We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.
The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.
The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10 per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmml mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmml. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4IB-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 104 copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophila.The rRNA genes (rDNA) in the ciliated protozoan Tetrahymena thermophila provide an excellent opportunity for studying DNA replication in a eucaryotic system. In the somatic nuclei of T. thermophila, the rDNA is a relatively short (21-kilobase pair [kb] somally integrated rDNA copy in each haploid genome. During macronuclear development, the rDNA is excised from the chromosome and converted into a 21-kb palindromic molecule. The palindrome is made up of two inversely oriented copies of the micronuclear rDNA sequence. The extrachromosomal rDNA acquires telomeres (1) and is amplified to a copy number of about 104 molecules per macronucleus. These maturation events are completed prior to the onset of vegetative growth, approximately 18 h after conjugation is initiated. The macronuclear rDNA is maintained at a high copy number throughout vegetative cell divisions. Figure 1A diagrams one-half of an rDNA palindrome. The origin of replication is within the 5' nontranscribed spacer (5'NTS), upstream from the transcription initiation site (Fig. 1A) (4). Two nuclease-hypersensitive regions within the 5'NTS (15), domains 1 and 2, have very similar sequences and contain evolutionarily conserved repeated sequence elements (5, 11). By mutational analysis, one of these repeated elements (type I) was implicated in the activation of rDNA replication (8).We have previously shown that rDNA from a T. thermophila inbred strain C3 (C3 rDNA) has a replicative advantage over rDNA from inbred strain B (B rDNA), causing C3 rDNA to completely replace B rDNA in C3/B heterozygotes during vegetative growth (8)....
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