Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1998-04-22
2000-07-11
Ketter, James
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435471, 4352581, C12P 2102
Patent
active
060871245
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a process for expressing a heterologous DNA in a novel expression system or host.
Ciliates are single-cell, animal eukaryotes. They possess virtually all the typical properties of eukaryotic cells and at the same time offer the advantage that they can be cultured like prokaryotes. This means that genetically identical clones can be raised from individual cells by means of vegetative replication. In connection with this, very high cell densities can be achieved in continuous or batch culture in the case of some species. Furthermore, like most eukaryotes, ciliates can reproduce sexually. The sexual reproduction, termed conjugation in the case of the ciliates, can be induced by bringing cells of different mating type (mating types are "multiple sexes") into contact under suitable conditions. This property offers the advantage that ciliates can be crossed like higher eukaryotes and strains can, for example, thereby be produced which are homozygous for selected features.
In addition to the features which are typical for most eukaryotes, ciliates possess a number of structural and functional peculiarities which make them particularly suitable both for fundamental cell-biological research and for biotechnological applications. Thus, two types of cell nucleus, which are differentiated in differing ways, are present in almost all ciliate cells: small, transcriptionally inactive, usually diploid micronuclei, and DNA-rich macronuclei which are usually very large. The micronuclei have functions which are in the main generative, i.e. haploid gamete nuclei are formed from them by meiosis during the course of conjugation. The gamete nuclei of two conjugation partners can fuse to form zygote nuclei and produce a new cell generation harboring genetically recombined micronucleus genomes. The zygote nuclei of the new generation produce new macronuclei by means of division. The macronuclei control all the somatic processes of the cells. Their genome is transcribed permanently. Many kinds of drastic elimination and reorganization events take place during the course of macronucleus development. In some species, up to 98% of the micronucleus genome is eliminated, intervening sequences are excised from genes, and coding regions can be rearranged in a completely novel manner ("gene scrambling"). In all ciliates which have been examined in this regard, the genes in the macronucleus are to a greater or lesser extent strongly amplified. Depending on the gene and ciliate species under consideration, the copy numbers can be as high as several millions.
In the very recent past, the molecular biological peculiarities of ciliate genomes have led to some sensational discoveries. Thus, for example, self-splicing introns (ribozymes) were found for the first time in highly amplified ciliate genes (Cech, T. R., B. L. Bass (1986): Ann. Rev. Biochem. 55, 599-629). The structure of telomeres, the terminal structures of linear DNA molecules, and of the telomerases which synthesize them, were likewise elucidated for the first time in ciliates (Blackburn, E. H. (1991): Nature 350, 569-573). It was subsequently found that these fundamental processes and structures, which were initially discovered in ciliates owing to their unusual genome structure, are also characteristic of almost all other eukaryotes; it is just that they are much more difficult to discover and investigate in other eukaryotes.
The peculiarities of the ciliates which have been outlined make the use of these organisms seem particularly rewarding for biotechnological purposes as well. Summarized briefly, the following reasons support this assumption: cultures, like prokaryotic microorganisms, at high cell density and with relatively short generation times. prokaryotes lack, for example in the areas of DNA replication, of transcription and processing, of translation, of cytoskeletal structure and membrane structure, of endocytic and exocytic processes, etc. from the common genealogical tree at a late stage, their enzymes, their structural and membrane p
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Kiy Thomas
Steinbruck Gunther
Hoechst Research & Technologies GmbH & Co. KG
Ketter James
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