DNA Restriction Enzymes

Publication Title: 
Cell

The amplification of ribosomal DNA during development of the somatic macronucleus in Tetrahymena thermophila was analyzed by genetic and molecular biological techniques. We have identified an alternate form of the rDNA, structurally distinguishable from the wild-type by an extra cutting site for Bam HI in its nontranscribed spacer. The altered rDNA was inherited in crosses in a simple Mendelian fashion, consistent with the presence of only one rRNA gene copy per haploid genome in the micronucleus.

Author(s): 
Pan, W. C.
Orias, E.
Flacks, M.
Blackburn, E. H.
Publication Title: 
Cell

The germ-line micronuclear genome of the ciliate Tetrahymena thermophila contains approximately 10(2) chromosome-internal blocks of tandemly repeated C4A2 sequences (mic C4A2). This repeated sequence is the telomeric sequence in the somatic macronucleus. Each of six cloned micC4A2 was found to be adjacent to a conserved 30 bp sequence, which we propose is the terminal inverted repeat of a family of DNA elements (the Tel-1 family).

Author(s): 
Cherry, J. M.
Blackburn, E. H.
Publication Title: 
Cell

We have constructed a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA to a yeast vector. Structural features of the terminus region of the Tetrahymena rDNA plasmid maintained in the yeast linear plasmid include a set of specifically placed single-strand interruptions within the cluster of hexanucleotide (C4A2) repeat units. An artificially constructed hairpin terminus was unable to stabilize a linear plasmid in yeast.

Author(s): 
Szostak, J. W.
Blackburn, E. H.
Publication Title: 
Nucleic Acids Research

To investigate the developmentally programmed telomere addition that accompanies chromosome fragmentation during macronuclear differentiation in Tetrahymena thermophila, five representative telomeric regions from the macronucleus were cloned and characterized in detail. The sequences adjacent to the telomeric (C4A2:T2G4) repeats on these five macronuclear ends had no significant sequence homology or shared secondary structure.

Author(s): 
Spangler, E. A.
Ryan, T.
Blackburn, E. H.
Publication Title: 
The Journal of Biological Chemistry

The chromatin structure of the 3'-nontranscribed spacer of the linear rRNA gene molecules of Tetrahymena thermophila was examined. This region includes the transcription termination site, two sets of recently identified conserved spacer repeats (Type IV and V repeats (Challoner, P. B., Amin, A. A., Pearlman, R. E., and Blackburn, E. H. (1985) Nucleic Acids Res. 13, 2661-2680], and the terminus of the molecule. Using sensitivity to nucleases as a probe, a unique chromatin structure was found in this rDNA region.

Author(s): 
Budarf, M. L.
Blackburn, E. H.
Publication Title: 
The Journal of Biological Chemistry

The chromatin structure of the 3'-nontranscribed spacer of the linear rRNA gene molecules of Tetrahymena thermophila was examined. This region includes the transcription termination site, two sets of recently identified conserved spacer repeats (Type IV and V repeats (Challoner, P. B., Amin, A. A., Pearlman, R. E., and Blackburn, E. H. (1985) Nucleic Acids Res. 13, 2661-2680], and the terminus of the molecule. Using sensitivity to nucleases as a probe, a unique chromatin structure was found in this rDNA region.

Author(s): 
Budarf, M. L.
Blackburn, E. H.
Publication Title: 
Molecular and Cellular Biology

We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila. To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events. This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome.

Author(s): 
Howard, E. A.
Blackburn, E. H.
Publication Title: 
Proceedings of the National Academy of Sciences of the United States of America

We transformed the ciliate Tetrahymena thermophila by microinjection of circular plasmids containing the ribosomal RNA gene (rDNA). In the somatic macronucleus of Tetrahymena, the rDNA is in the form of linear palindromic molecules. The rDNA molecules from the C3 strain have a replication advantage over rDNA from both B strain and the C3 rDNA mutant rmm1. We constructed two circular plasmids carrying replication origin sequences from C3 rDNA and a point mutation (Pmr) in the 17S rRNA gene that confers resistance to the antibiotic paromomycin.

Author(s): 
Yu, G. L.
Hasson, M.
Blackburn, E. H.
Publication Title: 
Nucleic Acids Research

To investigate the developmentally programmed telomere addition that accompanies chromosome fragmentation during macronuclear differentiation in Tetrahymena thermophila, five representative telomeric regions from the macronucleus were cloned and characterized in detail. The sequences adjacent to the telomeric (C4A2:T2G4) repeats on these five macronuclear ends had no significant sequence homology or shared secondary structure.

Author(s): 
Spangler, E. A.
Ryan, T.
Blackburn, E. H.
Publication Title: 
Nucleic Acids Research

We have investigated the occurrence of methylated adenine residues in the macronuclear ribosomal RNA genes of Tetrahymena thermophila. It has been shown previously that macronuclear DNA, including the palindromic ribosomal RNA genes (rDNA), of Tetrahymena thermophila contains the modified base N-6-methyladenine, but no 5-methylcytosine. Purified rDNA was digested with restriction enzymes Sau 3AI, MboI and DpnI to map the positions and levels of N-6-methyladenine in the sequence 5' GATC 3'.

Author(s): 
Blackburn, E. H.
Pan, W. C.
Johnson, C. C.

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