What chromosome is SCIDS on
Construction of targeted recombineering plasmids The position of the lac promoter:: gfp insertion site is given in base pairs with respect to the coordinate system origin ( 32). MATERIALS AND METHODS Strains and plasmidsīacterial strains and plasmids used in this study are listed in Supplementary Table S1. We identified several factors that impact upon gene expression, including processes that are involved with chromosome structuring and organization. We observed substantial position-dependent variation of promoter activity that is mediated at the point of transcription and is unrelated to gene dosage. coli by inserting a transcription reporter cassette at different targeted positions in the genome. Here we have re-addressed chromosomal position effects in E. Only a handful of similar studies have been conducted in bacterial systems, with all attributing minor differences in gene expression to gene dosage effects: the correlation between the increase in gene expression and the proximity of the gene to the origin of replication ( 23–31). Furthermore, expression levels were reflective of the local chromosomal environment: being attenuated in lamina binding domains and areas of compaction, and enhanced when located proximal to active genes. ( 22) analyzed ∼27 000 reporter gene integrations in mouse embryonic stem cells and demonstrated that expression varied across the genome by more than 1000-fold. In eukaryotic systems, it is well established that the expression of individual genes can be greatly affected by chromosomal position ( 18–21). These foci are located in discrete areas of the cell, indicating that some transcription events occur at particular locations and that the specific chromosomal position and spatial organization of genes may be important for maintaining control of gene expression ( 13, 16). Transcriptionally active regions are thought to be extruded to the periphery of the nucleoid where they are engaged by transcription foci, dense in RNA polymerase (RNAP) ( 11–17). Termed transcriptionally silent Extended Protein Occupancy Domains (tsEPODs), these domains overlap with regions bound by NAPs, but neither the precise protein organization nor whether tsEPODs contain predominantly poor promoters, or active promoters silenced by the associated proteins, is known.ĭespite being highly compacted, the nucleoid remains accessible for cellular processes such as transcription and replication. Highly proteinaceous transcriptionally silent regions of the chromosome have been identified as potential organizational hubs that may insulate the topologically isolated loops and macrodomains ( 10). All NAPs influence DNA conformation: with some binding predominantly within one macrodomain and others binding ubiquitously throughout the genome ( 6–9). Organization of the nucleoid is mediated by DNA supercoiling, macromolecular crowding and by a number of nucleoid associated proteins (NAPs), although the precise impact of each on the overall structure is not fully understood. Comprised of chromosomal DNA, protein and RNA, the nucleoid in Escherichia coli and Salmonella is arranged into topologically isolated loops, each ∼10 kb in length, which are further organized into four spatially isolated, structured macrodomains and two non-structured regions ( 3–5).
The nucleoid is a highly compact and organized structure occupying the majority of the intracellular cytoplasmic space in most bacteria ( 1, 2). Taken together, our findings identify a tier of gene regulation above local promoter control and highlight the importance of chromosome position effects on gene expression profiles in bacteria. Rather, the differences in gene expression occur predominantly at the level of transcription and are mediated by several different features that are involved in chromosome organization. These effects were not due to differences in gene copy number, caused by partially replicated genomes. At some positions, expression was more than 3-fold higher than at the natural lac promoter locus, whereas at several other locations, the reporter cassette was completely silenced: effectively overriding local lac promoter control. coli lac promoter driving expression of gfp, varies by ∼300-fold depending on its precise position on the chromosome.
Here, using Escherichia coli K-12, we demonstrate that expression of a reporter gene cassette, comprised of the model E. In eukaryotes, the location of a gene on the chromosome is known to affect its expression, but such position effects are poorly understood in bacteria.