Ori sequences

Prokaryotic Bacterial plasmid Inducible or constitutive Dmg resistance Bacterial origin (ori) sequence needed for DNA rephcation"... 

Eukaryotic Yeast plasmid or integration into host chromosome by homologous recombination Transient or permanent Amino acid requirement in autotrophic strain heavy metal induction of resistance gene Yeast ori sequence constitutive or inducible promoter transcription terminator... 

Eukaryotic Mammalian cell-transient expression Transient expression None A higher copy number can be achieved using ori sequence that responds to factors in recipient cells Strong constitutive or inducible promoter polyadenylation signal intron sequences... 

DnaA protein 52,000 1 Recognizes ori sequence opens duplex at specific sites in... 

Most origins have quasi-palindromic nucleotide sequences, perhaps so that DNA can be looped out from the main duplex as is shown in Fig. 27-18A and B. The lengths of ori sequences vary, as does the complexity of their possible folding patterns. Plasmids have been constructed which not only contain the E. coli origin, but are dependent upon that origin for their own replication.3823 Study of those plasmids indicate... 

Smith, R. H., Spano, A. J. and Kotin, R. M. (1997). The Rep78 gene product of adeno-associated vims (AAV) self-associates to form a hexameric complex in the presence of AAV ori sequences. J. Virol. 71, 4461-4471. 

Figure 2.9. Physical and genetical map of the mitochondrial genome unit of wild-type yeast (strain A). Some restriction sites are indicated. Circled numbers indicate the location of ori sequences 1-8 (arrowheads point in the direction cluster C to cluster A sec Fig. 2.8). Black and dotted areas correspond to exons and introns of mitochondrial genes, respectively. Thin radial lines ending in small circles indicate tRNA genes. White areas correspond to long AT spacers embedding short GC clusters. (Modified from de Zamaroezy et al.,...

It should be noted (i) that some ori sequences display one orientation and some the opposite one on the wild-type genome (ii) that ori 2 and 7 as well as ori 3 and 4 are close to each other and tandemly oriented (iii) that ori 4 is absent in a wild-type strain (iv) that ori sequences containing the 7 cluster were found only once (ori 4), or not at all (ori 6, ori 7) in extensive screenings of spontaneous petite genomes (see Table 2.1). 

Ori° petites, lacking a canonical ori sequence, were also found, although very rarely. An investigation of the mitochondrial genomes of eight such ori° petites (Goursot et al., 1982) has revealed that their repeat units contain, instead of canonical ori sequences, one or more orf or ori surrogate sequences. These orP sequences are a subset of GC clusters char-... 

Figure 2.12. Primary structure of the ori sequences and their flanking regions. Regions of ori sequences are indicated by thick lines for GC clusters A, B and C and thin lines for AT stretches p, s, I and r. The positions of GC clusters a in ori 4, p in on 4 and 6, y in ori 4, 6 and 7, and S in on 1 are given r sequences are indicated by heavy line boxes r and r sequences by broken line boxes. Other boxes indicate sequences /a, Ip, /g, rz, the excision. sequences of the repeat units of petites a-l/lR/1/26, a-l/lR/14, a-3/1/33, a-3/1/5, anda-l/IR/Z] and the initiation triplet of the open reading frames of ori 1, 2, 3 and 5. (From de Zamaroczy et al., 1983).

Figure 2.13. A. Comparison of ori sequences of mitochondrial genomes from yeast (de Zamaroczy et al., 1981) and HeLa cells (Crews et al., 1979). Homology of potential secondary structures is found for the inverted repeats in the cluster A - cluster B region arrows indicate the base changes found in this region in different petite genomes. Homology of primary structure is found for cluster C. B. Comparison of the two ori sequences the arrows indicate the inverted repeats of the A-B region, the broken line corresponding to the looped-out sequence bp, base pairs. (From de Zamaroczy et al., 1981).

A functional evidence that ori sequences are indeed involved in the replication of the mitochondrial genome came from crosses of spontaneous petites, characterized in their mitochondrial genome and their suppressivity, with wild-type cells (de Zamaroczy et al., 1979 1981 Goursot et al., 1980). In the suppressivity test, petite mutants are crossed with... 

That ori sequences also act as transcription initiation sites is indicated by three results on petite genomes (Baldacci and Bernard , 1982). 

Exceptions to this repeat-unit-size rule (see Section 2.3) were found, however, even when the parental mitochondrial genomes carried the same ori sequence (compare the supppressivities of petites bl7/b20, bll/b7, a 1/7/8. f in/20 of Fig. 2.16 and the predominance in crosses of b7 over bll, of b over hp5, of b 13/1 over a-23/3 see also Rayko et al., 1988). This indicates that noncoding, intergenic sequences flanking ori sequences also play a role in the modulation of replication efficiency. Since in different petites such sequences differ in primary structure, size, and position relative to ori sequences, this modulation is likely to take place through an indirect effect on DNA and nucleoid structure (Rayko et al., 1988 see Fig. 2.21). 

While each repeat unit of the mitochondrial genomes of petites al/l R/l and Zl contains a complete ori sequence, those of petites 26 and 14 contain ori sequences which had lost 11 and 27 bp, respectively, in the excision process. In the case of petite 26. the deletion comprises GC duster A in the case of petite 14, it comprises also part of the neighbouring sequence p (dc Zamaroezy et al., 1981 1984 Figs. 2.12 and 2.17). In the complete ori... 

The orf mitochondrial genome of petites 14 and 26, which lack a normal feature of the ori sequence, the A-B fold (a feature which is perfectly conserved in all ori sequences de... 

Figure 2.19. Potential secondary structure of the A-B fold of the ori sequence present in the repeat units of mitochondrial genomes from petites al/lR/1 and Z1 (ori ) and of the replacement folds that can be formed in the oril sequence present in the mitochondrial genomes of petites 14 and 26. In the case of petite 14, the residual nucleotides from the partially deleted p stretch (/ A) can generate a hairpin structure with nucleotides from the preceding repeat unit, but the stem, only formed by 13 A/T nucleotides, carries different terminal and side loops compared to the A-B fold. In the case of petite 26, the upper part of the stem and terminal loop are identical to those of the A-B fold, but the lower part is replaced by three A T pairs (three nucleotides are derived from the preceding repeat unit). The sequence involved in the structure shown (GC clusters A and B, sequences p and are those indicated in Fig. 2.13. (From Goursot et al., 1988).

It should be pointed out that the repeat unit of petite Zl extends 80 bp to the left of cluster A and only 40 bp to the right of sequence r, whereas those of petites 26 and 14 extend 115 bp to the right of sequence r (Fig. 2.18) in all cases, however, these extensions to the left of ori sequences are just made of AT spacer. Effects of flanking regions of ori sequences on the replicative ability of the latter are known (Rayko et al., 1988 see Section 2.5), but they are small compared with the different suppressivities exhibited by petites 14 and 26 relative to petite Zl. There is, therefore, no doubt that these differences are due to the deletions in the ori sequences of petites 14 and 26. 

Interestingly, the conclusion that DNA secondary structure is required for ori activity in vivo was also reached for bacteriophage G4, where a strong temperature-dependent impairment of replication was found after introducing by site-directed mutagenesis point mutations which destabilize intra-strand base-pairing in the ori sequence (Lambert et al., 1987). 

The extremely high homology of the eight canonical ori sequences indicates that they arose as the result of duplication and translocation events. More precisely, we proposed (see Fig. 2.20) that the canonical ori sequences derive from a primitive ori sequence (probably made of only a monomeric cluster C and its flanking sequences r and r) through (i) a series of duplications and inversions generating clusters A and B and (ii) an expansion process producing the AT stretches of ori sequences. It is possible that the ori sequences arc folded in a tertiary structure, as in the hypothetical model of Fig, 2.21. 

E- igurc 2,20, Hypothetical scheme for the evolutionary construction of ori sequences. (Irom dc Zamaroezy... 

Figure 2.21. Hypothetical superfolding of ori sequences from the mitochondrial genome of yeast, (a, a ) Superfolding of ori 3 and ori 5 base-pairing interactions take place between sequences /a and ra as well as between flanking sequences Zp. sequence is supposed to loop out. In ori 4 and ori 6, the size of the loop (132 bp) between sequences Zp and C would be considerably extended (by 70 bp) because of the insertion of clusters p and y. This does not occur, however, if these clusters take the configuration shown in (a), (b) Folding of ori I. In the case of ori 1, Zp cannot fold upon itself, but can interact with sequence r this interaction may also take place in ori 2, The overall result is no change in the size of the loop. Only in the case of ori 7, the loop would be larger by 30 bp, the size of cluster y. (From de Zamaroezy et al., 1984).

These results are similar to the in vivo data on the effect of growth temperature on the structure and function of ori sequences in the mitochondrial genome of yeast (Goursot et al., 1988 see Part 2). In this case too, increasing temperature from 28°C to 33°C led to the melting of AT stems in the ori sequence of some petite mutants and to the consequent decrease of replicative ability. These results demonstrate direct (reversible) temperature effects on the secondary (and tertiary) structures of DNA and RNA. 

---