Mapping autonomously replicating sequence elements in a 73-kb ...

c Indian Academy of Sciences 

RESEARCH ARTICLE

Mapping autonomously replicating sequence elements in a 73-kb region of chromosome II of the fission yeast, Schizosaccharomyces pombe VINAY KUMAR SRIVASTAVA* and DHARANI DHAR DUBEY† Department of Biotechnology, V. B. S. Purvanchal University, Jaunpur-222001, India

Abstract Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm of fission yeast chromosome II, we have mapped five new ARS elements using systematic subcloning and transformation assay. 2D analysis of one of the ARS plasmids that showed highest transformation frequency localized the replication origin activity within the cloned genomic DNA. All the new ARS elements are localized in two clusters in centromere proximal 40 kb of the region. The presence of at least six ARS elements, including the previously reported ars727, is suggestive of a higher origin density in this region than that predicted earlier using a computer based search. [Srivastava V. K. and Dubey D. D. 2007 Mapping autonomously replicating sequence elements in a 73-kb region of Chromosome II of the fission yeast. Schizosaccharamyces pombe. J. Genet. 86, 139–148]

Introduction In eukaryotic cells, the process of DNA replication is confined to S phase of the cell cycle during which it initiates at multiple sites called origins. In yeasts, specific small DNA sequences, known as autonomously replicating sequence (ARS) elements (reviewed in Campbell and Newlon 1991), function as replication origins. Plasmids containing ARS elements transform yeast cells at high frequency (Hsiao and Carbon 1979; Stinchcomb et al. 1979) and replicate once in each cell cycle suggesting that they share regulatory controls with chromosomal replication (Zakian and Scott 1982; Fangman et al. 1983). Although ARS elements function invariably as origins where in plasmids, only a subset of them is active as chromosomal origins (Linskens and Huberman 1988; Dubey et al. 1991; Newlon et al. 1993; Friedman et al. 1997). The efficiency and timing of firing of different ARS elements vary greatly— weak ARS elements fire infrequently while the strong ones are active in most cell cycles. Similarly, some ARS elements fire early in S phase while *Deceased † For

correspondence. E-mail: [email protected], [email protected].

others are late. The factors controlling efficiency and timing of initiation of different origins are not well understood. 100–200-bp long S. cerevisiae ARS elements are composed of two essential functional domains: domain A containing an 11 bp conserved sequence, the ARS consensus sequence ACS, which binds to a complex of six proteins, the origin recognition complex ORC (Bell and Stillman 1992), and a broad A + T rich domain B which flanks domain A but shows no sequence similarity. ORC has homologues in other eukaryotic organisms including fission yeast, Drosophila, Xenopus, mouse and humans (reviewed in Dutta and Bell 1997; Bell 2002). In S. cerevisiae, it is now largely known that how different replication factors bind to ORC in a cell cycle specific manner to initiate DNA replication (reviewed in Kelly and Brown 2000). While replication proteins and the basic replication mechanisms seem to be conserved among eukaryotes, their replication origins are not. For example, a successful ARS assay is not available for higher eukaryotes and plasmids containing 10 kb or larger host DNA fragments can be maintained autonomously irrespective of the sequence content (Mechali and Kearsey 1984). Hence it is difficult to say whether specific sequence elements are used to specify

Keywords. Fission yeast; Schizosaccharomyces pombe; ARS element; replication origins. Journal of Genetics, Vol. 86, No. 2, August 2007

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Vinay Kumar Srivastava and Dharani Dhar Dubey origins or higher level of regulation — such as chromatin organization, and nuclear associations are more important. In the fission yeast S. pombe, which share many similarities with higher eukaryotic cells, ARS elements are several fold larger (600 bp–1.5 kb) than that of S. cerevisiae, and they seem to have no functional conserved DNA sequences except that all of them are rich in A + T. Of the limited number of ARS elements mapped in S. pombe, only a few have been analysed in detail (reviewed in Masukata et al. 2004). The available data suggest that S. pombe ARS elements (i) mostly function as weak chromosomal origins, (ii) are clustered more often than they are in budding yeast (iii) contain stretches of asymmetric As and Ts which are important for origin function, and (iv) some of them may contain sequence elements controlling the timing of initiation (Dubey et al. 1994; Wohlgemuth et al. 1994; Masukata et al. 2004; Yompakdee and Huberman 2004). Some time ago, two laboratories mapped a total of 332 (Raghuraman et al. 2001) and 429 (Wyrick et al. 2001) origins in S. cerevisiae genome using two different approaches. The data obtained by them were strongly supported by the preexisting data available from extensive ARS and origin mapping studies in this organism (Newlon et al. 1993; Shirahige et al. 1993; Friedman et al. 1997; Yamashita et al. 1997; Poloumienko et al. 2001). Unfortunately, in S. pombe, ARS/origin studies have been mostly confined to shotgun methods and the only published report on systematic functional mapping of ARS elements and their origin activities in a continuous stretch of genomic DNA comes from Okuno et al. (1997). Here, we report the mapping of five new ARS elements in a 73-kb region of S. pombe chromosome II. The plasmid origin function of one of the newly mapped ARS elements is demonstrated by neutral/neutral 2D origin mapping technique.

Materials and methods Construction of plasmid subclones

Three cosmid clones c17D1, c11C11, and c3B8 (from the German Resource Centre, RZPD and the Sanger Centre, UK), which altogether contained a ∼73-kbp region from the 972h− strain of S. pombe were used for subcloning. The subcloning was done in pRS306 (Sikorski and Hieter 1989). Amplification and screening of recombinant plasmid was carried out in a rec− E. coli strain, DH5α as described in Sambrook et al. (1989). The clones were confirmed by insert size, restriction at internal sites or by sequencing. Yeast transformation assay

For ARS assay, D18 S. pombe cells (ura4-D18 leu1–32), containing deletion of ura4 gene were used. To make them competent, the lithium acetate method of Gietz et al. (1992), was used. Briefly, cells were grown to mid log phase in minimal media (supplemented with minerals, vitamins and 150 mg/l each of adenine, leucine, uracil) were harvested, rinsed 140

twice with sterile water, once with 0.1 M lithium acetate solution (made in TE, pH 7.5) and finally, suspended in 0.1 M lithium acetate at a concentration of 2 × 109 cells/ml. For each experiment, 200 ng plasmid DNA (prepared using Qiagen kit), 10 μg heat denatured salmon sperm DNA (Sigma, cat # D1626) and 50 μl (1 × 108 cells) of competent cells were mixed thoroughly followed by addition of 300 μl 40% PEG 4000 solution (4 ml of 50% PEG 4000 + 0.5 ml of 1 M lithium acetate + 0.5 ml of 10 × TE, pH 7.5). All the components were mixed by vortexing at high speed for 1 min and incubated for 30 min at 30◦ C with vigorous agitation. 35 μl of DMSO (100%) was then added and mixed with the cells by gentle inversion of tubes followed by heat shock at 42◦ C for 15 min. After spinning for 10 s, supernatant was discarded and the cells were suspended in 500 μl of sterile 1× TE buffer (pH 7.5). Cells equivalent to 1 ng and 5 ng plasmid DNA were plated onto uracil lacking minimal medium plates (uracil-MMA) and incubated for 5–7 days at 30◦ C before colony counting and taking photographs. Isolation of DNA from yeast cells and N/N 2D gel analysis

A colony of D18 cells transformed with pRC8 was transferred to fresh tube containing 10 ml minimal medium, grown to mid log phase and used to isolate total DNA using the method of Hoffman and Winston (1987). 8 μg of total DNA digested with ScaI was used for 2D gel electrophoresis as described earlier (Brewer and Fangman 1987; Huberman 1999). For first dimension electrophoresis, 0.4% agarose gel (Sigma) in 1× TAE containing 0.1 μg/ml ethidium bromide was used and the electrophoresis was performed in dark at 1 V/cm for 13–14 hours in 1× TAE buffer. For the second dimension, 1.1% gel in 1× TBE containing 0.5 μg/ml ethidium bromide was used and electrophoresis was carried out at 6 V/cm for 4.0 hours in cold room using precooled 1× TBE running buffer containing 0.5 μg/ml ethidium bromide. The DNA was transferred onto a nylon membrane (Hybond- XL), using alkaline transfer buffer (0.4 M NaOH and 1M NaCl) and cross linked in a UV cross linker (Techne) at 1200 μ J/cm2. 100 ng gel purified DNA fragment was labelled with α32 PdATP (3000 Ci/m.mol or 50 μ Ci) using DNA labelling kit from Zonaki, (CCMB, hyderabad). Labelled DNA was purified from unincorporated dNTPs by passing through sephadex G-50 column. Prehybridization and hybridization was done at 65◦ C in Church buffer (1% BSA, 1mM EDTA, 7% SDS, 250mM sodium phosphate buffer, pH7.2) using probe to a concentration of ∼ 3 × 107 Cpm/ml. The membrane was washed once in 2 × SSC, 0.2% SDS for 5 min at RT and then at 52◦ C. Depending on the counts it was further washed with next wash solutions (1× SSC / 0.2% SDS and 0.2 × SSC / 0.2% SDS), saran-wrapped, exposed to Phosphor screen for two h. before scanning using PhosphorImager (Molecular Dynamics, USA; model no. 475). The autoradiogram was captured as 8-bit TIFF file and the image was transferred from Macintosh computer to personal computer

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Mapping ARS elements in fission yeast using ISO9660 program and processed using Adobe Photoshop 6.0 to adjust the signal to desired specific intensity.

Results Figure 1 shows the diagram of the 73-kb region of long arm of chromosome II of S. pombe which is contained in three overlapping cosmid clones c17D1 (20.0 kb), c11C11 (22.5 kb) and c3B8 (33.4 kb). Nucleotide sequence data downloaded from the Sanger Centre was used to prepare restriction map for a number of commonly used enzymes and

the resulting information was used to obtain gel fractionated fragments of suitable sizes for subcloning. Total 50 clones (named as pRC1–pRC50) containing inserts ranging in size from ∼ 500 bp to 7 kb (locations shown in figure 1) were made in pRS306. Table 1 shows details of these clones including their size, their locations in respective cosmid and in the 73-kb region and the confirmatory digestions with restriction enzymes. pRS306 contains URA3 gene of S. cerevisiae that complements for the ura4 gene of S. pombe and is a suitable vector for transformation assays using S. pombe.

Figure 1. Diagram showing the 73-kb region (blue line) of S. pombe chromosome II and the locations of the subclones with (thick black lines) or without (thin black lines) ARS activity. The arrows above the line show the location, sizes and directions of genes along with their names. The sizes of intergenic regions are shown in base pairs.

Table 1. Details of the 50 clones from the ∼73-kb region (nucleotides 3247161–3319979) of S. pombe chromosome II. Clones pRC1 to pRC12 were constructed from cosmid c17D1, pRC13 to pRC32 from cosmid c11C11 and pRC33 to pRC50 from cosmid c3B8. The names of restriction enzymes are abbreviated as B-Bam HI; Bg-BglII; RI-EcoRI; RV-EcoR V; H-HindIII; Hc-HincII; Ps-PstI; Pv-PvuII; S-SalI; X-XhoI; Xb-XbaI. Fragments released on Location in 73-kb Fragment plasmid digestion with region (starting from the Clone (sizes in bp) Position in cosmid enzymes shown in bracket cen. end) pRC1 1268 RI 55–1323 3936 + 1475 + 338 (Pv) 55–1323 pRC2 2485 H 1670–4155 1641 + 844 + V (H/Xb) 1670–4155 pRC3 1103 H 4155–5258 608 + 495 + V (H/RI) 4155–5258 pRC4 1329 + 344 RI 4650–5979–6323 721 + 608 + 344 + V (H/RI) 4650–5979–6323 Journal of Genetics, Vol. 86, No. 2, August 2007

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Vinay Kumar Srivastava and Dharani Dhar Dubey pRC5 pRC6

5979–6323 5258/bp–∼9.8 kb

No internal site ∼3.5 kb + 721 + 344 + V (RI/H)

5979–6323 5258 bp–9.8 kb

pRC7 pRC8

344 RI 4.6 kbp H (6915 bp H fragment containing deletion from nucleotide ∼9.8 kb– ∼12158 bp) 1571 Bg ∼600 S/H

5621–7192 9282–∼9.8 kb

5621–7192 9282 bp–9.8 kb

pRC9

3.0 kb H

5258–5621 + 7192– ∼9.8 kb 6323–9.8 kb 6323–12701

5250 + 358 + 344 (RI) 3936(V) + (∼600 × 2 = 1200 + 445) = 1.65 kb (I) Cloned as doublet and confirmed by Pv digestion 793 + 2253 + V (H/X) + 7192 bp −9.8 kb 1299 + 2253 + V (RI/X) 3399 + 1680 + 1299 + V (RI/X) 1494 + 465 + V (RI/H) 844 + 546 + V (Bg/RI)

pRC10 3.5 kb RI/H pRC11 6378 RI pRC12 1959 H pRC13 1430 RI

pRC14 3203 H

12236–14195 1–1344 (86-bp cosmid vector +1–1344 bp insert) 1–3203

pRC15 1859 H/RI

1344–3203

pRC16 2770 RI pRC17 1254 H pRC18 816 H

1344–4114 3203–4457 4457–5273

pRC19 1254 + 816 H

3203–5273

pRC20 1664 H

5307–6971

pRC21 3063 + 4618 RI

4192–7255

+ 27

9928–14546

pRC22 2099 H pRC23 2673 RI

6971–9070 7255–9928

pRC24 397 H pRC25 1308 H

9647–10044 10044–11352

pRC26 867 H pRC27 4618 RI

11352–12219 9928–14546

pRC28 2585 H pRC29 1965 + 678 H

12219–14804 17371–19336–20014

pRC30 678 H pRC31 7083 RI

19336–20014 14546–21629

pRC32 871 RI

21629–22500 (Rt RI site in cosmid) 537–1537 1537–3820 7137–9281 7987–10459 14227–14799 15276–17403 16306–21068

pRC33 pRC34 pRC35 pRC36 pRC37 pRC38 pRC39

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1000 B/X 2283 X/B 2144 H 2472 B/X 572 H 2127 RI 4762 H

1859 + 1344 + V (RI/H)

5258–5621 bp 6323 bp–9.8 kb 6323–12701 12236–14195 18231–19575 bp (Left RI site in cosmid vector) 18231–21434 (Left H site in cosmid vector) 19575–21434

3.37 kb + 1654 + 1152 bp (RI/Ps) 1859 + 911 + V (RI/H) 911 + 265 + 78 + V (RI/H) 3200+1227+402+136+125 (Hc) 911 + 816 + 265 + 86 + V (RI/H) 2923 + 1458 + 1073 + 591 (H/RV) 1664 + 816 + 284 + 265 + 34 + V + 2327 + 1308 + 867 + 116 (H/RI) 1815 + 284 + V (RI/H) 1815 + 421 + 281 + 156 + V (RI/H) --853 + 455 + V (2713 + 1654) + ∼2500 + 600 (H/Ps) --2327 + 1308 + 867 + 116 + V (RI/H) 2214 + 371 + V (H/Bg) 1403 + 562 + 2713 + 1654 + 678 (H/Ps) --2348 + 1965 + 1615 + 678 + 258 + 207 + 12 + V (RI/H) 777 + 94 + V (RI/B)

25202–27301 25486–28159

5381 (S) 2017 + 4647 + V (RI/B) 1294 + 850 + V (B/H) 3393 + 2050 (Ps/B) 429 + 143 + V (B/H) 1097 + 938 + 92 + V (RI/H) 2447 + 2315 + V (B/H)

39954–40954 40954–43237 46554–48698 47404–49876 53644–54216 54693–56820 55723–60485

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19575–22345 21434–22688 22688–23504 21434–23504 23538–25202 22423–25486 28159–32777

27878–28275 28275–29583 29583–30450 28159–32777 30450–33035 35602–37567–38245 37567–38245 32777–39860 39860–40731

Mapping ARS elements in fission yeast pRC40 pRC41 pRC42 pRC43

2862 B 346 B/X 1795 H 2213 B

18621–21483 21575–21921 21068–22863 21575–23788

pRC44 pRC45 pRC46 pRC47 pRC48 pRC49

1757 X/B 1703 H 567 B/X 2108 RI 2595 H 2595 + 514 H

23788–25545 25911–27614 27735–28302 27607–29715 28238–30833 27614–28128 + 28238–30833 30833–31466

pRC50 633 H

1630 + 700 + 532 + V (RI/B) --942 + 853 + V (H/X) 1334 + 5260 (#5) 879 + 5715 (#15) (Xb) 6138 (H) 1539 + 157 + 7 + V (RI/H) 4838 + 100 (H) 1477 + 631 + V (H/RI) 1477 + 1118 + V (RI/H) 2179 + 514 + 352 + 64 + V (B/H) No internal site

58038–60900 60992–61338 60485–62280 60992–63205 63205–64962 65328–67031 67152–67719 67024–69132 67655–70250 67031–67545 + 67655–70250 70250–70883

Figure 2. Photographs showing transformation efficiency of some plasmid clones from the 73-kb region. Plates are showing the transformants that appeared 7 days after transformation. Cells equivalent to 5-ng DNA were plated. pars3002 and pRS306 were used as positive and negative controls, respectively. Determination of ARS activity of the subclones derived from 73-kb region

The ARS activity of these subclones was tested by their ability to transform S. pombe D18 (ura4−) cells. After transformation, cells equivalent to 1 ng and 5 ng DNA were plated onto uracil lacking MMA plates. Colonies that emerged after 5 days of incubation at 30◦ C were counted after 6–7 days and the transformation frequencies were calculated separately for both 1ng and 5 ng plates for each clone. pars3002, a plasmid containing ars3002 (Dubey et al. 1994; Zhu et al. 1994) was used as the positive control while no DNA as well as pRS306 (vector) which lacks an ARS element (Sikorski and Hieter 1989) were used as negative controls. While pRS306 produced only few tiny colonies, which did not grow even after longer incubation, pars3002 produced many colonies

of large and medium sizes. The transformation frequency of 0.2 × 105 colonies/μg DNA was arbitrarily taken as the cutoff point for ARS activity. Initially, all the clones derived from a cosmid were used for transformation in two sets of experiments. Finally, the transformation efficiencies of the selected clones from all three cosmids were compared together in two different sets of experiments. Although here only the data from 5 ng experiments are shown in figure 2 and tables 2 and 3, the results from all the experiments were considered while attributing ARS activity to a clone. Of the initial eight clones, pRC1–pRC6, pRC11 and pRC12, obtained from cosmid c17D1, only pRC2, pRC6 and pRC11, showed high transformation frequency above the cutoff value while pRC1, pRC4, pRC5 and pRC12 showed no transformation and pRC3 showed a very low frequency (0.007 ± 0.0048 × 105 colonies/μg DNA). The insert in pRC6

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Vinay Kumar Srivastava and Dharani Dhar Dubey Table 2. Transformation frequencies (No. of colonies × 105 /μg DNA) of plasmid subclones derived from three cosmids. Subclones of cosmid c17D1 pars3002 pRS306 pRC1 pRC2 pRC3 pRC4 pRC5 pRC6 pRC11 pRC12

Transformation frequency 0.826 ± 0.527 0.0 ± 0.0 0.0 ± 0.0 0.14 ± 0.156 0.007 ± 0.0048 0.0 ± 0.0 0.0 ± 0.0 0.653 ± 0.173 0.94 ± 0.285 0.0 ± 0.0

ars3002 pRS306 pRC6 pRC7 pRC8 pRC9 pRC10

1.305 ± 0.245 0.0 ± 0.0 0.995 ± 0.445 0.986 ± 0.2015 0.895 ± 0.95 0.049 ± 0.016 0.0875 ± 0.05

Subclones of cosmid c11C11 pars3002 pRS306 pRC13 pRC14 pRC15 pRC16 pRC17 pRC18 pRC19 pRC20 pRC21 + 27 pRC22 pRC23 pRC25 pRC26 pRC28 pRC29 PRC30 pRC31 pRC32

Table 3. Transformation frequencies of selected clones from the 73-kb region. Serial No. 1. 2. 3. 4. 5. 6. 7. 8. 9.

Clones pars3002 pRS306 pRC2 pRC6 pRC7 pRC8 pRC11 pRC21 + 27 pRC31

Transformation frequency (no. of colonies × 105 /μg DNA; 5ng experiment) 0.694 ± 0.006 0.00 ± 0.0 0.525 ± 0.245 0.782 ± 0.168 1.04 ± 0.45 1.476 ± 0.443 0.73 ± 0.229 0.55 ± 0.025 0.42 ± 0.06

was further subcloned as pRC7, pRC8, pRC9 and pRC10. The former two of them showed high transformation frequency while the later two did not (table 2; figure 2). The locations of the five fragments showing high transformation frequency in plasmids is shown as thick lines in figure 1. It should be mentioned here that pRC6, which was initially cloned as a 6.9-kb HindIII fragment, released an insert of only 4.6-kb on cutting with HindIII. A series of double digestions and sequencing from the centromeric end confirmed that the reduced size of the insert was due to deletion of ∼2.3 kb at telomere proximal end. It may be noted that removal of a 1571-bp BglII sub fragment (cloned as pRC7) from pRC6 totally knocked out its ARS activity as shown by the greatly reduced transformation frequency of the resulting clone, pRC9 (figure 1, table 1 and 2). pRC10, which lacked part of the 1571-bp BglII fragment resulted into a remarkably reduced transformation frequency (table 1 and 2). These results clearly demonstrate that ARS activity is confined to the 1571-bp BglII fragment. 144

Transformation frequency 0.736 ± 0.495 0.0 ± 0.0 0.0 ± 0.0 0.138 ± 0.153 0.0 ± 0.0 0.17 ± 0.12 0.0 ± 0.0 0.023 ± 0.0145 0.029 ± 0.013 0.13 ± 0.0 0.69 ± 0.0 0.0 ± 0.0 0.03 ± 0.0 0.033 ± 0.013 0.02 ± 0.0 0.22 ± 0.0 0.0 ± 0.0 0.03 ± 0.0 0.31 ± 0.0 0.0 ± 0.0

Subclones of cosmid c3B8 pars3002 pRS306 pRC33 pRC34 pRC35 pRC38 pRC39 pRC40 pRC42 pRC43 pRC44 pRC45 pRC47 pRC48 pRC49 pRC50

Transformation frequency 0.736 ± 0.493 0.0 ± 0.0 0.0 ± 0.0 0.0667 ± 0.04 0.001 ± 0.0 0.0076 ± 0.004 0.098 ± 0.069 0.037 ± 0.043 0.0045 ± 0.0025 0.1015 ± 0.0724 0.012 ± 0.0 0.023 ± 0.0 0.111 ± 0.046 0.0633 ± 0.0316 0.1 ± 0.01 0.0

Although the ∼600-bp SalI/ HindIII sub fragment of the 4.6-kbp HindIII fragment, which was cloned most probably as a doublet in pRC8 (figure 1; table 1), also transformed yeast cells at a high frequency comparable to that of pars3002 and pRC6 (figure 2; table 2), it is unlikely to contain a complete ARS element as the presence of its single copy in pRC9 failed to yield high transformation frequency. It is, however, possible that the ∼600-bp SalI/ HindIII fragment is part of another ARS element a part of which was lost by the deletion of ∼2.3-kb-telomere-proximal region of the original 6.9-kb HindIII fragment. The observation that the 6378 bp EcoRI fragment cloned in pRC11 yields high transformation frequency while its centromere proximal 3.5kb EcoRI/ HindIII fragment (pRC10) yields greatly reduced number of transformants strongly suggests the presence of another ARS element at the telomere proximal end of the 6378-bp EcoRI fragment between nucleotide positions 9282 and 12,701. The precise boundaries of this ARS element, however, remain to be defined. Of the 20 clones, namely pRC13 to pRC32 (table 1) from cosmid c11C11 region, 18 were used for transformation assay. Their average transformation frequencies as determined from three experiments are shown in table 2. Seven of them (pRC13, pRC15, pRC17, pRC22, pRC26, pRC29 and pRC32) showed no transformation activity, as only a few tiny colonies were seen on the plates while six other clones (pRC18–pRC20, pRC23, pRC25 and pRC30) showed transformation frequencies lower than 0.1 × 105 colonies/μg. Of the remaining clones, pRC14 and pRC16 produced a small number of medium and large colonies while pRC28 produced a large number of small (uncountable) colonies and a small number of medium and large colonies. Since only medium and large colonies were counted the transformation

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Mapping ARS elements in fission yeast frequency of all these three clones were very close to each other and to the cutoff value (table 2). Clone pRC21 + 27 which contained two EcoRI fragments of 3063 bp and 4618 bp (table 1) showed highest transformation frequency followed by pRC31. Although the 3063-bp EcoRI fragment has not been cloned separately, since none of the five overlapping fragments from this region (cloned in pRC17–pRC20 and pRC22; figure 1; table 1) produced significant number of transformants, this fragment is unlikely to have any ARS activity. Therefore, the transformation efficiency of pRC21+27 seems to be due to the 4618-bp EcoRI fragment. Three clones pRC25, pRC26 and pRC28 contain parts of the 4618bp EcoRI fragment (figure 1). Of these, only pRC28 shows some transformation activity suggesting the presence of ARS activity near the central part of the 4618-bp EcoRI fragment. pRC31, the other clone, which showed significant transformation activity, contains a 7083-bp-EcoRI fragment. Since pRC29 and pRC30, which contain two HindIII fragments from telomere proximal end of this clone, did not transform S. pombe cells, the ARS activity is likely to be located in the centromere proximal region of the 7083-bp EcoRI fragment. From cosmid c3B8 eighteen plasmid clones (pRC33 to pRC50; table 1) were constructed and 14 were used for the transformation experiments. Surprisingly none of the tested sub clones from cosmid c3B8 showed high transformation activity (table 2). Since Maundrell et al. (1988) have characterized ars727 earlier, its cloning and characterization was ignored. After cosmid-wise screening of the clones for ARS activity, all the clones showing high transformation frequency were further used to transform D18 cells for the final comparison of their efficiencies. These clones are listed in table 3. Seven of the nine clones finally tested pRC2, pRC6, pRC7, pRC8, pRC11, pRC21 + 27 and pRC31, repeatedly showed high efficiency of transformation confirming their ARS activity. To name the newly identified ARS elements, nomenclature strategy of Dubey et al. (1994) was adopted. Thus, the 2485-bp HindIII fragment cloned as pRC2 was named ars2006. Four of the above mentioned clones pRC6, pRC7, pRC8 and pRC11 were confined to ∼ 7.5-kb-region (from nucleotide position 5258–12701 in the 73-kb-region) and two fragments in this region seemed to contain ARS activity. The 1571-bp BglII fragment cloned in pRC7 was named ars2007 while the other relatively poorly defined ARS element which was confined to the telomere proximal half of the 6378-bp EcoRI fragment cloned in pRC11 (part of it also cloned as doublet in pRC8) was named as ars2008. Similarly, the 4618-bp EcoRI fragment (cloned in pRC21 + 27) was named as ars2009 and the 7083-bp EcoRI fragment as ars2010. Thus, a total of five new ARS elements were identified from the 73-kb region, which are listed in table 4 and their locations are shown in figure 1. Localization of plasmid replication origin

In earlier studies in yeasts and other lower eukaryotes, it

Table 4. The five newly identified ARS elements Plasmid clones pRC2 pRC7 pRC11 pRC27 pRC31

Insert 2485 bp HindIII 1571 bp BglII 6378 bp EcoRI 4618 bp EcoRI 7083 bp EcoRI

Name ars2006 ars2007 ars2008 ars2009 ars2010

has been shown by using N/N 2D technique that replication origins can be mapped specifically at or near ARS elements in plasmids as well as on chromosomes (Brewer and Fangman 1987; Linskens and Huberman 1988). Using this technique, nonlinear replication intermediates (RI) of different shapes (formed due to replication of a DNA fragment by internal or external origin(s)), can be separated from each other as well as from nonreplicating linear molecules making it possible to localize replication origins within a few hundred basepairs. It has also been shown that a correlation exists between efficiency of the ARS element and the strength of its origin activity (Okuno et al. 1997). Since pRC8 showed the highest transformation frequency among all the other plasmids made in present study, we chose it for 2D analysis. DNA prepared from D18 cells transformed with pRC8 was digested with ScaI and subjected to N/N 2D gel electrophoresis as described in materials and methods.

Figure 3. (A) Diagram showing the structure of pRC8, which probably contains a doublet of ∼600 bp region of pars2008. (B)The location of the ARS element and flanking-plasmid sequences in 4.33-kb ScaI fragment of pRC8. The thick black bar marks the position of the probe. (C) Autoradiograph showing N/N 2D migration pattern of the 4.33kb ScaI fragment detected by the probe shown in (B).

Selection of ScaI enzyme was based on the fact that the bubble signal from a restriction fragment can only be obtained, if the origin is present within its central 2/3rd region. As shown in figure 3, ScaI digestion of pRC8 releases a fragment of 4.33-kb, which contains the 1200-bp genomic DNA

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Vinay Kumar Srivastava and Dharani Dhar Dubey insert near the center flanked by 1340-bp and 1790-bp vector DNA sequences on its right and left side, respectively. To avoid cross hybridization with replicating genomic copy of the insert, a 1.9-kb vector derived PvuI fragment (fragment c in figure 3) was used as a hybridization probe. The autoradiogram presented in figure 3 shows a strong ’bubble’ arc and a faint ‘Y’ arc signal. The 1 N spot is saturated even in a 2 hour exposure time. Presence of a full-length strong ‘bubble’ arc signal suggests the presence of most of the initiation events at or, near the center of the fragment. This result clearly demonstrates presence of a strong origin within the genomic DNA insert in plasmid pRC8.

Discussion To map and characterize ARS elements in a 73-kb region (from nucleotide 3247161–3319979) of the long arm of S. pombe chromosome II, overlapping clones were prepared from the entire region in pRS306. pRS306 is a shuttle vector which contains S. cerevisiae URA3 gene which complements for S. pombe ura4 gene. These clones were used to transform ura4− S. pombe cells for ARS selection. A total of five new ARS elements, ars2006–ars2010, were identified of which three, ars2007, ars2009 and ars2010, have been found to be associated with weak chromosomal origins using N/N 2D technique (Dubey and Srivastava, manuscript in preparation). Earlier, a number of investigators have successfully used the systematic approach of first mapping the ARS elements followed by their functional analysis by 2D techniques to characterize DNA replication in long stretches of yeast genomic DNA (Friedman et al. 1997; Okuno et al. 1997; Yamashita et al. 1997; Poloumienko et al. 2001). The spacing between ARS elements mapped in this study seems to vary from 2 kb (between ars2006 and ars2007 or ars2007 and ars2008) to 16 kb (between ars2008 and ars2009), and all these five ARS elements have been found to be located in the left, centromere proximal 40-kb portion of the 73-kb region while the right, telomere proximal 33-kb portion seems to have no new ARS element except the earlier reported ars727 (Maundrell et al. 1988). It is important to mention here that due to the presence of gaps in some regions and insufficient overlapping in some other regions, the possibility of missing some ARS elements cannot be ruled out. For example, the intergenic region between two divergent transcripts, c17D1.07 and c17D1.08, which has been found to possess a weak origin activity (Dubey and Srivastava, manuscript in preparation) and which escaped test for ARS activity due to unavailability of a suitable clone, is very likely to turn out to be an ARS element because all origins mapped till date in yeasts correspond to ARS elements. Thus including ars727 and the weak origin associated ’likely ARS’, at least seven ARS elements are present in this 73-kb region. This shows an average distribution frequency of one ARS element per 10 kb in this region. A comparable analysis of a 100-kb region in a previous study from the short arm (left 146

arm) of chromosome II showed the presence of five ARS elements with the average inter ARS spacing of 20 kb (Okuno et al. 1997). In this study also the inter ARS distances varied greatly more than 40 kb between ars2004 and ars2005, 9 kb between ars2003 and ars2004 and 5 kb between ars2001 and ars2002. Two earlier studies, which used random sub cloning and transformation strategy to find out ARS elements in S. pombe genomic DNA, have reached to different conclusions regarding their frequency. While Maundrell et al. (1988) estimated an average frequency of one ARS per 20 kb, the Calos group (Caddle and Calos 1994; Wohlgemuth et al. 1994) deduced a distribution frequency of one ARS element per 55 kb. Since the latter group identified a number of additional clones with a reduced transformation frequency, which they thought could represent fragmented ARSs due to cutting at enzyme sites present inside the ARS elements, they suspected that the frequency of one ARS per 55 kb was an underestimation and the real number could be higher. In a recent study, Segurado et al. (2003) predicted the presence of 384 ORIs in S. pombe genome based on computer-assisted identification of distinctive A+T rich islands of up to 1 kb. On functional analysis of a subset of these islands most of them were found active as chromosomal origins. A comparison of our results with their data shows that ars2007 and ars2010 correspond to AT-rich islands 2101 and 2102 of Segurado et al. (2003). However, of the seven ARS elements reported here, they could map only three AT islands in the 73-kb region. Thus the results of the present study provide a significantly higher density of ARS elements in the 73-kb region as compared to other studies. Since present study and the study by Okuno et al. (1997) are confined to mapping ARS elements in a limited region from different parts of the genome, they probably represent only specific genomic regions. It is, therefore, possible that the frequency of ARS elements varies greatly in different parts of S. pombe genome. If the high density of ARS reported in the present study represents most of the genome, the total number of ARS elements in this organism may exceed all the earlier estimates. Similar variations in spacing between ARS elements and their regional distribution frequencies have also been reported in S. cerevisiae in which large chromosomal regions (Newlon et al. 1993; Poloumienko et al. 2001) or entire chromosomes (Shirahige et al. 1993; Friedman et al. 1997; Yamashita et al. 1997) have been extensively studied for ARS activity. Thus, the two yeasts, S. pombe and S. cerevisiae, show a wide variation in spacing of ARS elements. The ARS elements mapped in the present study are distributed in such a way that ars2006, ars2007 and ars2008 lie close to each other making one cluster while, ars2009 and ars2010 form another cluster of ARS elements. This ARS clustering seems to be similar to the ars 1-1 cluster (Wohlgemuth et al. 1994), the cluster of three ARS elements in the ura4 origin region (Dubey et al. 1994), and the ARS clusters in centromeres (Smith et al. 1995 reviewed by Masukata et al. 2004). Although clustering of ARS elements

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Mapping ARS elements in fission yeast has also been observed in S. cerevisiae genome, e.g. a cluster of ARS302 and ARS303, another of ARS317, ARS318 and ARS320, on chromosome III (Poloumienko et al. 2001), a cluster of two ARS elements, ARS601 and ARS602, present in the telomere proximal region on the left arm of chromosome VI (Yamashita et al. 1997), it seems to occur more frequently in S. pombe genome, at least in the regions containing a high density of ARS elements. Detailed analysis of chromosomal origin activity of each of these ARS elements will be required to see if clustering is associated with their reduced chromosomal origin activity as generally observed for majority of origins in S. pombe (Masukata et al. 2004). Acknowledgements The authors are grateful to the Principal, Kutir PG College Chakkey, Jaunpur-222146, where this work was started and largely completed and to Joel A. Huberman, Rajiva Raman, Lalji Singh and K. VijayRaghavan for extending laboratory facilities to carryout part of the work. We thank Aditya Singh Pratihar for help in preparing figures. This work was supported by a research grant (#SP/SO/D39/95) to DDD from the DST, New Delhi, India.

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