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M C Yao

Publications and source records attributed to M C Yao.

At least 19 recordsLinked to original sources
PubMed1991

Short inverted repeats at a free end signal large palindromic DNA formation in Tetrahymena.

Large palindromic DNAs are formed in many cell types, but their molecular mechanism is unknown. During nuclear differentiation in Tetrahymena, the ribosomal RNA genes (rDNA) are converted from a single integrated copy to an extrachromosomal head-to-head palindrome. Using in vitro mutagenesis and Tetrahymena transformation, we show that two properties of the rDNA are necessary and sufficient for palindrome formation. The first is a pair of 42 bp inverted repeats found at the rDNA's 5' end. Its inverted symmetry, but not specific sequence, is important. The second is a free end next to the repeats. It is normally created by chromosome breakage in vivo, but can also be provided by restriction endonuclease cutting before transformation. We also demonstrate that the ability to form palindromes is not restricted to developing nuclei, but is present in vegetative cells as well. This process may represent a general mechanism for palindrome formation in eukaryotes.

Animals↗
PubMed1991

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

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 approximately 1% of injected 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 injected 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.

Amino Acid Sequence↗
PubMed1991

A mutation in the large subunit ribosomal RNA gene of Tetrahymena confers anisomycin resistance and cold sensitivity.

Anisomycin, an antibiotic that specifically inhibits the peptidyl transfer function of eukaryotic ribosomes, has been used to select resistant mutants in Tetrahymena thermophila. A mutation conferring anisomycin resistance (an-r) has been localized to a 1.2-kb fragment of the large subunit ribosomal RNA (rRNA) gene by transformation via microinjection. A single base pair change was detected within this region. Nine independently isolated an-r mutants had the same base pair change. T. thermophila strains that are homozygous for this mutation are cold sensitive, unable to mate and grossly abnormal in cell morphology.

Animals↗
PubMed1990

The controlling sequence for site-specific chromosome breakage in Tetrahymena.

Site-specific chromosome breakage occurs in many ciliated protozoa during nuclear differentiation. We have determined the cis-acting sequence that controls this process in Tetrahymena thermophila. The Tetrahymena ribosomal RNA gene is bounded by two breakage sites. Injection of this gene into developing macronuclei leads to breakage at these sites. Deletion analysis has localized the sequences essential for breakage to a 28 bp region that includes a 15 bp sequence (Cbs) known to be present in other breakage sites. Insertions of Cbs allow breakage to occur at new sites, which is accompanied by elimination of surrounding DNAs and formation of telomeric sequences, as it is at natural sites. Thus, Cbs is the necessary and sufficient sequence signal for chromosome breakage in Tetrahymena.

Animals↗
PubMed1990

A programmed site-specific DNA rearrangement in Tetrahymena thermophila requires flanking polypurine tracts.

During macronuclear development in ciliates, precise deletion events eliminate thousands of specific DNA segments. Each segment is bounded by a unique pair of short direct repeats, but no other common feature has been reported. To determine the critical cis-acting sequences, we developed an in vivo system for analyzing this process in Tetrahymena. We show that sequences essential for recognition and excision of one such region are located within the 70 bp of DNA flanking either side of it. Three authentic splice sites and one cryptic site are each adjacent to an unusual polypurine tract (5'-A5G5) situated 40-50 bp distal to each terminal repeat. Removal of this tract or substitution of 3 bp within it abolishes splicing to the adjacent site. The normal chromosomal environment and the integrity of the eliminated sequence are not required for its removal. We believe the polypurine tract is a signal essential for excision of this sequence.

Animals↗
PubMed1989

Sequence microheterogeneity is generated at junctions of programmed DNA deletions in Tetrahymena thermophila.

Regulated DNA deletions are known to occur to thousands of specific DNA segments in Tetrahymena during macronuclear development. In this study we determined the precision of this event by examining the junction sequences produced by three different deletions in many independent caryonidal lines. 0.9 kb deletions in region M produce at least 3 types of junction sequences, of which two have been determined and found to be different by 4 bp. The alternative 0.6 kb deletions in this region are much less variable. 1.1 kb deletions in region R, known from a previous study to be slightly variable, produce two types of junction sequences which are different from each other by 3 bp. Thus, developmentally regulated deletions in Tetrahymena can produce sequence microheterogeneity at their junctions. This process contributes significantly to the diversification of Tetrahymena's somatic genome.

Animals↗
PubMed1989

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

The free, linear macronuclear ribosomal RNA genes (rDNA) of Tetrahymena are derived from a unique copy of micronuclear rDNA during development. We have injected cloned copies of the micronuclear rDNA that have been altered in vitro into developing macronuclei and obtained transformants that express the paromomycin-resistant phenotype specified by the injected rDNA. In most cases, these transformants contain almost exclusively the injected rDNA which has been accurately processed into macronuclear rDNA. Mutants with a 119 bp insertion at three points in the transcribed spacers and at two points in the 26S rRNA coding region were tested. Cells containing these spacer mutant rDNAs are viable, although one of them grows slowly. This slow-growing line contains the insertion between the 5.8S and 26S rRNA coding regions and accumulates more rRNA processing intermediates than control lines. One of the 26S rRNA mutants failed to generate transformants, but the other did. These transformants grew normally, and produced 26S rRNA containing the inserted sequence. A longer insertion (2.3 kb) at the same four points either abolished transformation or generated transformants that retained at least some wild-type rDNA. This study reveals that some rRNA sequences can be altered without significantly affecting cell growth.

Animals↗
PubMed1989

Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.

Animals↗
PubMed1988

Transformation of Tetrahymena thermophila with hypermethylated rRNA genes.

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

Animals↗
PubMed1988

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

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. Allis, and M.-C. Yao, Proc. Natl. Acad. Sci. USA 81: 7383-7387). 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.

Animals↗
PubMed1987

A conserved nucleotide sequence at the sites of developmentally regulated chromosomal breakage in Tetrahymena.

Chromosomal breakage occurs at hundreds of specific sites in Tetrahymena, including the two ends of the unique ribosomal RNA genes, during the development of the somatic macronucleus. We have identified a 15-nucleotide sequence that occurs widely in the germinal micronuclear genome and is associated exclusively with chromosomal breakage sites. When copies of this sequence were cloned and analyzed, they were found in all cases to be located at or very near sites of breakage. This general rule is further supported by the observation that in a different site in which a single nucleotide substitution is found within this sequence, no chromosomal breakage occurs. The complete sequence structure of one of the breakage junctions has also been determined in both the germinal DNA and the two somatic DNA termini. This structure reveals that the 15-nucleotide conserved sequence is located within a 54-nucleotide region that is removed following chromosomal breakage.

Animals↗
PubMed1987

Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila.

DNA deletion by site-specific chromosome breakage and rejoining occurs extensively during macronuclear development in the ciliate Tetrahymena thermophila. We have sequenced both the micronuclear (germ line) and rearranged macronuclear (somatic) forms of one region from which 1.1 kilobases of micronuclear DNA are reproducibly deleted during macronuclear development. The deletion junctions lie within a pair of 6-base-pair direct repeats. The termini of the deleted sequence are not inverted repeats. The precision of deletion at the nucleotide level was also characterized by hybridization with a synthetic oligonucleotide matching the determined macronuclear (rejoined) junction sequence. This deletion occurs in a remarkably sequence-specific manner. However, a very minor degree of variability in the macronuclear junction sequences was detected and was shown to be inherent in the mechanism of deletion itself. These results suggest that DNA deletion during macronuclear development in T. thermophila may constitute a novel type of DNA recombination and that it can create sequence heterogeneity on the order of a few base pairs at rejoining junctions.

Animals↗
PubMed1986

Sequence characterization of Tetrahymena macronuclear DNA ends.

Tetrahymena is a ciliated protozoan which has two nuclei: a micronucleus, which maintains the genetic continuity of the cell, and the macronucleus which is derived from the micronucleus after sexual conjugation. A macronuclear DNA library was constructed to contain DNA ends. A probe containing C4A2 repeats which are known to be present at macronuclear DNA ends (1) was used to screen the library. Three clones were characterized by sequencing, restriction enzyme mapping and Bal 31 digestion. The data indicate that these three clones represent macronuclear DNA ends which were generated by DNA fragmentation during macronuclear formation. The sequencing data at the C4A2 repeat junction show a conserved sequence of five nucleotides, TTATT. Sequences further away show no obvious homologies except that they are highly enriched in AT. This structure is quite different from the subtelomeric sequences of other organisms.

Animals↗
PubMed1986

Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes.

The ribosomal RNA genes (rDNA) of Tetrahymena thermophila macronucleus exist as free linear 21-kilobase molecules that contain replication origins and telomeres. A mutation in this gene confers resistance to the antibiotic paromomycin. We have isolated rDNA from such a mutant (strain p2f), microinjected it into the macronucleus of a sensitive strain, and obtained drug-resistant cells at a frequency of 1-3%. The transformed cells have a distinct and stable phenotype. The rDNA of the transformants contains the expected sequences of the mutant rDNA as determined by oligonucleotide hybridization. rDNA from a different inbred line (C3-368), which contains heteromorphic restriction sites, has also been used for injection, and the results confirm the fact that the injected rDNA is indeed present in the transformants. Injection of rDNA from the C3 strains also increases the transformation frequency 5- to 10-fold and leads to the total replacement of the resident rDNA of the B-inbred strains. This is presumably due to the replication dominance of rDNA from the C3 strains over that of the B strains. Using this method, we have also been able to transform developing cells, at similar frequencies, by microinjecting into the macronuclear anlagen.

Alleles↗
PubMed1985

Macronuclear DNA of Tetrahymena thermophila exists as defined subchromosomal-sized molecules.

Using the method of orthogonal-field-alternation gel electrophoresis, we have resolved the macronuclear DNA of Tetrahymena thermophila into a series of distinct bands. Using electrode switching intervals ranging from 10 to 70 seconds we have resolved DNA bands ranging in size from about 21 kb up to and beyond the size of yeast chromosomes VII and XV. Hybridization of Southern blots from these gels to both unique and repetitive DNA sequences shows that the macronuclear genome of T. thermophila has a precise organization. The unique sequences tested each hybridize to only one band of macronuclear DNA and the hybridization patterns seem to be identical in several inbred strains examined.

Animals↗
PubMed1985

Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA.

Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development. In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction. The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development. The breakage event is associated with DNA elimination and telomeric sequence addition. Similar results were also found previously for the 3' junction of this gene. These results could explain how the extrachromosomal rDNA is first generated. Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found. These sequences might serve as signals for site-specific breakage. In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene. The repeats are 42 nucleotides long and are separated by 28 nucleotides. The existence of this structure provides a simple explanation for the formation of the palindromic rDNA.

Base Sequence↗