Dataset Information

Developmental expression of surface antigen genes in the parasitic ciliate Ichthyophthirius multifiliis.

ABSTRACT: A 1.2-kilobase (kb) cDNA encoding a major surface antigen of the holotrich ciliate Ichthyophthirius multifiliis (an obligate parasite of fish) has been isolated and used as a probe to examine the expression of immobilization antigen (i-antigen) genes in this system. The cDNA encodes a predicted protein of 394 amino acids with a tandemly repeated structure characteristic of the i-antigens of the related free-living ciliates Paramecium and Tetrahymena. As shown by Northern hybridization analysis with both total and poly(A)+ RNAs, the 1.2-kb cDNA recognizes distinct transcripts of 1.6 and 1.9 kb which are differentially expressed through the parasite life cycle. During the transition from the host-associated trophozoite stage to the infective tomite stage, steady-state levels of the 1.9-kb RNA undergo a marked increase of greater than or equal to 50-fold, while the 1.6-kb transcript increases only slightly. The absolute amounts of RNA encoding the i-antigen have been quantitated and were found to reach extremely high levels equivalent to approximately 6% of the poly(A)+ RNA of I. multifiliis tomites. Southern hybridization analysis with I. multifiliis genomic DNA suggests that at least two genes encode the i-antigen transcripts. In experiments to examine the effects of temperature on the expression of I. multifiliis i-antigen genes, levels of the 1.6- and 1.9-kb transcripts were found to remain relatively constant in cells maintained at different temperature extremes. These studies indicate that genes encoding i-antigens of I. multifiliis are developmentally regulated, and they suggest the existence of alternative mechanisms for the control of surface antigen expression in ciliates.

SUBMITTER: Clark TG

PROVIDER: S-EPMC49501 | biostudies-other | 1992 Jul

REPOSITORIES: biostudies-other

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Developmental expression of surface antigen genes in the parasitic ciliate Ichthyophthirius multifiliis.

Clark T G TG McGraw R A RA Dickerson H W HW

Proceedings of the National Academy of Sciences of the United States of America 19920701 14

A 1.2-kilobase (kb) cDNA encoding a major surface antigen of the holotrich ciliate Ichthyophthirius multifiliis (an obligate parasite of fish) has been isolated and used as a probe to examine the expression of immobilization antigen (i-antigen) genes in this system. The cDNA encodes a predicted protein of 394 amino acids with a tandemly repeated structure characteristic of the i-antigens of the related free-living ciliates Paramecium and Tetrahymena. As shown by Northern hybridization analysis w ...[more]

PMID: 1631132

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Project description:BackgroundTetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.ResultsFunctional and full length human intestinal alkaline phosphatase was expressed by T. thermophila using a codon-adapted gene containing the native signal-peptide and GPI (Glycosylphosphatidylinositol) anchor attachment signal. HiAP activity in the cell extract of transformants suggested that the hiAP gene was successfully expressed. Furthermore, it was demonstrated that the enzyme was modified with N-glycosylation and localized on the surface membrane by the C-terminal GPI anchor. A C-terminally truncated version of hiAP lacking the GPI anchor signal peptide was secreted into the medium as an active enzyme. In a first approach to establish a high level expression system up to 14,000 U/liter were produced in a time frame of two days, which exceeds the production rate of other published expression systems for this enzyme.ConclusionsWith the expression of hiAP, not only a protein of commercial interest could be produced, but also a reporter enzyme that offers the possibility to analyze T. thermophila genes that play a role in the regulation of protein secretion. Additionally, the fact that ciliates do not secrete an endogenous alkaline phosphatase provides the possibility to use the truncated hiAP as a reporter enzyme, allowing the quantification of measures that will be necessary for further optimization of the host strains and the fermentation processes.

Dataset Information

Developmental expression of surface antigen genes in the parasitic ciliate Ichthyophthirius multifiliis.

Publications

Developmental expression of surface antigen genes in the parasitic ciliate Ichthyophthirius multifiliis.

Similar Datasets

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