Biomedical subjects

Thawornchai Limjindaporn

Publications and source records attributed to Thawornchai Limjindaporn.

4 recordsLinked to original sources

PubMed2006

Impaired interaction of alpha-haemoglobin-stabilising protein with alpha-globin termination mutant in a yeast two-hybrid system.

Alpha-thalassaemia caused by alpha-globin gene termination codon mutations (alphaT-globin) has been explained by their inherent mRNA instability and by oxidative damage arising from the presence of membrane-bound alphaT-globin chains. To better understand the latter phenomenon, a yeast two-hybrid system was used to assay the interaction between alphaT-globin and its molecular chaperone, alpha-haemoglobin-stabilising protein (AHSP) and impaired binding of alphaT-globin with AHSP compared with alpha(wild-type)-globin was observed.

Blood Proteins↗

PubMed2003

Nitrogen metabolism and virulence of Candida albicans require the GATA-type transcriptional activator encoded by GAT1.

Nitrogen acquisition and metabolism is central to microbial growth. A conserved family of zinc-finger containing transcriptional regulators known as GATA-factors ensures efficient utilization of available nitrogen sources by fungi. GATA factors activate expression of nitrogen catabolic pathways when preferred nitrogen sources are absent or limiting, a phenomenon known as nitrogen catabolite repression. GAT1 of Candida albicans encodes a GATA-factor homologous to the AREA protein of Aspergillus nidulans and related transcription factors involved in nitrogen regulation. Two observations implicated GAT1 in nitrogen regulation. The growth of mutants lacking GAT1 was reduced when isoleucine, tyrosine or tryptophan were the sole source of nitrogen. Secondly, when cultured on a secondary nitrogen source, gat1Delta mutants were unable to activate expression of GAP1, UGA4 or DAL5, which were shown to be nitrogen regulated in C. albicans. This regulatory defect did not prevent filamentation of gat1Delta mutants in nitrogen repressing or non-repressing conditions, demonstrating that nitrogen catabolite repression does not influence dimorphism. The mutants were, however, highly attenuated in a murine model of disseminated candidiasis. Attenuation was not associated with any diminution of growth in serum or ability to utilize serum amino acids. The results indicate an important role for nitrogen regulation in the virulence of C. albicans.

Amino Acid Sequence↗

PubMed2003

Simultaneous cloning of open reading frames into several different expression vectors.

Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.

Bacterial Proteins↗

PubMedDate not supplied

High-throughput cloning of Campylobacter jejuni ORfs by in vivo recombination in Escherichia coli.

A rate-limiting and costly step in many proteomics analyses is the cloning of all of the ORFs for an organism into technique-specific vectors. Here, we describe the generation of a Campylobacter jejuni expression clone set using a high-throughput cloning approach based on recombination in E. coli. The approach uses native E. coli recombination functions and requires no in vitro enzymatic steps or special strains. Our results indicate that this approach is an efficient and economical alternative for high-throughput cloning.

Campylobacter jejuni↗