EcoRI sites

EcoRI methylase E. coli Transfer CH3 from S-adenosylmethionine to adenines in EcoRI sites... 

Fig. 1.5. Mechanism of cloning into the EcoRI site. N represents any nucleotide and N is its complement. (For discussion see text.)...

Fig. 4.2. Nucleotide sequences adjacent to the EcoRI site in M13mp2, the Bam HI site in M13mp2/Bam and the Hindlll site in M13mp2/HmdIII. Both the latter vectors are derived from the original M13mp2 by the inclusion of an 18-base pair fragment (in bold) which contributes 6 additional codons but does not alter the reading frame. M13mp5 is functionally equivalent to M13mp2/HindIII.

The array of restriction endonuclease cleavage sites which has been inserted near the N-terminus of the /3-galactosidase gene occupies positions 6231 (EcoRI site) to 6238 (centre of the HaeIII site) cf. page 134, chapter 4). A restriction map of the closely related M13mp8 is given in Appendix 8. 

Approximately how many EcoRI sites would you expect to find in the E. coli chromosome ... 

EcoRI recognizes and cleaves at a sequence-specific six-base-pair site on DNA. Considering that each site can be occupied by one of four correctly orientated base pairs, the enzyme would cleave a random sequence once every 46 (or 4,096) base pairs. The E. coli chromosome has a size of 4.6 x 106 base pairs. Thus, there would be about 4.6 x 106/4,096, or approximately 1,000, EcoRI sites on the chromosome. 

Figure 1. DNA sequence and translation product of 14-1 cDNA clone. The EcoRI sites at the 5 and 3 ends were generated by oligonucleotide linkers.

Figure 1.2-8 shows the scheme where EcoRI and BamHI sites alternate. In reality, EcoRI sites and BamHI sites do not always alternate, and this will lead... 

Figure 1.2-8. The main idea of the slalom approach. Numbers designate identical end sequences in different libraries that can be joined by a computer program in a contig of overlapping clones. B represents BamHI, and R denotes EcoRI sites. Small horizontal arrows show short sequences flanking EcoRI sites. The BR library plays a role of a linking library where EcoRI replaces Notl. The ordinary R library plays the role of a jumping library where EcoRI replaces Notl.

What would happen if the restriction fragment had an internal EcoRI site ... 

Prepare the template for transcription by linearizing approx 5 jg of vector/insert DNA m a final volume of 20 pL containing IX incubation buffer and 10 U restriction endonuclease for at least 1 h at 37 C. Use Hindlll for the vector system pSPT18, EcoRI for pSPT19 (4), and Hindlll for DNA probes cloned into the EcoRY site of pBluescript II KS. This will result in the linear order vector DNA, T7 promoter, insert ( hybridization probe), cleavage site of the endonuclease used. Store the template at -20 C (see Notes 3-5). 

The mRNA from the low CO -grown wild-type cells was then used to make a cDNA library. The cDNA was then cloned into the EcoRI site of the pUC-18 plasmid and these recombinant plasmids were used to transform E. coli strain JM83. Colonies carrying C. reinhardtii DNA that is expressed under low CO, conditions were screened for by differential hybridization to P-polyadenylated mRNA from both low C02-grown, wild-type cells and high C02-grown, CIA-5 cells (Figure 4). Transformants that preferentially hybridized to the wild type mRNA have been selected and will be further characterized. 

Fig. 8. Genomic organization of poly(ADP-ribose) polymerase. The alignment of similar EcoRI sites present in the genomic and cDNA map is illustrated together with cDNA probes used to order the EcoRI fragments. The approximate location in the genomic map for the PsH cDNA site is also shown. (Separate scales have been used to construct the genomic and cDNA maps.) b. Base. (Taken from Ref. 3).

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