Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila.

Sweeney, Rosemary; Yao, Meng-Chao

doi:10.1002/j.1460-2075.1989.tb03454.x

Cited by 45 publications

(41 citation statements)

References 29 publications

(14 reference statements)

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“…Upon introduction of pD5H8 into the new MAC, the rDNA is excised from the vector and forms an rDNA chromosome. At the 39 nontranscribed region of the rDNA, we can insert a sequence of interest (in this study, we used M-IES and its flanking sequences) without disturbing the functions of the rDNA (Sweeney and Yao 1989). The unique NotI site was made in the middle of M-IES where we can insert the target DNA sequence ( Figure 1B).…”

Section: Codel Occurs At Many Target Locimentioning

confidence: 99%

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

Hayashi¹,

Mochizuki

2015

Genetics

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Tetrahymena is a useful eukaryotic model for biochemistry and molecular cell biology studies. We previously demonstrated that targeted ectopic DNA elimination, also called co-Deletion (coDel), can be induced by the introduction of an internal eliminated sequence (IES)-target DNA chimeric construct. In this study, we demonstrate that coDel occurs at most of the loci tested and can be used for the production of somatic gene KO strains. We also showed that coDel at two loci can be simultaneously induced by a single transformation; thus, coDel can be used to disrupt multiple gene loci in a single cell. Therefore, coDel is a useful tool for functional genetics in Tetrahymena and further extends the usefulness of this model organism.KEYWORDS Tetrahymena; DNA elimination; RNAi; gene knockout T HE ciliated protozoan Tetrahymena thermophila can grow at an exceptionally high rate (its doubling time is approximately 2 hr) and can reach a high density (a few million cells per milliliter) under simple and inexpensive culture conditions (reviewed in Orias et al. 2000). In combination with robust genetic manipulation methods (reviewed in Chalker 2012), Tetrahymena is a useful model eukaryote for biochemistry and molecular cell biology studies (reviewed in Collins and Gorovsky 2005).Three strategies for loss-of-function genetic studies have been established for this organism. The first strategy is a germline knockout (KO), in which one of the two gene copies in the diploid micronucleus (MIC) is replaced with a drug-resistance gene by homologous recombination, and two heterozygous strains are then sexually crossed to obtain a homozygous germline KO strain (Cassidy-Hanley et al. 1997). The second strategy is somatic KO, in which one of the 45 copies of a gene in the polyploid macronucleus (MAC) is replaced with a drugresistance gene by homologous recombination, and "phenotypic assortment" is used to obtain cells with the drug-resistance gene at all loci (Merriam and Bruns 1988). Phenotypic assortment is possible because MAC chromosomes are randomly segregated into daughter cells in vegetative cell division and a stepwise increase of the drug in culture selects for cells that have more drug-resistance genes. The final strategy is gene knockdown (KD) by RNA interference (RNAi), in which a construct expressing long hairpin RNA that is complementary to the gene of interest is introduced into a nonessential locus in the MAC and small RNAs produced from the hairpin RNA posttranscriptionally silence the expression of the gene (HowardTill and Yao 2006).Although the first two gene KO strategies have been reliably used to study individual gene functions, they are not suitable for high-throughput genetic screening because the integration of a KO construct into the MIC germline occurs at very low efficiency and the phenotypic assortment process used in somatic KOs require the careful control of drug concentrations in each strain and culture step. Moreover, it takes 1 month to obtain KO strains using these methods. For germline KO, t...

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Section: Codel Occurs At Many Target Locimentioning

confidence: 99%

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

Hayashi¹,

Mochizuki

2015

Genetics

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“…Mating T. thernophila cells can be transformed via either microinjection (42,51) or electroporation (14) with a micronuclear rDNA vector that confers paromomycin resistance (pm-r) (51). This rDNA is converted to macronuclear form and can completely replace the resident rDNA when transformants are maintained under selection (41,51).…”

Section: Fig 1 Positions Of Prominent Variable Regions Prominent Vmentioning

confidence: 99%

An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.

1994

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Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably.Although much of the secondary structure of rRNA is evolutionarily conserved, these portions are interspersed with discrete variable regions at conserved positions within the structure (18). The more prominent ones have been identified as expansion segments (7,46), divergent domains (27), or variable regions (17). The variability of some regions is such that closely related organisms such as mice and rats show many sequence differences (26), and sequence differences are even detected in different copies of the rRNA genes (rDNAs) from a single organism (25). These regions are absent in most eubacteria and archaebacteria and present in most eukaryotes (19,46), although exceptions to this rule have recently been found (4, 10, 43). The functional role of variable regions has not been identified. While their absence from most eubacteria suggests that they are dispensable, their almost universal presence in eukaryotes implies that they may be important in these organisms.Two models for the evolution of rRNA both imply that variable regions initially had no functional role. Some rRNAs are fragmented (2,5,12,13,15,36,39,45,47) (44). Comparison of rRNA sequences from diverse phyla offers support for the secondary structure model of most of the LSU rRNA but is uninformative for variable regions because they are so divergent. Secondary structure models for variable regions have been proposed based on the assumption that, in addition to having a low free energy, a model must be structurally similar for all species (26). Comparing the sequences of closely related species has recently produced more-refined models (12,20).No functional role has been defined for any variable region. However, some pertinent evidence does exist. The D2 and D12 regions of the LSU rRNA in Tetrahymena thennophila tolerate insertion mutations with no apparent diminution of rRNA function in vivo, while insertions in the D8 region totally obliterate rRNA function (40a, 41). In Saccharomyces cerevisiae, rRNAs beari...

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“…pD5H8 was created by recombining two modified versions of 94701: the 5' half of one in which all but 199 bp of the 5' flanking sequences was deleted by exonuclease III digestion (Tt947-400) {Yao et al. 1990), and the 3' half of the other, which contains a polylinker at its 3' spacer region (947H8) (Sweeney and Yao 1989), into which fragments of interest can be inserted.…”

Section: Plasmid Constructionmentioning

confidence: 99%

A distant 10-bp sequence specifies the boundaries of a programmed DNA deletion in Tetrahymena.

Godiska

James²,

Yao³

1993

Genes & Development

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Programmed DNA deletion occurs at thousands of specific sites in most ciliates studied. To understand the mechanism of this prominent DNA rearranging process, we analyzed one of the deletion elements {the M-element) in Tetrahymena by making specific mutations in cloned DNAs and testing their effects on rearrangement in vivo. We found that a 10-bp polypurine sequence {5'-AAAAAGGGGG1 plays a crucial role. This sequence is located at a short distance {-45 bpl outside of the element on either side. Removal of it abolishes the deletion process. Moving it short distances away causes the deletion boundary to move with it. Insertion of this sequence into a site within the element induces new boundaries to form near the insertion site. Sequence analysis reveals that all new boundaries created are 41-54 bp away from the sequence. Thus, this sequence is necessary and sufficient to determine the boundaries of DNA deletion, and it does so from a short distance outside of the element. These results offer a possible explanation for the control of DNA deletion in ciliates and suggest that a new type of mechanism for site-specific DNA rearrangements is involved.

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Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila.

Cited by 45 publications

References 29 publications

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.

A distant 10-bp sequence specifies the boundaries of a programmed DNA deletion in Tetrahymena.

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