Multiple redundant sequence elements within the fission yeast ura4 replication origin enhancer - PubMed (original) (raw)
Multiple redundant sequence elements within the fission yeast ura4 replication origin enhancer
S M Kim et al. BMC Mol Biol. 2001.
Abstract
Background: Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized.
Results: Systematic deletion of consecutive segments of approximately 50, approximately 100 or approximately 150 bp within the enhancer and its adjacent core origin (ars3002) revealed that several of the approximately 50-bp stretches within the enhancer contribute to its function in partially redundant fashion. Other stretches within the enhancer are inhibitory. Some of the stretches within the enhancer proved to be redundant with sequences within core ars3002. Consequently the collection of sequences important for core origin function was found to depend on whether the core origin is assayed in the presence or absence of the enhancer. Some of the important sequences in the core origin and enhancer co-localize with short runs of adenines or thymines, which may serve as binding sites for the fission yeast Origin Recognition Complex (ORC). Others co-localize with matches to consensus sequences commonly found in fission yeast replication origins.
Conclusions: The enhancer within the ura4 origin cluster in fission yeast contains multiple sequence motifs. Many of these stimulate origin function in partially redundant fashion. Some of them resemble motifs also found in core origins. The next step is to identify the proteins that bind to these stimulatory sequences.
Figures
Figure 1
The ura4 gene and its nearby cluster of replication origins. The diagram is based on the nucleotide sequence of S. pombe cosmid c330 (EMBL accession number SPCC330). The numerical scale indicates nucleotide sequence position within SPCC330. Cut sites for selected restriction enzyme are shown in blue. The locations of the two characterized (ura4 and pmp20) and uncharacterized (SPCC330.07c and SPCC330.08) open reading frames and their putative directions of transcription are shown by the magenta arrows. Replication origins are indicated by green boxes. Lighter green boxes are more efficient chromosomal replication origins than darker green boxes [7,8,9]. The replication enhancer (RE) is shown in yellow.
Figure 2
Nucleotide sequence of the modified extended ars3002 showing the boundaries of the ~50-bp deletions. The nucleotide sequence with gray background is the modified extended version of ars3002, cloned into the vector pUra4script (see Materials and Methods). The flanking nucleotide sequences with white background show portions of the vector sequence. Yellow highlighting indicates the inserted _Cla_I linker, and light orange highlighting indicates the modified _Eco_RI site. The sequence with light gray background corresponds to core ars3002 (nucleotides 19,312 to 20,125 of SPCC330; see legend to Fig. 1), while the sequence with dark gray background corresponds to the replication enhancer (nucleotides 20,126 through 20,523 of SPCC330). Each numbered orange or green line indicates the boundaries of the corresponding ~50-bp deletion.
Figure 3
Effects of deleting stretches of ~50-bp from (a) extended ars3002 or (b) core ars3002. In (a), transformation frequencies (blue bars) and mean colony sizes (gold bars) are shown relative to those of modified extended ars3002 (sequence in Fig. 2). Each bar represents the mean of four independent experiments, and the error bars show standard deviations. Controls include intact, extended ars3002 (Ext), intact core ars3002 (Core), and vector alone (Vector; pUra4script [10]). Panel (b) shows our previously published [10] transformation frequency results for deletions Δ1-Δ14 in core ars3002, in the absence of the enhancer. Here the values are relative to intact core ars3002, which is a much less efficient origin than extended ars3002 (see panel (a)). In panel (b), the results are the averages of two experiments, and the error bars show the data range.
Figure 4
Correlation between plasmid retention rate and colony size. Groups A, B and C consist of deletion mutants producing large, medium and small colonies, respectively. See the text for details. Images of colonies on Petri plates were photographed at the same magnification 5 days after transformation. Plasmid retention rates are shown with standard deviations and number of transformants tested (in parentheses).
Figure 5
Effects of deleting stretches of (a) ~100 bp and (b) ~150 bp from extended ars3002. See the legend to Fig. 3a for details.
Figure 6
Nucleotide sequence of deletion regions 15-22 showing near matches to S. pombe replication origin consensus sequences. The sequence shown here is the same as in the bottom portion of Fig. 2. The light green 11-bp sequence is a 10/11 match to the consensus sequence of Maundrell et al. [13]. The blue 30-bp sequence is a 20/30 match to the consensus sequence of Clyne and Kelly [18]. The 14-bp stretch highlighted in light magenta consists of 3 overlapping 11/12 matches to the consensus sequence of Zhu et al. [9].
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