Supporting information for Slesarev et al. (2002) Proc. Natl. Acad. Sci. USA99 (7), 4644–4649. (10.1073/pnas.032671499)

Methods

Sequencing directly from cells. Plasmid clones of Mka DNA were grown in 1 ml of LB medium in deep 96-well plates with vigorous shaking. Fifty microliters of cell cultures was transferred into a 96-well PCR plate and centrifuged. Pellets were washed once with 150 µl of 10 mM Tris· HCl (pH 8.0). Five microliters of reaction mixture containing 2 µl of big dyes premix, 1 µl of Fimer, 0.1 µl of ThermoFidelase 2, and 1.9 µl of water was added to each pellet. The following Fimers were used: forward (5'-gggttttcccagtcacgacgUs1tgta-3') and reverse (5'-acaggaaacagctaUs1gaccat-3'), where Us1 is a NaOH derivatized 2'-succinimido-2'-deoxyuridine. Cycle sequencing conditions were as follows: initial denaturation at 95°C for 2 min, then 400 cycles with denaturation at 95°C for 5 sec, annealing at 55°C for 20 sec and extension at 60°C for 1 min. Unincorporated dyes were removed by centrifugation through Sephadex G-50 columns in 96 well format. Sequencing reactions were vacuum dried, resuspended in 3 µl of loading buffer, and 1 µl was loaded on a gel. We performed several thousand sequencing reactions on plasmids using a few microliters of overnight Escherichia coli clone cultures. The success rate of this procedure was about 30% and average quality read length was 300 bases. The procedure can be further improved and, given its simplicity and the fact that it does not require plasmid isolation, could become an alternative to available procedures for plasmid isolation and sequencing. However, even in its current stage, this method is effective for the identification of genes of interest in new organisms.

Sequencing of genomic DNA. Reaction volume was 10 µl. We used 1 µg of DNA per reaction. Sequencing reaction was carried out in 384-well plates. For 96 reactions, 106 µg of genomic DNA was mixed in a tube with 10.6 µl of ThermoFidelase 2, 10.6 (l of 1 mM 7-deaza-dGTP, 424 µl of big dye terminator mix (version 2 or 3). Water was added to bring the volume up to 954 µl. Nine microliters of this premix was dispensed into every well. One microliter of a corresponding Fimer was added to each well by multichannel pipette from the stock plate. Plates were sealed and placed in a thermocycler. Cycling conditions were as follows: denaturation for 2 min at 95°C, then 200 cycles with denaturation for 5 sec at 95°C, annealing for 30 sec at 55°C, and extension for 2 min at 60°C. Reactions were purified as described above, dried, and resuspended in 2 µl of loading buffer; 1.5 µl was loaded on a gel.

Sequencing from λ clones.λ clones were purified by using the Wizard λ kit (Promega). DNA was eluted in 100 ml of water. The yield varied from clone to clone. For end sequencing, we used forward (5'-cttatctgcttctcatagagUs1cttg-3') and reverse (5'-agaggttcattactgaacacUs1cgtc-3') Fimers. Reactions contained 2 µl of template DNA, 4 µl of big dyes, 1 µl of Fimer, and 0.1 µl of ThermoFidelase 2. Total reaction volume was 10 µl. Cycle sequencing conditions were: initial denaturation for 2 min at 95°C, followed by 100 cycles with denaturation for 5 sec at 95°C, annealing at 55°C for 30 sec, and extension at 60°C for 4 min.

Origin of replication. Identification of the origin of replication (ORI) in archaeal genomes is a notoriously hard problem. However, the ORI region of P. abyssi has been recently detected by using a combination of computational and experimental approaches (1), and ORI regions for other species of Pyrococci and M. thermoautotrophicum were predicted on the basis of similar nucleotide sequence features and genome contexts (1, 2). We applied similar computational techniques to predict the ORI region of M. kandleri. First, cumulative GC skew analysis (3) was used after removing all coding regions from the genome sequence. Several local minima were detected, with the global minimum located in positions 900,000--910,000 (Fig. 6). The adjacent region of the M. kandleri genome includes two long intergenic spacers (>800 bp): 884,531--885,746 and 894,161--895,399. The spacer 884,531--885,746 is surrounded by several genes implicated in DNA replication and repair, namely the genes for fused histones H3 and H4), ERCC4-like helicase and nuclease, ATPase involved in DNA replication, and a TIP49-type DNA helicase. All these genes are located within a 30-gene vicinity of this spacer. Such an enrichment in replication-related genes is typical of other archaeal ORI sites. Furthermore, context analysis of the spacer 884,531--885,746 revealed numerous direct and inverted repeats, again resembling the experimentally characterized and predicted ORI sites from Pyroccocus and M. thermoautotrophicum (1, 2). No similarity was found between this spacer and other intergenic spacers from the M. kandleri genome, whereas weak similarity (region of local similarity 30 bp in length with 9 mismatches, E = 0.03) was detected between this spacer and the experimentally confirmed Pyrococcus ORI. In contrast, the spacer 894,161--895,399 showed significant similarity to several other spacers from the M. kandleri genome, but no local similarity to the Pyrococcus ORI. These observations identify the spacer 884,531--885,746 as a candidate ORI site of M. kandleri.

References:

1. Myllykallio, H., Lopez, P., Lopez-Garcia, P., Heilig, R., Saurin, W., Zivanovic, Y., Philippe, H. & Forterre, P. (2000) Science288, 2212--2215.

2. Lopez, P., Philippe, H., Myllykallio, H. & Forterre, P. (1999) Mol. Microbiol.32, 883--886.

3. Grigoriev, A. (1998) Nucleic Acids Res.26, 2286--2290.