E. Coli Chromosome High-throughput Screening Model Of Interaction

The eukaryote cell nucleus is a strict compartmentalization architecture containing diverse subnuclear organelles. During metaphase of cell mitosis, different chromosomes occupy discrete and relatively stable position, chromosome territories. Recently, the comprehensive intrachromosomal and interchromosomal interactions have been reported in eukaryote, which can regulate gene expression efficiently.As a classic genetic model, Escherichai coli have some specific gene regulation features. The E. coli genetic substances almost accumulate in the nucleoids structure. The nucleoid, which is absent of nuclear membrane, comprise of the nucleoid- associated protein, DNA and RNA. The active RNA polymerase almost focus on the nucleoid domain, while not smearing everywhere in the bacteria cell. Unlike eukaryote, different genes can consist of the operon which is the basical structure unit for gene regulation.Several operons regulated by the same transcription factor can comprise of the regulon. Some time, the operons belong to the same regulon are dispersed in the chromosome and far away from each other. Some studies indicate that the dispersed genes can colocalize together and form DNA loops during gene expression. But the molecule mechanism of long-range interaction, particularly the detail of three dimension structure of the nucleoid and its impact on genes expression remained elusive.In fact, the dissection of the nucleoid high-order structure of E. coli will greatly help to reveal the molecular mechanism of gene regulation that bacteria response to different growth conditions or signals. It wills also greatly faciliate the modification and improvement of some features special useful for the heterologous recombinant expression in E. coli.To systematially study the long-range chromosome interction and the genome 3D structure, we developed a new high throughput genome-wide assay, GCC-Bac (Genome Conformation Capture:Bacterial Version) which is based on the principle of proximal ligation and chromosome conformation capture. First of all, the E. coli K-12 MG1655 (ATCC 47076) was chosen as the research material. During the log phase growth, the E. coli was fixed by formaldehyde. And then the cross-linked DNA was broken by the ultrasonic method. Subsequently the crosslinked and broken DNA ends were filled and subjected into intramolecular ligation at very low DNA concentration. After the crosslinking was reversed, the DNA was cloned into the vectors by the T/A clone method. Some clones were picked up and sequenced by traditional Sanger methods. From the 216 clones, we obtained 60 clones each of which comprises of different gene locus partners. The bioinformatics analysis of the limited direct cloning and sequencing results revealed that many different operons are regulated by the same transcription factor. These proofs of principle experiments indicated our GCC-Bac assay works very well.Secondly, to obtain the genome-wide chromosome interaction infromation, the technique need to be combined with the second, even the third generation seqeuncing technology. For this purpose, the biotin-tag is incorporated into the cross-linked DNA ends, and then blunt-ended and intramolecular ligation was carried out under the much diluted conditons. The intramolecular ligation products were enriched by the streptavidin-biotin pulldown and used as the solexa sequencing samples. Limited cloning and sequencing results showed that many different genes occupying different chromosome location can be regulated by the same transcription factor, which is very similar as that of the direct clone sequencing. Several DNA sequences contain many consensuses binding motif. Bioinformatics analysis of sequencing data set showed that a few coding genes belonging to the same metabolism pathway interact with each other, which is concordant with known experiement results. We also find many rRNA genes, coding genes and regulation genes can interact with each other, which strongly indicated that these genes share the same regulation protein complex or colocalize at the same subcellular sturcuture. These results also agree with the special phenomenon that transcription and translation is coupled simultaneously in prokaryote. In addition, some pseudogenes are found to interact with coding genes. As the functions of most pseudogenes are not known, the bioloigical significance of these interactions remains elusive.In conclusion, our limited cloning, sequencing and the bioinformatics analysis of the sequencing results indicaetd that using the proximal ligation as well as the directly biotin labeling of sonicated DNA ends, we have successully developed a high throughput method: GCC-Bac which can be robustly integrated into the traditional Sanger, the second even the third generation sequrencing platforms. The establishment of GCC-bac will greatly facilitate the study of long-range chromosome interaction, the 3D stucture of nucleoid and its impact on gene regulation of E. coli and other microorganisms. It might be also extremely helpful for the revealing of the general folding principle of all the genome.