Molecular cloning and expression of a Tetrahymena pyriformis ubiquitin fusion gene coding for a 53-amino-acid extension protein

Abstract

The genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTU11), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.

Microtubule cytoskeleton perturbation induced by taxol and colchicine affects chaperonin containing TCP-1 (CCT) subunit gene expression in Tetrahymena cells

“…This mRNA species was never described as being speci¢cally induced under stress conditions in T. pyriformis cells. On the other hand, the 0.75 kb ubiquitin fusion gene transcripts coding for 53 amino acid extension protein [33] remained almost unaltered during the treatment with the referred antimitotic agents. The e¡ects on the induction of hsp70 and ubiquitin genes by colchicine and taxol are not comparable both with respect to time and to the extent of the expression induced by heat shock.…”

“…When hsp70 [26] and actin oligonucleotide (5P-CTCTGGGAGCATCGT-CACCAGC-3P) [31] probes were used, hybridization conditions were those described in [32]. Hybridizations using TU20 gene as probe were done essentially as described by Neves et al [33].…”

“…The homologous DNA probe encoding T. pyriformis TpCCTK subunit was prepared from pTp7 plasmid [24], TpCCTR was prepared from pTpH2.3 plasmid [22], whereas TpCCTQ subunit and LTT1 tubulin were prepared from pTpE3 and IB1 plasmid, respectively, as described in [21]. The ubiquitin probe was prepared from pTU20 plasmid [33]. Fragments were labeled using the Megaprime DNA labeling system (Amersham) and [K-32 P]dATP (3 000 Ci/mmol) (Amersham International plc).…”

“…The e¡ects on the induction of hsp70 and ubiquitin genes by colchicine and taxol are not comparable both with respect to time and to the extent of the expression induced by heat shock. However, our results unequivocally show that these genes are [30]; TpCCTK^V2.9 kb EcoRI fragment from TpCCTK1.1 [23]; TpCCTO^V4.3 kb SphI fragment from TpCCTO6.1.1; TpCCTQ^V2.2 kb EcoRI fragment from pTpE3 plasmid [21]; TpCCTR^V2.3 kb HindIII fragment from TpCCTR11.3 [22]; actin^a speci¢c oligomer probe (see Section 2); hsp70^a speci¢c oligomer probe [26]; ubiquitin^0.23 kb HindIII fragment from pTU20 plasmid [33]. The results shown are representative of three to ¢ve independent experiments that gave similar results.…”

cDNA cloning of a tetraubiquitin gene, and expression of ubiquitin-containing transcripts, in aleurone layers of Avena fatua

“…Two types of gene within this class have been identified, carboxy extension protein (CEP) 52 and CEP 76-80. Ubiquitin CEPs are expressed primarily during rapid cell growth and have been identified in, amongst others, Nicotiana tabacum [ 10], Hordeum vulgate [9], Arabidopsis thaliana [6], L ycopersicon escuIentum [12], Chlamydomonas reinhardtii [5], Saccharomyces cerevisiae [23 ], Dictyostelium [20], Tetrahymena-Pyriformis [21] and Homo sapiens [17]. The second class (polyubiquitins) consists ofubiquitin repeats fused together in a contiguous head-totail fashion.…”