Biomedical subjects

Alberto Martinez

Publications and source records attributed to Alberto Martinez.

7 recordsLinked to original sources

PubMed2005

New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis.

To facilitate glucocorticoid-inducible transgene expression from the pOp promoter in Arabidopsis the ligand-binding domain of a rat glucocorticoid receptor (GR LBD) was fused to the amino terminus of the synthetic transcription factor LhG4 to generate LhGR-N. Fusions bearing the GR LBD at other positions in LhG4 exhibited incomplete repression or inefficient induction. LhGR-N was stringently repressed in the absence of exogenous glucocorticoid but was fully activated by addition of 2 microm dexamethasone which resulted in 1000-fold increase in GUS reporter activity. Half maximal induction was achieved with 0.2 microm dexamethasone. Reporter transcripts were detectable within 2 h of dexamethasone application and peaked 4-10 h later. Neither LhGR-N nor dexamethasone affected seedling development although ethanol retarded development when used as a solvent for dexamethasone. The efficiency of the pOp target promoter was improved 10- to 20-fold by incorporating six copies of the ideal lac operator with sufficient inter-operator spacing to allow simultaneous occupancy. Introduction of the TMV Omega sequence into the 5'UTR resulted in a further 10-fold increase in dexamethasone-inducible reporter activity and an increase in the induction factor to 10(4). Although promoters containing the TMV Omega sequence exhibited slightly increased basal expression levels in the absence of dexamethasone, stringent regulation of the cytokinin biosynthetic gene ipt was achieved with all promoters. Despite the severity of the induced ipt phenotypes, transcripts for the KNOX homoeodomain transcription factors BREVIPEDICELLUS and SHOOTMERISTEMLESS were not significantly increased within 48 h of dexamethasone application to seedlings.

Arabidopsis↗

PubMed2005

Improved transcriptional activators and their use in mis-expression traps in Arabidopsis.

The synthetic transcription factor LhG4 has been used in numerous mis-expression studies in plants. We show that the sequence encoding the LhG4 activation domain, derived from Saccharomyces cerevisiae GAL4, contains several cryptic polyadenylation signals in Arabidopsis. The GAL4-derived sequence was modified according to preferred Arabidopsis codon usage, generating LhG4AtO which was faithfully transcribed in Arabidopsis plants. In protoplasts, LhG4AtO achieved maximum transactivation of the pOp promoter with 10-fold less input DNA than LhG4. The same methods were used to compare 10 other LhG4 derivatives that carried alternative natural or synthetic activation domains. Lh214 and Lh314, which contain synthetic activation domains comprising trimers of a core acidic activation domain, directed threefold more GUS expression from the pOp promoter with 20-fold less input DNA than LhG4. In contrast, when expressed from the CaMV 35S promoter in transgenic plants carrying a pOp-GUS reporter, Lh214 and Lh314 yielded transformants with substantially lower GUS activities than other constructs including LhG4AtO and LhG4 which performed similarly. When incorporated into an enhancer-trapping vector, however, LhG4AtO and Lh314 yielded enhancer traps with approximately twice the frequency of LhG4, suggesting that the modified activation domains offer improved performance when expressed from weaker transcription signals. To increase the number of LhG4 patterns available for mis-expression studies, we describe a population of enhancer-trap lines obtained with LhG4AtO in a pOp-GUS background. We show that enhancer-trap lines can transactivate an unlinked pOp-green fluorescent protein (pOp-GFP) reporter in the pattern predicted by staining for GUS activity.

Amino Acid Sequence↗

PubMed2005

Predictable activation of tissue-specific expression from a single gene locus using the pOp/LhG4 transactivation system in Arabidopsis.

The pOp/LhG4 transcription factor system was used to determine whether the synthetic pOp promoter, integrated at one position in the Arabidopsis genome, could be efficiently and faithfully activated by the heterologous transcription factor, LhG4, expressed in a variety of different patterns. This is a precondition for the development and exploitation of large collections of LhG4 activation lines that direct predictable tissue-specific expression of transgenes. We selected a pOp-GUS reporter insertion that was efficiently activated after crossing to an activator line that expressed the synthetic transcription factor LhG4 from the Cauliflower Mosaic Virus 35S promoter. This reporter line, pOp-GUS(g2), was then combined with activator loci that expressed LhG4 from one of seven different promoters, each with a different tissue specificity. pOp-GUS(g2) was activated faithfully in combination with six of these seven activator constructs, but generated an unexpected expression pattern in combination with the seventh construct, a fusion to a cyclin promoter (CYC-LhG4). The aberrant expression pattern could be attributed to the pOp-GUS(g2) insertion site, as the CYC-LhG4 activator lines directed the expected pattern of expression from a second pOp-GUS insertion. These results show that it is feasible to construct an activator collection in which LhG4 is expressed from diverse promoters or enhancer traps, but that individual pOp reporter loci can vary in their competence to respond to certain activator patterns. We discuss the implications for the design and use of mis-expression technology in Arabidopsis.

Journal Article↗

PubMed2004

Isoforms of cyclic AMP response element binding proteins in Drosophila S2 cells.

Activation or inhibition of the cyclic AMP (cAMP)-protein kinase A (PKA) pathway can ultimately regulate the transcription of a variety of genes. In vertebrates, the best characterized nuclear targets of PKA are the 'cAMP response element' (CRE) binding proteins (CREB). Differences in the transcriptional response to this pathway between cells and tissues can be based on the presence of distinct CREB isoforms. In this context, we have now investigated the presence of different dCREB transcripts in a stable, embryonic insect cell line, i.e., Drosophila Schneider 2 (S2) cells. In addition, we have studied the possible effect of cellular cAMP- and Ca2+ increases on the expression of a luciferase reporter in cells transfected with a CRE-containing reporter gene construct. In combination with recent data from the literature, our results indicate that the regulation of CRE-dependent gene expression shows some important differences between insects and vertebrates.

Amino Acid Sequence↗

PubMed2004

Functional and comparative analysis of two distinct ecdysteroid-responsive gene expression constructs in Drosophila S2 cells.

Inducible expression systems have proven to be of major interest when analysing the function of specific genes or when expressing cytotoxic proteins. In an effort to develop inducible switches allowing for flexible fine-tuning of gene expression levels in insect cells, we have compared the induction capacities of two Drosophila minimal promoters when linked to four consecutive ecdysone response elements. These minimal promoters, either containing a TATA-box or a downstream promoter element, drove the expression of a luciferase reporter gene. Potent induction capacities were observed with the insect moulting hormone, 20-hydroxyecdysone, and with ponasterone A, a plant ecdysteroid. The developed inducible switches further expand the repertoire of molecular tools for functional expression of proteins of interest in insect cells. In addition, the combination of an ecdysone switch with promoters that possess different structural elements can provide novel insights into ecdysteroid-induced transcription in an insect cell line.

Animals↗

PubMed2003

Closed fractures of the proximal tibia treated with a functional brace.

It generally is agreed that fractures of the proximal tibia, when treated with intramedullary nails, are more likely to present technical difficulties and to be associated with an increased rate of complications. This study reports on 108 closed fractures located in the proximal third of the tibia that were treated with functional braces. Nonunion occurred in 2.7% of the patients; the final displacement and shortening averaged 20% and 3.5 mm, respectively. Eighty-eight percent of the fractures healed with less than 6 degrees of angular deformity. It seems that functional braces for closed fractures of the tibia, located in its proximal third, are a viable therapeutic approach that offers satisfactory clinical and radiographic results in a high percentage of instances.

Adolescent↗

PubMed2002

Design, synthesis, and biological activity of hybrid compounds between uramustine and DNA minor groove binder distamycin A.

The design, synthesis, characterization, DNA binding properties, and cytotoxic activity of a novel series of hybrids, namely, a molecular combination of the natural antibiotic distamycin A and the antineoplastic agent uramustine, are reported, and the structure-activity relationships are discussed. This homologous series 29-34 consisted of the minor groove binder distamycin A joined to uramustine (uracil mustard) by suitable aliphatic carboxylic acid moieties containing a flexible polymethylene chain that is variable in length [(CH(2))(n)(), where n = 1-6). All the hybrid compounds in this series exhibit enhanced activity compared to both distamycin A and uramustine derivatives 22-27 used for conjugation, giving IC(50) values in the range 7.26-0.07 microM following a 1 h exposure of human leukemic K562 cells, with maximal activity shown when n = 6. The distance between the uramustine and distamycin frame is crucial for the cytotoxicity, with compounds having linker lengths of four to six being at least 20-fold more cytotoxic than linker lengths one to three. Taq polymerase stop experiments demonstrated selective covalent binding of uramustine-distamycin hybrids to A/T rich DNA sequences, which was again more efficient with compounds 32-34 with a longer linker length. Two consequences can be derived from our study: (a) the distamycin moiety directs binding to the minor groove of A/T rich DNA sequences and, consequently, is responsible for the alkylation regioselectivity found in footprinting studies; (b) the higher flexibility due to a longer linker between the distamycin and uracil moieties allows the formation of complexes with the mustard moiety situated more deeply in the minor groove and, hence, with better alkylating properties.

Anti-Bacterial Agents↗