Mário Henrique Bengtson

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Hunting for differentially expressed genes

Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Several methods have been used for this purpose, including differential hybridization, cDNA subtraction, differential display and, more recently, DNA chips. Subtractive hybridization has significantly improved after the polymerase chain reaction was incorporated into the original method and many new protocols have been established. Recently, the availability of the wellknown coding sequences for some organisms has greatly facilitated gene expression analysis using high-density microarrays. Here, we describe some of these modifications and discuss the benefits and drawbacks of the various methods corresponding to the main advances in this field.

Identification of genes regulated by oleic acid in Jurkat cells by suppressive subtractive hybridization analysis

In this study, the effect of oleic acid (50 μM) on gene expression of Jurkat cells (human T lymphocytes cell line) was examined using the suppressive subtractive hybridization approach. This technique allowed us to identify genes with higher or lower expression after cell treatment with oleic acid as compared to untreated cells. Oleic acid upregulated the expression of the translation elongation factor alpha 1 and ATP synthase 8 and downregulated gp96 (human tumor rejection antigen gp96), heat‐shock protein 60 and subtilisin‐like protein 4. These results suggest that oleic acid, at plasma physiological concentration, can regulate the expression of important genes to maintain the machinery that ensures cell functioning.