To determine which nucleotide sequences are most important for core origin and enhancer function, we constructed consecutive ~50-bp deletions throughout extended ars3002. To simplify production of these deletion mutants, we destroyed the EcoRI site between the core ars3002 and the enhancer and introduced a ClaI linker (see Materials and Methods). This manipulation did not cause any detectable change in plasmid origin activity (data not shown). All deletions were constructed in this altered plasmid. They are called Δ1-Δ22. Their locations are shown in Figure 2, which also shows the positions of the inactivated EcoRI site (light orange highlight) and of the introduced ClaI linker (yellow highlight). Note that Δ1-Δ15 are located in core ars3002 and correspond in position precisely to the ~50-bp deletions previously studied in this region [10].

Figure 3a shows the average values of four independent transformation experiments with constructs containing (from left to right): intact extended ars3002 (Ext), ~50-bp deletions Δ1-Δ22, core ars3002 (lacking the replication enhancer), and vector alone. All results were calculated relative to the values generated by extended ars3002. Blue bars indicate relative transformation frequency (number of colonies generated per 100 ng input DNA), and gold bars indicate the relative average areas of the transformed colonies, which is a reflection of colony growth rate. In general, the more efficiently a plasmid replicates, the greater the number of transformed colonies and the faster those colonies grow under conditions that select for plasmid maintenance [7, 10,11,12]. Notice that the replication efficiencies of all the deletions, except Δ10, are greater than those of intact core ars3002 (core, Fig. 3a), which was the subject of our previous study [10].

For comparison purposes, the results from our previous study of core ars3002 are displayed in Fig. 3b. In the previous study, we measured replication efficiency only by the transformation frequency assay (blue bars). The results were expressed relative to those obtained for intact core ars3002. Note that our new results (Fig. 3a) show that the transformation frequency of intact core ars3002 is only about 40% that of extended ars3002. Thus the heights of the blue bars in Fig. 3b are exaggerated compared to those in Fig. 3a. The previous transformation frequency results (Fig. 3b) revealed that the Δ4-Δ14 regions are important for the transformation activity of core ars3002, and the Δ8 and Δ10 regions are essential. Our new transformation frequency results (blue bars in Fig. 3a) show that in the context of extended ars3002 with its replication enhancer, none of the ~50-bp deletion regions within the core is individually essential for transformation activity, and only the Δ10 region is important.

As discussed in detail below, we have found that when plasmid origin efficiency is generally high, colony size measurements are more sensitive indicators of small differences in origin activity than are transformation frequency measurements. For that reason we interpret the colony size reductions produced by deletions Δ11-Δ14 (Fig. 3a) to indicate that these deletions cause a small decrease in plasmid origin activity, even though these deletions have no effect on transformation efficiency.

Thus the transformation frequency and colony size results show that sequences within the enhancer partially or completely suppress the negative effects of deletions Δ4-Δ14 on the plasmid origin activity of core ars3002. The simplest explanation of this observation is that sequences within the enhancer-in combination with other core sequences-are partially or fully competent to carry out each of the replication functions mediated by each of the ~50-bp regions within the core. Note that the enhancer must work in combination with the core, because the enhancer by itself has no plasmid origin activity [7].

It is interesting that deletion Δ15, which is part of the core but was not previously tested due to a cloning problem [10], has a slight stimulatory effect on colony size, implying that region 15 may be a mild suppressor of plasmid origin activity. The stimulatory effect of deleting region 15 is more obvious in the double and triple deletion analyses to be presented below (Fig. 5).

The results in Fig. 3a also indicate that there is considerable sequence redundancy within the enhancer itself. None of the ~50-bp deletions within the enhancer (Δ16-Δ22) reduces transformation frequency. Therefore none of these regions is uniquely essential for enhancer activity. However, some of the deletions (Δ16-Δ20) moderately reduce colony size, indicating that each of the corresponding regions (16-20) makes a modest unique contribution to enhancer activity.

In Fig. 3a we used both transformation frequency and colony size to evaluate plasmid origin activity. Transformation frequency is the standard assay for plasmid origin activity in S. pombe [9, 12, 13]. Colony size measurement is usually more reproducible than transformation frequency measurement (compare the error bar sizes in Fig. 3a), but colony size measurement is a newer assay and not yet so widely used [7, 11, 12]. Furthermore, the results obtained with the two assays are not identical. Two of the mutants studied in Fig. 3a (Δ19 and Δ20) produce elevated transformation frequencies combined with slightly reduced colony sizes. Other mutants (Δ11-Δ14, Δ16) yield normal transformation frequencies and significantly reduced colony sizes. Only in the cases of Δ10 and core ars3002 are transformation frequency and colony size simultaneously reduced.

For these reasons, it seemed important to check the validity of colony size measurement by comparing it to an alternative well-established indicator of plasmid origin activity-plasmid retention rate under selective conditions (reviewed in [12]). Plasmid retention rate is the proportion of cells containing plasmid during exponential growth in selective medium. To simplify our measurements, we divided the mutants into three groups based on the relative colony size data in Fig. 3a. The mutants in group C (Δ10, Δ13 and Δ14) generated the smallest colonies (20-38 % of the area of those generated by the control, which was intact extended ars3002). The mutants in group B (Δ7, Δ11, Δ12, Δ16-Δ20) produced larger colonies (50-79 % of control). The rest of the mutants were in group A (colony size >80 % of control). Then we selected three mutants from each group and tested plasmid retention rate in selective medium for two to five independent transformants for each of the selected mutants.

Our results reveal a correlation between colony size (panels on the left side of Fig. 4; see also the relevant gold bars in Fig. 3a) and plasmid retention rate (right column in Fig. 4). The plasmid retention rates of the 3 tested mutants in group A (largest colonies) are 60-70%, for group B (medium-size colonies) they are 38-65%, and for group C (smallest colonies) they are 17-29%. This correlation suggests that colony size measurement provides a valid assay of plasmid origin activity in fission yeast. Note, however, that none of the tested group A mutants had a plasmid retention rate as high as that of extended ars3002 (98%), despite the fact that both their transformation frequencies and their colony sizes were equal to or slightly greater than those of extended ars3002 (Fig. 3a). We do not have an explanation for this discrepancy. It is possible that plasmid retention rate is more sensitive than colony size or transformation frequency to small differences in the plasmid origin activity of highly efficient replication origins. We also do not have an explanation for the fact that the Δ7 mutation led to colony sizes in the medium (group B) range but plasmid retention rates in the higher (group A) range (Fig. 4).

In the case of origins of somewhat lower (but still high) replication efficiencies, these and previous results [7, 11] suggest to us that colony size may be a more sensitive indicator of plasmid origin activity than is transformation frequency. These results also suggest that transformation frequency may be a good indicator of plasmid origin activity only when replication efficiency is relatively low. Consider, for example, deletions Δ11-Δ14. In Fig. 3a, where the effects of these deletions were measured in the context of extended ars3002 (with enhancer; efficient replication), these deletions had no effect on transformation frequency but did reduce colony size. Fig. 3b shows that when these same deletions were assayed in the absence of the enhancer (so overall replication efficiency was much lower), each deletion significantly reduced transformation frequency [10]. Qualitative results presented in our previous publication [10] showed that each deletion also reduced colony size. Thus colony size and transformation frequency measurements seem to vary together when replication efficiency is sufficiently low.

Because deletions of stretches of ~50 bp within the enhancer had no effects on transformation frequency and only mild effects on colony size (Fig. 3a), we also tested ~100-bp deletions (double deletions of adjacent ~50-bp stretches, Fig. 5a) and ~150-bp deletions (triple deletions of adjacent ~50-bp stretches; Fig. 5b) in the enhancer region.

The data in Figs. 3a and 5 suggest that regions 16-20 all make individual contributions to enhancer activity. Although individual deletion of region 21 does not have a measurable effect (Fig. 3a), loss of region 21 along with region 20 (Fig. 5a) does significantly reduce enhancer activity. Regions 15 and 22 appear to suppress enhancer activity. Although their individual loss does not have much effect-just a slight stimulation of colony size (Δ15) or transformation frequency (Δ22)-their loss is capable of totally suppressing the negative effects of simultaneous deletion of adjacent sequences (Δ16 and Δ17 or Δ20 and Δ21, respectively; Figs. 3a and 5).

Region 16 is the ~50-bp region whose deletion most affects enhancer activity (Fig. 3a). However, deletion of region 16 by itself affects only colony size. In contrast, deletions of two ~100-bp stretches (Δ16-17 and Δ20-21) severely reduce both colony size and transformation frequency (Fig. 5a). Two ~150-bp deletions (Δ16-18 and Δ19-21) have even more severe effects, reducing both transformation frequency and colony size to the levels of the enhancerless core, within experimental error (Fig. 5b). The progressively more severe phenotypes of single, double, and triple deletions indicate partial redundancy of function between the three ~50-bp stretches of which each of the two essential ~150-bp regions is composed.

Escherichia coli DH5α cells were used for cloning mutated ars3002 plasmids. The S. pombe strain ura4-D18 (ura4-D18 leu1-32 end1 h^-) [21] was used as the recipient strain for transformation assays. Cells were grown at 30°C in EMM [22] supplemented with uracil and leucine (150 mg each per liter) when not under selection or leucine (150 mg per liter) when under selection for uracil prototrophy.

To construct the plasmid, pUra4script:ars3002(ext), which contains the extended ~1.2 kbp ars3002 [7], the ~1 kbp EcoRI fragment containing the replication enhancer (Fig. 1) was cut and blunt-ended by treatment with Klenow polymerase (New England Biolabs) and dNTPs. The gel-purified fragment was ligated with ClaI linker and then cut with ClaI and HincII to produce the ~400-bp fragment containing the enhancer [7]. The gel-purified ~400-bp fragment was inserted into the ClaI and HincII sites of p3002PCR (the plasmid containing core ars3002 generated by PCR; [10]). The resulting plasmid was then cut with EcoRI, blunt-ended with Klenow polymerase and dNTPs, and self-religated, in order to destroy the EcoRI site at the boundary between core ars3002 and the enhancer that was regenerated during the process of ClaI linker addition. The insert sequences were confirmed by sequence analysis.

Deletions Δ1-Δ14 are the same as previously described [10]. To evaluate these deletions in the context of extended ars3002, the ~400-bp ClaI-HincII fragment containing the replication enhancer (from pUra4script:ars3002(ext)) was inserted between the ClaI and HincII sites of the deletion constructs Δ1-Δ14 previously described [10]. For deletions Δ15-Δ22, the two-step PCR procedure and cloning strategy described previously [10, 11] was employed. All primer sequences are available upon request.

Double and triple deletions were constructed according to the same two-step PCR and cloning strategy, except that the choices of internal (forward and reverse) primers for the first amplification step were different. For example, the rightward-pointing (forward) primer used in constructing Δ16 and the leftward-pointing (reverse) primer used in constructing Δ15 were chosen as internal primers at the first step PCR to obtain the double deletion, Δ15-16, and so on.

S. pombe D18 cells lacking the ura4 gene were transformed [23] with equal amounts of DNA (100 ng) from the plasmids under test. All analyses were performed as described previously [7, 10], except that transformation plates were analyzed after 4-5 days, since the growth rates of colonies transformed with enhancer-containing plasmids are faster than those with core ars3002 alone (which was used in the previous experiment [10]). All results shown are an average of four independent experiments with standard deviation shown as error bars.

To determine the percentage of exponentially growing cells containing plasmid, the transformants were grown in selective medium (EMM-uracil) to 1.2-1.5 × 10⁷ cells/ml. Then 500 cells were plated onto selective and non-selective (EMM+uracil) plates to determine the fraction of cells containing plasmid.