CLONING AND CHARACTERIZATION OF THE GENE ENCODING THE HIGHLY EXPRESSED RIBOSOMAL PROTEIN L3 OF THE CILIATED PROTOZOANTETRAHYMENA THERMOPHILA. EVIDENCE FOR DIFFERENTIAL CODON USAGE IN HIGHLY EXPRESSED GENES

Abstract

We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena. The codon usage of the L3 gene is highly biased. A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias. Class A comprises r-protein genes and a number of other highly expressed genes. Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1. Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases. Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions. The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena.

Expression, secretion and surface display of a human alkaline phosphatase by the ciliate Tetrahymena thermophila

“…In order to express the full-length precursor of hiAP in ciliates we used a codon adapted artificial gene, according to the codon bias for highly expressed genes in T. thermophila [19,20]. The complete hiAP cDNA was cloned into the rDNA ori based pH4T2 vector [21,22].…”

Secretion of functional human enzymes by Tetrahymena thermophila

“…Aim of this study was to show that T. thermophila is capable of expressing a human enzyme which is secreted under the control of an endogenous T. thermophila derived precursor peptide. The codon bias of ciliates has been analysed in detail [ 13 , 15 , 16 ]. In summary it is quite different from human genes.…”

“…Cloning of the expression modules and mutagenesis were performed with standard techniques. To ensure a translation of the fusion proteins a synthetic, codon optimised human DNaseI gene was used [ 13 , 15 ]. The synthesis was performed by a solid phase process described in German Patent DE 19812103.2 by the company ATG biosynthetics, Merzhausen Germany.…”

“…All three protozoans Tetrahymena thermophila, Ichthyophthirius multifiliis as well as the malaria parasite Plasmodium falciparum belong to the alveolates, a distinct phylogenetic group that includes ciliates, apicomplexans and dinoflagellates. They are characterised by both very AT-rich genomes and an unusual codon usage [ 13 - 16 ]. However, so far the expression of functional mammalian or human proteins in T. thermophila remains to be shown.…”

Analysis of Expressed Sequence Tags (ESTs) in the Ciliated Protozoan Tetrahymena thermophila

“…The ESTs that matched T. thermophila SERH3 and the three GRL genes were the only ones that gave no matches to genes in other organisms. Other EST matches to Tetrahymena gene products included ribosomal protein L3 (Larsen et al 1999), histone H4 (Bannon et al 1984), phosphoglycerate kinase (Vohra et al 1992), all from T. thermophila , and elongation factor EF‐1α from T. pyriformis (Kurasawa et al 1992). EST 201 matched the T. thermophila actin gene, albeit imperfectly.…”