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David González-Ballester

Publications and source records attributed to David González-Ballester.

4 recordsLinked to original sources
PubMed2005

Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA.

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.

Animals↗
PubMed2005

Ammonium transporter genes in Chlamydomonas: the nitrate-specific regulatory gene Nit2 is involved in Amt1;1 expression.

Ammonium transport is a key process in nitrogen metabolism. In the green alga Chlamydomonas, we have characterized molecularly the largest family of ammonium transporters (AMT1) so far described consisting of eight members. CrAmt1 genes have an interesting transcript structure with some very small exons. Differential expression patterns were found for each CrAmt1 gene in response to the nitrogen source by using Real Time PCR. These expression patterns were similar under high and low CO2 atmosphere. CrAmt1;1 expression was characterized in detail. It was repressed in both ammonium and nitrate medium, and strongly expressed in nitrogen-free media. Treatment with a Glutamine synthetase inhibitor released partially repression in ammonium and nitrate suggesting that ammonium and its derivatives participate in the observed repressing effects. By studying CrAmt1;1 expression in mutants deficient at different steps of the nitrate assimilation pathway, it has been shown that nitrate has a double negative effect on this gene expression; one related to its reduction to ammonium, and a second one by itself. This second effect of nitrate was dependent on the functionality of the regulatory gene Nit2, specific for nitrate assimilation. Thus, NIT2 would have a dual role on gene expression: the well-known positive one on nitrate assimilation and a novel negative one on Amt1;1 regulation.

Algal Proteins↗
PubMed2005

Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas.

The existence of mutants at specific steps in a pathway is a valuable tool of functional genomics in an organism. Heterologous integration occurring during transformation with a selectable marker in Chlamydomonas (Chlamydomonas reinhardtii) has been used to generate an ordered mutant library. A strain, having a chimeric construct (pNia1::arylsulfatase gene) as a sensor of the Nia1 gene promoter activity, was transformed with a plasmid bearing the paramomycin resistance AphVIII gene to generate insertional mutants defective at regulatory steps of the nitrate assimilation pathway. Twenty-two thousand transformants were obtained and maintained in pools of 96 for further use. The mutant library was screened for the following phenotypes: insensitivity to the negative signal of ammonium, insensitivity to the positive signal of nitrate, overexpression in nitrate, and inability to use nitrate. Analyses of mutants showed that (1) the number or integrated copies of the gene marker is close to 1; (2) the probability of cloning the DNA region at the marker insertion site is high (76%); (3) insertions occur randomly; and (4) integrations at different positions and orientations of the same genomic region appeared in at least three cases. Some of the mutants analyzed were found to be affected at putative new genes related to regulatory functions, such as guanylate cyclase, protein kinase, peptidyl-prolyl isomerase, or DNA binding. The Chlamydomonas mutant library constructed would also be valuable to identify any other gene with a screenable phenotype.

Ammonia↗