Cloning of regulatory sequences mediating guard-cell-specific gene expression

This report describes the use of promoter trap lines from the model plant Arabidopsis thaliana to clone regulatory sequences that mediate guard-cell-specific reporter gene expression. Stomatal guard cells represent a highly differentiated cell type within the epidermis of green tissues of higher plants. They control the stomatal aperture in response to different endogenous and environmental signals in order to optimize carbon fixation while minimizing water loss. We screened available promoter trap lines for guard-cell-specific activation of a beta-glucuronidase (uidA) reporter gene in order to obtain marker lines for guard-cell development and to gain access to regulatory pathways leading to gene expression which is restricted to this cell type. From two lines identified we successfully cloned upstream regulatory sequences. For one line, guard-cell-specific promoter activity was confirmed by re-introducing the uidA gene, fused to the newly identified regulatory sequences, into the Arabidopsis nuclear genome. However, DNA sequences downstream of the uidA/T-DNA insertion sites in the original promoter trap lines revealed no obvious coding regions in the corresponding orientation, indicating that we have probably identified 'cryptic' promoters, being active in guard cells.     

Yeast proteins can activate expression through regulatory sequences of the amdS gene of Aspergillus nidulans

The upstream regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence known to be important in setting both basal and derepressed levels of expression. We have investigated whether the CCAAT-binding HAP2/3/4 complex of the yeast Saccharomyces cerevisiae can recognise this sequence in an amdS context. Sequences from the 5′ region of amdS were cloned in front of the CYCI-lacZ fusion gene bearing a minimal promoter and transformed into wild-type and hap2 strains of yeast. This study has indicated that amdS sequences are capable of promoting regulated expression of the fusion gene in response to carbon limitation. The yeast HAP2/3/4 complex can recognise the amdS CCAAT sequence and activate expression from this sequence. In addition, the results indicate that other yeast proteins can also regulate expression from the A. nidulans amdS 5′ sequences under carbon-limiting conditions.     

Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase

Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. We have performed ple rescue experiments with modified DTH transgenes. Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila.     

Secretion of foreign proteins mediated by chicken lysozyme gene regulatory sequences.

Exploitation of the insulating properties of the complete chicken lysozyme gene domain may facilitate the production of transgenic chicken bioreactors with the capacity to deposit valuable proteins in the egg white. Chimeric genes consisting of the chicken lysozyme gene regulatory sequences and sequences encoding foreign proteins could be inserted randomly into the chicken genome and retain appropriate expression levels. The research reported here established that chicken lysozyme gene regulatory sequences can be used to direct the production and secretion of green fluorescent protein (used as a reporter protein) in transiently transfected chicken blastodermal cells. Attempts to verify these findings in transgenic hens are currently in progress. To provide a rapid means of generating constructs encoding other foreign proteins under the control of lysozyme gene regulatory sequences that can facilitate the secretion of heterologous proteins in vivo, a generic lysozyme gene regulatory scaffold was created using a poxvirus-mediated gene targeting system.     

Polymorphism in regulatory gene sequences

The extensive polymorphism revealed in non-coding gene-regulatory sequences, particularly in the immune system, suggests that this type of genetic variation is functionally and evolutionarily far more important than has been suspected, and provides a lead to new therapeutic strategies.     

Polymorphism in regulatory gene sequences

The extensive polymorphism revealed in non-coding gene-regulatory sequences, particularly in the immune system, suggests that this type of genetic variation is functionally and evolutionarily far more important than has been suspected, and provides a lead to new therapeutic strategies.     

Yeast proteins can activate expression through regulatory sequences of the amdS gene of Aspergillus nidulans

The upstream regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence known to be important in setting both basal and derepressed levels of expression. We have investigated whether the CCAAT-binding HAP2/3/4 complex of the yeast Saccharomyces cerevisiae can recognise this sequence in an amdS context. Sequences from the 5 region of amdS were cloned in front of the CYCI-lacZ fusion gene bearing a minimal promoter and transformed into wild-type and hap2 strains of yeast. This study has indicated that amdS sequences are capable of promoting regulated expression of the fusion gene in response to carbon limitation. The yeast HAP2/3/4 complex can recognise the amdS CCAAT sequence and activate expression from this sequence. In addition, the results indicate that other yeast proteins can also regulate expression from the A. nidulans amdS 5 sequences under carbon-limiting conditions.     

High level expression of proteins using sequences from the ferritin heavy chain gene locus

An expression vector has been generated using a gene highly expressed under conditions found in a typical fed-batch bioreactor process. The ferritin heavy chain (HC) gene exhibits higher levels of expression in the late stages of a fed-batch bioreactor than in the early stages. This property was considered advantageous for an expression vector, since the maximal cell density would coincide with maximal expression. The rat ferritin HC genomic region was isolated and converted into an expression vector where large segments of 5' and 3' flanking regions were included in an attempt to recreate the same high level of expression in stably transfected cells. Expression from the resulting ferritin HC vector was compared to vectors containing the commonly used strong promoters, CMV IE, and SV40 early promoter/enhancer, in the generation of stable transfectants. The ferritin HC vector was able to generate cell lines with significantly higher expression levels than those under the control of the viral promoters.     

P-cats: prediction of catalytic residues in proteins from their tertiary structures

SUMMARY: P-cats is a web server that predicts the catalytic residues in proteins from the atomic coordinates. P-cats receives a coordinate file of the tertiary structure and sends out analytical results via e-mail. The reply contains a summary and two URLs to allow the user to examine the conserved residues: one for interactive images of the prediction results and the other for a graphical view of the multiple sequence alignment. AVAILABILITY: P-cats is freely available at http://p-cats.hgc.jp/p-cats CONTACT: kino@ims.u-tokyo.ac.jp     

Identification of partially linear structure in additive models with an application to gene expression prediction from sequences

The additive model is a semiparametric class of models that has become extremely popular because it is more flexible than the linear model and can be fitted to high-dimensional data when fully nonparametric models become infeasible. We consider the problem of simultaneous variable selection and parametric component identification using spline approximation aided by two smoothly clipped absolute deviation (SCAD) penalties. The advantage of our approach is that one can automatically choose between additive models, partially linear additive models and linear models, in a single estimation step. Simulation studies are used to illustrate our method, and we also present its applications to motif regression.     

RVP-net: online prediction of real valued accessible surface area of proteins from single sequences

SUMMARY: RVP-net is an online program for the prediction of real valued solvent accessibility. All previous methods of accessible surface area (ASA) predictions classify amino acid residues into exposure states and named them buried or exposed based on different thresholds. Real values in some cases were generated by taking the mid points of these state thresholds. This is the first method, which provides a direct prediction of ASA without making exposure categories and achieves results better than 19% mean absolute error. To facilitate batch processing of several sequences, a standalone version of this tool is also provided. AVAILABILITY: Online predictions are available at http://www.netasa.org/rvp-net/. Standalone version of the program can be obtained from the corresponding author by E-mail request.