Modulating the body's networks could become mainstream therapy for many health issues.
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Edited by Carl R. Woese, University of Illinois at Urbana–Champaign, Urbana, IL, and approved February 15, 2007 (received for review January 9, 2007)
Article Figures & SI
Figures
- Fig. 1.
Characterization of the Aeropyrum pernix replication origins by two-dimensional (2D) gel electrophoresis. (A) Alignment of the UCM sequences located centrally in the two A. pernix and three Sulfolobus origins (St, Sulfolobus tokodaii; Sac, S. acidocaldarius; Ss, S. solfataricus). (B) Cartoon representing the A. pernix genome, indicating the position of the two origins relative to the initiator genes Cdc6-1 and Cdc6-2. (C and D) Representation of the two origin loci. Ovals denote the origin locations. Gene ID numbers are indicated; ORFs above the midline are transcribed left to right, and those below the line are transcribed from right to left. (E) Illustration of the species of replication intermediates detectable at the origins. (F–K) 2D gel examination of the A. pernix origin loci. DNA isolated from asynchronous replicating A. pernix cells was digested with restriction enzymes to produce the fragments indicated in C and D and subjected to the 2D procedure. G and J display bubble arc intermediates that are indicative of an active bidirectional origin of replication within the central third of the restriction fragment.
- Fig. 2.
A hybrid arrangement of Sulfolobus origin associated gene homologues is observed at the A. pernix oriC2 locus. Genes located at two S. solfataricus initiation sites, oriC1 and oriC3, are positioned together at the A. pernix oriC2. Homologous genes are denoted by color, and the gene ID numbers are indicated. Transfer-RNA genes (blue) and viral integration sites (black) are also highlighted.
- Fig. 3.
Organization and orientation of the oriC3 loci in three Sulfolobus species. Colored hatching represents homologous regions of the genome in the three species. The direction of the hatching indicates the orientation of the homologous regions relative to the rest of the genome. A 21.5-kb intrafragment translocation is indicated by the yellow hatching. Origins are represented by ovals. The magnified segment of the S. acidocaldarius region illustrates the oriC3 proximal genes. S. solfataricus gene homologues are also displayed. ORF colors denote gene function: red, copG; green, WhiP; and yellow, probable stress response-related genes. HSP60*, thermosome α subunit belonging to the HSP60 family.
- Fig. 4.
Organization of WhiP and its relationship to eukaryal Cdt1. (A) Sequence alignment of archaeal WhiP proteins [Sac, S. acidocaldarius (GenBank accession no. YP_256028); Sto, S. tokodaii (GenBank accession no. NP_377180); Mse, M. sedula (GenBank accession no. ZP_01600793); Sso, S. solfataricus (GenBank accession no. NP_342366); Ape, A. pernix (GenBank accession no. NP_148313); Hbu, H. butylicus (GenBank accession no. YP_001013395)] with human Cdt1 residues 296–546 (GenBank accession no. EAW66763) and yeast Saccharomyces cerevisiae Cdt1 residues 354–604 (GenBank accession no. NP_012580). The alignment was generated by using ClustalW and colored in Jalview (30). (B) Cartoon depicting the two conserved DNA-binding domains in the S. acidocaldarius WhiP ORF. These domains, of the “winged-helix” DNA-binding domain superfamily (SSF46785), were identified by using InterProScan (version 14.0; www.ebi.ac.uk/InterProScan).
- Fig. 5.
The Winged-Helix Initiator Protein (WhiP) binds S. solfataricus oriC3. DNaseI footprinting analysis of WhiP, Cdc6-1, Cdc6-2, and Cdc6-3 interactions with oriC3 is shown. The position of previously described 12-bp inverted repeats (ir) and Sso0866 and Sso0867 ORFs are indicated at the side of the panel. (A) 0, 85, 100, and 130 nM WhiP on the oriC3 upper strand. (B and C) Cooperative effects of WhiP on Cdc6 binding on the oriC3 upper and lower strands, respectively. (B) Lanes 1, 3, 6, and 9: no protein; lanes 2, 4, 7, and 10: 130 nM WhiP, 500 nM Cdc6-1, 250 nM Cdc6-2, and 100 nM Cdc6-3, respectively; lanes 5, 8, and 11: 130 nM WhiP plus 500 nM Cdc6-1, 250 nM Cdc6-2, or 100 nM Cdc6-3, respectively. (C) Lanes 1, 4, 7, and 10: no protein; lanes 2, 3, 5, 8, and 11: 125 nM WhiP, 150 nM WhiP, 1,000 nM Cdc6-1, 200 nM Cdc6-2, and 75 nM Cdc6-3, respectively; lanes 6, 9, and 12: 150 nM WhiP plus 1,000 nM Cdc6-1, 200 nM Cdc6-2, or 75 nM Cdc6-3, respectively. (D) Cartoon summarizing the footprinting at oriC3. The open boxes labeled M denote regions of modification rather than discrete footprints.
Tables
Gene ID Known or predicted motif/function SAC1385 SSO0921 ST1204 Partial homology to HSP60 SAC1386 SSO0922 ST1203 Hsp20 SAC1388 SSO0925 ST1201 FeS assembly ATPase SufC SAC1389 SSO0927 ST1200 FeS assembly protein SufB SAC1390 SSO0928 ST1199 FeS assembly protein SufD SAC1399 SSO0859 ST1256 Homology to bacterial universal stress protein (USP) SAC1401 SSO0862 ST1253 Thermosome α subunit SAC1403 SSO0865 ST1251 CxxC thioredoxin motifs SAC1404 SSO0866 ST1250 Homology to HSP70 C terminus SAC1407 SSO0870 ST1246 Homology to heat-inducible transcription repressor (hrcA) - Table 2.
Probabilities of the conserved domains in three Sulfolobus species and A. pernix
Species N residues P C residues P S. acidocaldarius 1–95 9.9 × 10−6 166–233 1.1 × 10−2 S. tokodail 33–101 9.4 × 10−6 179–246 2.4 × 10−3 S. solfataricus 21–91 1.2 × 10−5 159–226 1.1 × 10−2 A. pernix 54–172 1.9 × 10−7 225–292 3.5 × 10−3
Data supplements
Robinson and Bell. 10.1073/pnas.0700206104.
Supporting Information
Files in this Data Supplement:
SI Figure 6Fig. 6. Putative replication origins in H. butylicus and M. sedula, harboring the UCM element. (A) Cartoon representing the H. butylicus genome, indicating the position of the two putative origins. The magnified regions display the ORFs (including gene ID numbers) flanking the origin sites and reveal the position of the UCM (black rectangle). Open arrows indicate the position and direction of ORB elements. UCM and ORB nucleotide sequences are presented. (B) Two putative origins of replication in the M. sedula genome. These loci are equivalent to SsoriC3 (MseoriC1) and SsoriC1 (MseoriC2). Gene ID numbers are indicated. Black rectangles and open arrows depict the location of the UCM and ORBs, respectively, and the motif sequences are presented.
Extrachromosomal element capture and the evolution of multiple replication origins in archaeal chromosomes
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