting several regions located upstream of its coding region. A gel mobility shift assay carried out with crude extracts of cells of R. etli has been used to show that a DNA-protein complex is formed in the R. etli recA promoter region in vitro. Analysis of the minimal region of the recA promoter giving rise to this DNA-protein complex revealed the presence of an imperfect palindrome corresponding to the sequence TTGN11CAA. Site-directed mutation of both halves of this palindrome indicated that both motifs, TTG and CAA, are necessary for both normal DNA-protein complex formation in vitro and full DNA damage-mediated inducibility of the recA gene in vivo. However, the TTG motif seems to be more dispensable than the CAA one. The presence of this same palindrome upstream of the recA genes of Rhizobium meliloti and Agrobacterium tumefaciens, whose expression is also regulated in R. etli cells, suggests that this TTGN11CAA sequence may be the SOS box of at least these three members of the Rhizobiaceae.     

Relation between mRNA expression and sequence information in Desulfovibrio vulgaris: combinatorial contributions of upstream regulatory motifs and coding sequence features to variations in mRNA abundance

The context-dependent expression of genes is the core for biological activities, and significant attention has been given to identification of various factors contributing to gene expression at genomic scale. However, so far this type of analysis has been focused either on relation between mRNA expression and non-coding sequence features such as upstream regulatory motifs or on correlation between mRNA abundance and non-random features in coding sequences (e.g., codon usage and amino acid usage). In this study multiple regression analyses of the mRNA abundance and all sequence information in Desulfovibrio vulgaris were performed, with the goal to investigate how much coding and non-coding sequence features contribute to the variations in mRNA expression, and in what manner they act together. Using the AlignACE program, 442 over-represented motifs were identified from the upstream 100bp region of 293 genes located in the known regulons. Regression of mRNA expression data against the measures of coding and non-coding sequence features indicated that 54.1% of the variations in mRNA abundance can be explained by the presence of upstream motifs, while coding sequences alone contribute to 29.7% of the variations in mRNA abundance. Interestingly, most of contribution from coding sequences is overlapping with that from upstream motifs; thereby a total of 60.3% of the variations in mRNA abundance can be explained when coding and non-coding information was included. This result demonstrates that upstream regulatory motifs and coding sequence information contribute to the overall mRNA expression in a combinatorial rather than an additive manner.     

Coordinate genetic control of yeast fatty acid synthase genes FAS1 and FAS2 by an upstream activation site common to genes involved in membrane lipid biosynthesis.

A systematic search for upstream controlling elements necessary for efficient expression of the yeast fatty acid synthase genes FAS1 and FAS2 revealed identical activation sites, UASFAS, in front of both FAS genes. The individual element confers, in a heterologous yeast test system, an approximately 40-fold stimulation of basal gene expression. The UASFAS motifs identified have the consensus sequence TYTTCACATGY and function in either orientation. The same sequence motif is found in the upstream regions of all so far characterized yeast genes encoding enzymes of phospholipid biosynthesis. In gel retardation assays, a protein factor, Fbf1 (FAS binding factor), was identified which interacted with UASFAS. The UASFAS motif proved to be an inositol/choline responsive element (ICRE) conferring strict repression by exogenous inositol and choline on a heterologous reporter gene. Its core sequence perfectly matches the CANNTG motif typical of basic helix-loop-helix DNA-binding proteins. In contrast to the individual UASFAS element, the intact yeast FAS promoters are not significantly influenced by inositol and choline, and thus allow nearly constitutive fatty acid synthase production. Available evidence suggests that additional cis- and trans-acting elements, other than UASFAS and Fbf1, are involved in this constitutive FAS gene expression.