A physical map of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius 7 chromosome

A chromosomal map of the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius 7 was constructed with four restriction enzymes: NotI, BssHII, RsrII, and EagI. The map indicated that the chromosome is a single circular DNA of 2,760 +/- 20 kb (mean +/- standard error of the mean). rRNA genes were also mapped. They were located at one site in the genome.

Digestion of the DNA in agarose blocks and electrophoresis. Agarose blocks were washed with TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA) for 3 h on ice with six buffer exchanges and then equilibrated with suitable enzyme buffer for 1 h with a buffer exchange. Each agarose block was treated with 10 to 20 U of restriction enzyme at the appropriate temperature for 2 to 4 h. PFGE was performed on a 1.2% agarose gel in 0.5 x TEB buffer (44.5 mM Tris base, 44.5 mM boric acid, 1 mM EDTA) at 5.7 V/cm by using a hexagonal system (30) at 16°C for an appropriate period, depending on the fragment size. Lambda DNA ladders and the chromosomal DNA of Saccharomyces cerevisiae S288C were prepared as described in references 1 and 5, respectively. Preparation of rRNA. Ribosomes were prepared in solution A, which contained 20 mM Tris-HCl, pH 7.6, 10 mM Mg acetate, and 20 mM NH4C1. Cells were suspended in solution A and lysed by passage through a French pressure cell. After the addition of DNase, the cell lysate was cleared by centrifugation at 30,000 x g for 30 min. Ribosomes were recovered from the cleared lysate and washed once with solution A by centrifugation at 180,000 x g for 3 h. The precipitate was suspended in solution B, which contained 10 * Corresponding author.
mM Tris-HCl, pH 7.6, and 0.2 mM Mg acetate. Subunits were separated in a 10 to 30% sucrose density gradient in buffer B by centrifugation in a Beckman SW28 rotor for 16 h at 21,000 rpm. RNA was purified by phenol extraction and recovered by ethanol precipitation. The rRNA was further purified by gel electrophoresis. Southern blot analysis. Southern blot analysis was done according to the standard procedure (18) as described previously (29). A Fujix Bio-imaging Analyzer was used to detect weaker isotope signals. For isotopic labeling of probes, DNA bands were cut out after electrophoresis and DNA was extracted by using phenol from low-meltingtemperature agarose gel (Bio-Rad) or by using glass milk (Bio 101, Inc.) from normal agarose gel.
Alternatively, the low-melting-temperature agarose blocks containing DNA bands were labeled directly. They were labeled by using a random primer labeling kit (Takara Shuzou). When agarose blocks were labeled directly, they were melted and 12 RI was mixed with 2 ,u of primer solution and annealed. Agarose was solidified again in the annealing procedure, and reaction buffer, deoxynucleoside triphosphate, [32P]dCTP, and the Klenow fragment of DNA polymerase I were added. The mixture was incubated at 37°C for 5 h. After the addition of 225 ,ul of TE buffer, gels were melted at 65°C and applied to a small gel filtration column (Sephadex G50).
For preparation of 16S and 23S rRNA probes, RAV-2 reverse transcriptase (Takara Shuzou) was used instead of the Kienow fragment. The labeled mixtures were heat denatured at 100°C for 3 min, treated with RNase A, and extracted with phenol before gel filtration. 5S rRNA was dephosphorylated and labeled with polynucleotide kinase at its 5' end.
Restriction enzymes. About 20 restriction enzymes were tested considering the relatively low G+C content, 40% (29), of the organism. Among those tested, eight enzymes (4paI, FspI, NaeI, NarI, NruI, SmaI, MluI, and XhoI) yielded fragments greater than 200 kb in size, although there were more than 20 fragments. Four restriction enzymes (BssHII, EagI, NotI, and RsrII) yielded fewer than about 20 fragments and were used for construction of the physical map.
Estimation of the size of the S. acidocaldarius 7 chromosome. We have previously reported that the 8-base-recognition enzyme NotI cut the S. acidocaldanius 7 chromosomal DNA into two bands and that the sum of the fragment lengths was about 3,100 kb (29). The restriction enzymes BssHII, EagI, and RsrII yielded 6, 20, and 12 bands, respectively, with the fragment size ranging from 6 to 945 kb  Table 1). The size was estimated from the electrophoretic separation pattern with an appropriate pulse time for each fragment size. A lambda ladder size marker was used to estimate the sizes of all the fragments except NoM. The total lengths of the fragments were 2,812, 2,780, and 2,708 kb from BssHII, RsrII, and EagI digestion, respectively. The sizes of fragments resulting from BssHII-RsrII and BssHII-EagI double digestion are summarized in Table 2. The total lengths of these fragments were 2,782 and 2,728 kb, respectively. By averaging these five values, we estimated the chromosomal size to be 2,760 +-20 kb (mean + standard error of the mean).
Physical map of the S. acidocaldarius 7 chromosome. These gels were transferred to a membrane and analyzed by using several DNA fragments as probes. Results of these Southern blot analyses are summarized in Tables 1 and 2. Six BssHII fragments were primarily used as probes. Notl-linking clones (29) and RsrII bands were also used. Each of the four Notl-linking clones, pHNS7, pHNS7R, pXNC7, and pXNL7, harbors each arm of the two NotI-linking fragments, pHN7 and pXN7 (29).
Every fragment of the RsrII and EagI digests was detected by at least one of these probes. From these hybridization data and the estimated sizes of fragments, each fragment after double digestion was assigned to the original fragment resulting from single digestion ( Table 2). These results were used for restriction map construction. Every fragment was uniquely aligned in a circular form (Fig. 2). Because EagI recognizes the inner six bases d(CGGCCG) of the NotI recognition sequence d(GCGGCCGC), Not-linking clones could be used to align four EagI fragments. Although the order of a few fragments, i.e., R9/R1O, R11/R12, E1O/E19, E12b/E16/E18, and E14/E17, are still unsure, the position of each pair or triplet was uniquely determined.
Physical mapping of rRNA genes. Figure 3  a Sizes of the NotI fragments were determined by using S. cerevisiae and Schizosaccharomycespombe size markers described in reference 29 and may not be very accurate. I Probes used were BssHII and RsrII fragments of S. acidocaldarius 7 DNA and NotI-linking clones and rRNAs. Each of the NotI-linking clones, pXNC7, pXNL7, pHNS7 and pHNS7R, harbors each arm of the two NotI-linking fragments, pXN7 and pHN7 (29).
Southern blot analysis of the fragment after EcoRI, BamHI, HindIII, and XbaI digestion. Only one band was detected by rRNA probes in each lane. Accordingly, these rRNA genes are located within these fragments, i.e., 8.0-kb EcoRI, 11.5-kb BamHI, 9.0-kb HindIII, and 7.0-kb XbaI fragments. Additional bands were detected by using a 5S rRNA probe after BamHI digestion and XbaI digestion: a 1.0-kb BamHI band and a 4.3-kb XbaI band, respectively. Figure 4 shows that rRNA genes are located at or near the interface between E4 and El or between Bi and B3 within the R5 fragment. An additional band, designated E21, was also detected by 23S rRNA. The relative position of the band Original RsrII orEagI fragments were assigned on the basis of the size and the hybridization data. changed in different electrophoresis runs. The band position shown in Fig. 4A corresponds to a position between E10 and E12. The apparent size estimated here, 50 to 60 kb, could not be reconciled with the fact that 23S rRNA hybridized with only one band having a size ranging from 7 to 11 kb in Fig.  3. We believe that the band labeled E21 corresponds to the band that migrated at 1.4 kb after normal gel electrophoresis, as shown in Fig. 5.
After TaqI digestion, one strong 1.8-kb band and three weaker bands (1.3, 1.0, and 0.3 kb) were detected by 23S rRNA (Fig. 5). Signals were detected at 1.8 and 0.2 kb by 16S rRNA and at 1.8 and 0.4 kb by 5S rRNA. The 1.8-kb fragment that hybridized with 23S rRNA was different from the band detected by 16S and 5S rRNA, because the former fragment was cleaved by EagI, in contrast to the latter. None of these bands changed its mobility by the additional treatment with XbaI. These hybridization experiments showed that the genes are adjacent to one another and are a single copy. The position is indicated in Fig. 2. The relative order of rRNA genes will be analyzed precisely elsewhere. Circular chromosome in the last common ancestor. The A 23S 16S 5S recent molecular evolutional analysis of H+-ATPases (10) and other paralogous genes (12,27) revealed the position of the archaebacterial group in the evolutionary tree. The archaebacterial group is likely to be situated in the same branch as eukaryotes. The archaebacterial group is divided into two subgroups; one is of sulfur-dependent thermophiles, including S. acidocaldarius, and is named Crenarchaeota, and another is of methanogens, including halophiles and T. celer, and is named Euryarchaeota (27,28). Noll has reported the circular chromosomal map of T. celer (19). Circularity of the chromosome was also shown for M. voltae (23), H. mediterranei (17), and H. volcanii DS2 (7). These organisms belong to Euryarchaeota. We have clearly proved the circularity of the S. acidocaldarius 7 chromosome (29); the organism belongs to Crenarchaeota. The complete physical map obtained here supported the circularity of the chromosome. These findings suggest the general occurrence of circular chromosomes in archaebacterial species.
Increasing numbers of reports of eubacterial physical maps indicate the general occurrence of circular chromosomes (2, 4, 6, 8, 13-15, 20-22, 24, 25; for a review, see reference 16). Although there is the report of linear DNA in Borrelia burgdorfen, it is perhaps unique among eubacteria (9). The common occurrence of circular chromosomes in both eubacteria and archaebacteria supports our previous proposal that the last, or the most recent, common ancestor of all the living organisms on the earth had a circular chromosome (29). Thus, at the time when eubacteria and archaebacteria diverged, life on the earth had already developed the rigid DNA-based genetic system with circular chromosomal DNA.