Tandem arrayed ligation of expressed sequence tags (TALEST): a new method for generating global gene expression profiles.

We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. which rely on PCR.

Use of URA3 as a reporter of gene expression in C. albicans.

The C. albicans URA3 gene was tested as a reporter of gene expression. An integrating vector was constructed which contained ADE2 as a selectable marker together with a truncated form of URA3 lacking the first three codons. A DNA fragment containing the promoter and the first 90 codons of the C. albicans CEF3 gene was inserted into the unique XhoI site 5' to URA3 in order to provide an in-frame translational fusion. The functionality of the fusion gene was tested following integration of a single copy of the plasmid into the ADE2 locus. The fusion gene was shown to complement a ura3 deletion mutation and to produce orotidine 5'-monophosphate decarboxylase activity (OMP), which is encoded by URA3. Expression of the fusion gene was appropriately regulated by the growth rate and utilized the same transcriptional start sites as the native CEF3 gene. The results demonstrated that URA3 provides a sensitive and versatile reporter gene for use in C. albicans.

Isolation and sequence analysis of the gene for translation elongation factor 3 from Candida albicans.

Elongation factor 3 (EF-3) is a unique and essential component of the translational system in fungi. The gene, CEF-3, encoding elongation factor 3 has been isolated from the dimorphic fungus Candida albicans. A heterologous gene probe containing the coding region of the EF-3 gene from Saccharomyces cerevisiae (YEF-3) was used to screen three Candida albicans genomic DNA libraries. The nucleotide sequences of four partial clones were determined and combined for a full-length of 3,671 base pairs (bp). A continuous open reading frame (ORF) of 3,147 bp encoding a predicted protein of 1,049 amino acids and Mr of 116,739 daltons has been identified. A transcript of 3,400 nucleotides is seen in Northern blot hybridization of Candida albicans total RNA using a CEF-3 gene probe. The single locus CEF-3 gene maps to chromosome 5 in the genome. Comparison of the deduced amino acid sequences of CEF-3 and YEF-3 shows 77.6%. identity. A higher degree of identity, 86.5%, is found when comparing the carboxy-terminal portions of the two proteins. At the nucleotide level, comparison of the coding regions of the two genes exhibit 79% identity while the upstream and downstream regions show 46% and 40% identity, respectively.

Initial experience with the HEMOPUMP.

Preclinical and clinical trial investigations are now underway for a variety of ventricular assist devices at centers around the country. The current progress of the HEMOPUMP is encouraging, and survival rates show the potential for a successful outcome. This article describes the HEMOPUMP system, and discusses patient description and clinical experience with the system.