An intron in a ribosomal protein gene from <i>Tetrahymena</i>

Nielsen, Henrik; Andreasen, Per Hove; Dreisig, Hanne; Kristiansen, Karsten; Engberg, Jan

doi:10.1002/j.1460-2075.1986.tb04555.x

Cited by 41 publications

(10 citation statements)

References 36 publications

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“…Gene Cloning and DNA Manipulation. The cDNA clones were isolated from a T. thermophila (strain B1868VII) cDNA library provided by Per Hove Andreasen, and were believed to be enriched for ribosomal protein mRNAs (13 20-,ul drop) on Petri dishes and grown for 3 days. About 10% of cells in each drop were then replicated into media drops containing 50-100 ,ug of paromomycin per ml as described (16).…”

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Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.

Yao

1991

Proc. Natl. Acad. Sci. U.S.A.

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A method for transforming Tetrahymena has been established earlier, but its application has been limited because of the lack of selectable markers other than the rRNA-encoding DNA (rDNA). Mutations in the yeast ribosomal protein L29 gene (CYH2) are known that confer cycloheximide resistance. We have cloned and sequenced the homologue of this gene from both a wild-type and a cycloheximide-resistant (ChxA) strain of Tetrahymena. Surprisingly, a comparison shows that the ChxA mutation is not present in the CYH2 homologue. We therefore created the yeast mutations in the Tetrahymena gene by site-directed mutagenesis and used them to transform Tetrahymena either with or without linking to an rDNA vector. All clones transformed by the rDNA vector also became resistant to cycloheximide when the rDNA contained the engineered mutant genes. Without the rDNA vector, the mutant genes transform ='1 % of iu'ected cells to become resistant to cycloheximide. DNA analysis indicates that transformation occurs by replacement of the host sequence and not by random integration of the iu'ected sequence. The replacement occurs to some but not all copies of this gene in the polyploid macronuclear genome. Thus, transformation in Tetrahymena occurs by specific sequence replacement, and the injected mutant genes can serve as dominant selectable transformation markers in this organism.DNA-mediated transformation is an important tool for genetic studies of an organism. The ciliated protozoan Tetrahymena thermophila has been a favored model system for the studies of many interesting problems, including RNA splicing, telomere structure, gene regulation, and DNA rearrangements. To facilitate these and other studies, a method for transforming Tetrahymena was developed in this laboratory several years ago (1). It exploited the unique features of the ribosomal RNA genes (rDNA) of this organism, and used its drug-resistant alleles as selectable markers and its replication origin for maintenance in the cell. To date all reported transformation studies in Tetrahymena rely on the use of the rDNA. While the method has been instrumental in many studies, it also has serious limitations. For instance, the rDNA exists in the macronucleus as high-copy-number extrachromosomal molecules. In addition, they are transcribed actively by RNA polymerase I and are localized in nucleoli (reviewed in ref.2). The effects of these properties on the expression of other genes linked to it are not known, causing concerns over its use as a general vector.Our efforts to adapt foreign drug-resistant genes to serve as selectable markers in Tetrahymena had only limited success (unpublished results). Thus, we decided to explore the possibility of using native Tetrahymena genes for this purpose.

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Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.

Yao

1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Relationships between ribosomal proteins of different organisms can also be inferred from evolutionarily conserved rRNA domains (14); proteins from different organisms that each bind the same rRNA sequence or structure can be assumed to share a similar function and/or location in the ribosome (15)(16)(17)(18). Finally, deductions from DNA sequences have revealed amino acid homologies among ribosomal proteins from different organisms, homologies that suggest equivalent function (19)(20)(21)(22)(23)(24)(25).…”

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Functional substitution of mouse ribosomal protein L27' for yeast ribosomal protein L29 in yeast ribosomes.

Fleming

Belhumeur

Skup

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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A cDNA clone of mouse ribosomal protein L27' was shown previously to be 62% identical in amino acid residues to yeast ribosomal protein L29. The L27' cDNA was expressed in yeast to determine the ability of the mouse protein to substitute for yeast L29 in assembling a functional ribosome. In a yeast strain resistant to cycloheximide by virtue of a recessive mutation in the L29 protein, the murine cDNA did not produce a sensitive phenotype, indicating failure of the mouse L27' protein to assemble into yeast ribosomes. However, when the mouse L27' gene was expressed in cells devoid of L29 and otherwise inviable, the murine protein supported normal growth, demonstrating that mouse ribosomal protein L27' indeed was interchangeable with yeast L29. We conclude that mouse ribosomal protein L27' is assembled into ribosomes in yeast, but yeast L29 is assembled preferentially when both L29 and L27' are present in the same cell.The ribosome is an organelle whose constituent building blocks have been extensively catalogued. With identification of ribosomal components completed, attention has turned toward deducing the arrangement of the various protein and RNA moieties in the overall structure as well as assigning functions to individual or groups ofribosomal components. A particularly profitable means of determining the function of individual ribosomal components has been through genetic and biochemical analysis of mutants resistant or hypersensitive to inhibitors of protein synthesis (1, 2). Another approach has been in vitro reconstitution of ribosomes (3); omission of specific ribosomal proteins from reconstitution mixtures often results in absence of a specific function in the completed particles, thereby implicating the missing protein in that function (4)(5)(6). Although eukaryotic ribosomes have been reconstructed with groups ofribosomal proteins (7-9) or subunits (10)

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“…It seems unlikely that the deletions destroy or create protein-coding regions. Known protein-coding sequences in T. thermophila are <60% A+T (6,13,17,18,24,33), while the sequenced DNAs from region M are rather uniformly A+T rich, ranging from 76 to 85% A+T. All three deletion junctions contain the 5-bp sequence 5'-TAATT-3' arranged in the same orientation. In the case of the 0.6-kb deletion, the repeat is expanded to 8 bp as 5'-AATAATTG-3' ( Fig.…”

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Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila.

Austerberry¹,

Yao²

1988

Mol. Cell. Biol.

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Deletions of specific DNA sequences are known to occur in Tetrahymena thermophila as a developmentally regulated process. Deletions of a particular region (region M) were previously shown to be of two alternative sizes, 0.6 or 0.9 kilobases (kb) (C. F. Austerberry, C. D. Alis, and M.-C. Yao, Proc. Nati. Acad. Sci. USA 81: [7383][7384][7385][7386][7387]. In this study, the nucleotide sequences for both deletions were determined. These two deletions share the same right junction, but their left junctions are 0.3 kb apart. An 8-base-pair (bp) sequence is present at both junctions of the 0.6-kb deletion, but only 5 bp of this direct repeat are present at the left junction of the 0.9-kb deletion. Further comparison revealed a common 10-bp sequence near each of the two left junctions and a similar sequence in inverted orientation near the right junction. These sequences may play a role in the developmental regulation of the deletion process.Elimination of germ line-specific sequences from developing somatic nuclei, which was first observed (as "chromatin diminution") a century ago (10), has now been shown to occur in several species of organisms (reviewed in references 2, 3, 8, 9, 11, 22, 29, and 30). In the holotrichous ciliate Tetrahymena thermophila, 10 to 20% of the DNA sequences in the micronucleus (germ line nucleus) are selectively eliminated (35) from the somatic macronucleus (16). Most of this elimination is the result of internal deletion (12,34,36), which occurs in more than 5,000 specific DNA segments during a 2-h period of differentiation (4, 34).We have previously cloned and analyzed the DNAs containing two neighboring deletion sites in T. thermophila which we have named region R and region M (4, 5, 34). Deletion in region R consistently eliminates 1.1 kilobases (kb) of DNA; the sequences in this region are the only such sequences in T. thermophila reported to date (5). A surprising result from these studies was the absence of obvious sequence structures at or near the deletion junctions that might appear to be sufficient to account for the high efficiency and site specificity of this deletion (4, 5).To further understand DNA deletions we have characterized the sequence structure of the deletion in region M. Unlike the deletion in region R, this deletion occurs in two alternative ways, eliminating either 0.6 or 0.9 kb of DNA (4). Alternative DNA deletions have also been suggested for several other T. thermophila DNAs (19,31). Analysis of the region M sequence should provide not only a second example of a sequence involved in deletion in T. thermophila but also the molecular basis for alternative DNA deletions.To rule out the possibility that heterozygous alleles in the germ line are responsible for generating the two alternatively sized deletions in region M, we established cell lines with homozygous genomes from individual progeny (caryonides) of the second round of genomic exclusion matings (CU427 or CU428 x A*III) (1). Southern blots (28) of HindlIl-digested DNAs of these caryonidal lines were hybridi...

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An intron in a ribosomal protein gene from Tetrahymena

Cited by 41 publications

References 36 publications

Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.

Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.

Functional substitution of mouse ribosomal protein L27' for yeast ribosomal protein L29 in yeast ribosomes.

Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila.

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