Unidirectional deletion analysis of the ars2004 fragment.
To examine the regions required for autonomous replication of the ars2004 fragment, we first made a series of unidirectional nested deletions from either end of the 3.2-kb ARS fragment. ARS activity was examined by measuring the efficiency of transformation of a haploid S. pombe leu1 strain to Leu+ as described in the Materials and Methods.
As shown in Fig. , derivatives with deletions differing in length from the left end (N series) to position 860 formed transformants at the same efficiency as the parental plasmid, pARS2004. Further deletion derivatives (pN902, pN963, and pN1018) exhibited gradually reduced transformation efficiency and pN1186 gave no transformants. Deletions from the right end (X series) up to about a 1.7-kb segment did not affect transformation efficiency (pX1540). However, deletion of a further 150-bp segment (pX1404) completely abolished activity. These results pointed to the existence of an element required for ARS activity in a distinct region between positions 860 and 1540.
FIG. 1 Effects of deletions on autonomous replication of ars2004. Derivatives of pARS2004 with nested unidirectional deletions were constructed as described in Materials and Methods. Plasmid DNA was introduced into S. pombe HM123 cells, and the number of Leu (more ...)
To determine the minimum region sufficient for autonomous replication, the region right of position 1741 was removed from the N-series derivatives (HD series). pHD444, pHD714, pHD802, and pHD860 showed almost the same transformation efficiencies as pARS2004, although the pHD802 and pHD860 transformants grew slightly slower than the pARS2004 transformants. In contrast, pHD902 yielded no transformants. These results confirmed the presence of a crucial sequence element right of position 860. The difference between pHD902 and pN902 in replication efficiency suggested the region right of position 1741 to contain an element(s) compensating for lack of the region between 860 and 902. For more detailed analysis of the region required for ARS function, we used a 940-bp fragment from positions 802 to 1741, designated ars2004M.
Identification of regions essential for replication.
To identify functional regions required for autonomous replication of ars2004M, we deleted 50- to 200-bp internal segments and examined the effects on ARS activity (Fig. A). A derivative lacking segment B yielded no transformants. Deletion of segment D, E, F, or K greatly reduced transformation efficiency, and the resultant transformants grew very slowly. In contrast, deletion of segment A, C, G, H, I, J, or L had little effect on transformation efficiency. These results showed at least three distinct regions, I (from positions 894 to 934), corresponding to segment B, II (from 991 to 1156), containing segments D, E, and F, and region III (from 1487 to 1552), corresponding to segment K excluding segment J, to be important for autonomous replication of ars2004M. It should be noted that the boundaries for ARS activity detected by analyses of nested deletions from either end of the 3.2-kb ars2004 fragment are colocalized at regions I and III.
FIG. 2 Regions required for autonomous replication of the 940-bp ars2004 fragment. (A) Internal segments of 50 to 200 bp (A to L) were deleted from the 940-bp ars2004M fragment, and effects on transformation of HM123 cells were examined. Transformation efficiencies (more ...)
We then examined whether replication of the 3.2-kb ars2004 was dependent on regions I, II, and III. Lack of the 940-bp ars2004M segment from ars2004 abolished ARS activity (Table ). In contrast, deletion of any one of regions I, II, and III did not abolish ARS activity, although resultant transformants lost plasmids at frequencies of 8.4, 3.2, and 5.1% per generation, respectively, in all cases higher than the 2.2% for pARS2004 transformants. However, the derivative lacking both regions I and III yielded no transformants (Table ). Although derivatives lacking regions I and II or regions II and III yielded transformants, the transformants grew very slowly and the plasmid loss rates increased to 14 or 8.5% per generation, respectively (Table ). Thus, regions I and III are required for replication of ars2004 but lack of regions I and II or regions II and III can be partly compensated for by certain elements existing outside the ars2004M.
Role of regions I, II, and III in replication of ars2004
The nucleotide sequences of regions I, II, and III together with a schematic illustration of the ars2004 structure are shown in Fig. B and C. Region I is extremely rich in adenines and thymines, containing 8, 5, and 19 consecutive adenine residues in the upper strand. Region III consists of 11 repeats of TTTTA or variants. On the other hand, region II does not contain any long characteristic sequence but has short AT-rich sequences with intervening guanine- and cytosine-rich (GC-rich) sequences.
Requirement of A stretches in region I.
To evaluate the importance of adenine (A) stretches in region I, an 8-bp segment was serially replaced with the Sse
8387I recognition sequence (CCTGCAGG). Although the number of transformants after 4 days was not reduced by substitution, they grew significantly more slowly than with pARS2004M transformants. Since the growth rate of ARS plasmid transformants correlates with ARS activity (37
), the results indicated partial loss with the base substitutions. To detect reduction in growth rates of transformants quantitatively, the transformants were counted at 3 days instead of 4 days after transformation.
As shown in Fig. , all substitutions in region I (S896, S906, S916, and S926) reduced the transformation efficiency at 3 days to about 1/10 the parental value, while substitutions outside region I (S886 and S936) exerted only slight effects, indicating that all the A stretches in region I are required for efficient replication. The fact that none of the substitutions completely abolished the ARS activity suggested that the remaining A-stretch array retained substantial function.
FIG. 3 Effects of Sse8387I linker substitution in region I on autonomous replication. Various 8-bp sequences from positions 886 to 939 in ars2004M were replaced with an Sse8387I sequence. Derivatives carrying 10-bp insertions were made by recombining linker-substitution (more ...)
We next tested whether continuity of the A stretches in region I was required for the ARS activity, by inserting the Sse8387I site within or between A stretches. With a 10-bp insertion at position 906 (IS906), the transformation efficiency was reduced to one-fourth of the parental value (Fig. ). Insertion at position 916 (IS916) or 926 (IS926) also reduced ARS activity (Fig. ). These results suggested continuity of A stretches to be important for ARS activity. However, the transformation efficiency with IS906 was three times higher than that with S896, in which the left-most 8-bp A stretch was substituted (Fig. ). The value with IS926 was also three times higher than that with S926, suggesting that the sequestered 8-bp A stretches contribute to ARS activity.
Multiple elements in region II.
To determine the sequence element of region II required for ARS activity, effects of serial 8-bp substitutions were examined. With substitutions in a region from positions 1033 to 1087, the transformation efficiency after 3 days of incubation was reduced to about one-third of the parental value (Fig. ). Substitutions from positions 1139 to 1146 (S135, S1139, and S1143), and to a lesser extent 1120 to 1126, also caused reduction. Other substitutions did not significantly affect transformation efficiency. These results suggested that region II contains three subdomains extending from positions 1033 to 1087 (region II-1), 1120 to 1126 (region II-2), and 1139 to 1146 (region II-3). All substitution derivatives yielded almost the same number of transformants as pARS2004M after 4 days of incubation, showing that the 8-bp substitution in region II did not abolish but rather diminished its function.
FIG. 4 Effects of Sse8387I-linker substitution in region II on autonomous replication. Locations of linker substitutions are shown by rectangles below the sequence of region II. Transformation efficiencies relative to pARS2004M after 3 days of incubation are (more ...) Replacement of essential regions.
To evaluate the relative importance of regions I, II, and III in autonomous replication, we constructed derivatives carrying three copies of only one of these, replacing the other two. The derivative with two additional copies of region I at the positions of regions II and III gave about one-tenth as many transformants as the parental construct (pI-I-I in Fig. A). That with two additional copies of region III (pIII-III-III) yielded transformants as efficiently as pARS2004M. In contrast, the derivative with three copies of region II (pII-II-II) yielded no transformants (Fig. A).
FIG. 5 Effects of substitutions of region I, II, or III for the other two regions of ars2004M on ARS activity. (A) Two of three regions required for the ARS activity of ars2004M were replaced with copies of the other region. Transformation efficiencies relative (more ...)
Although the transformation efficiency with pI-I-I was much lower than that with pARS2004M itself, the ARS activity was greatly affected by clustering of region I fragments. A derivative carrying two tandem copies of the region I fragment at the region III site without region II (pI-Δ-Ix2) exhibited transformation efficiency three times higher than that of pI-I-I (Fig. C). Further elevation was observed with a derivative carrying two copies of the region I fragment at the region II site without region III (pI-Ix2-Δ). Moreover, the plasmid with three copies of the region I fragment at the region I site without regions II and III (pIx3-Δ-Δ) yielded transformants as efficiently as pARS2004M. These results showed closely located region I fragments to be much more effective than when separated.
Specific sequences required for autonomous replication.
To examine whether specific sequences or merely AT richness in region I has importance for autonomous replication, region I of pARS2004M was replaced with a synthetic (A/T)40
, or (AT/TA)20
fragment, with almost the same numbers of adenines and thymines. As shown in Fig. A, the derivative carrying (A/T)40
transformed as efficiently as pARS2004M. That with (A/T)40
in the opposite orientation had similar ARS activity (37a
). In contrast, the derivative with (AAT/TTA)13
yielded about 1/10 as many transformants (pAAT-II-III in Fig. A), and none were obtained with (AT/TA)20
(pAT-II-III in Fig. A). These results demonstrated that specific sequences rather than mere AT richness are required for ARS activity. Furthermore, replacement of region I with (AAAC/TTTG)10
reduced the transformation efficiency to about 1/10 of the parental value, showing that the presence of cytosine or guanine impairs the function of region I. From these results, we concluded that three or more consecutive A/T stretches without intervening guanine or cytosine are required for ARS function.
FIG. 6 Substitutions of artificial sequences for regions required for autonomous replication of ars2004M. (A) Region I of ars2004M was replaced with a synthetic (A/T)40, (AAAT/TTTA)10, (AAT/TTA)13, (AT/TA)20, or (AAAC/TTTG)10 fragment. Transformation efficiencies (more ...)
As shown in Fig. B, regions II and III could also be functionally replaced with (A/T)40 but not (AT/TA)20. Moreover, the derivative with (A/T)40 fragments at positions of regions I, II, and III yielded the same number of transformants as pARS2004M.
Minimum ARS fragments.
Since serial deletions of a 50- to 200-bp segment except for regions I, II, and III had little effect on ARS activity (Fig. ), we tested whether segments other than regions I, II, and III were dispensable for ARS activity. A derivative carrying a set of region I, region II, and region III fragments in the native order and direction yielded no significant transformants (pMI-II-III [Fig. ]), showing that the spacer regions are required. However, pMA40-II-III carrying (A/T)40 instead of the region I fragment yielded transformants at about one-third the level for pARS2004M. Insertion of additional copies of (A/T)40 increased transformation to almost the same efficiency as pARS2004M (pMT80-II-III and pMA120-II-III [Fig. ]), indicating that the functions of all spacer regions could be replaced by the presence of additional A stretches. A derivative carrying three copies of (A/T)40 alone did not transform efficiently (data not shown). Since we failed to construct a derivative with longer A stretches, it has not been determined whether an A stretch alone, if long enough, functions as an ARS element.
FIG. 7 Autonomous replication of ars2004M without spacer regions. Transformation efficiencies with derivatives of pYC11 carrying combinations of regions I, II, and III and (A/T)40 fragments without spacer regions of ars2004M were measured after 4 days of incubation. (more ...)
Our previous study demonstrated that efficient ARS fragments are maintained as monomeric plasmids in the transformants, while less efficient ARS fragments are present as multimeric plasmid forms (37
). We examined the minimum ARS plasmids lacking spacer regions for their form maintained as extrachromosomal elements. Plasmid DNA from the Leu+
transformants grown under selective conditions was separated by agarose gel electrophoresis and analyzed by Southern hybridization. The parental plasmid pARS2004M was maintained as monomers (37a
). Plasmids pMA40
-II-III, and pMA120
-II-III were maintained as monomers, although transformants that grew faster than others also contained dimer and trimer forms (38b
). These results confirmed that minimum ARS fragments lacking spacer regions are maintained as extrachromosomal elements.