E. coli genomic DNA

A DNA purification chip was constructed on PDMS. The chip consisted of mixing valves and a rotary mixer with fluidic and valve actuation channels. The chip had 26 access holes, 1 waste hole, and 54 valves to simultaneously carry out three purification procedures of E. coli genomic DNA. After removing DNA from the chip, the gene that encoded the prelipin protein peptidase-dependent protein D (ppdD) was PCR-amplified externally [915]. 

A Cft plot of E. coli genomic DNA is like that shown in Fig. 7-4. What are the physical units on the abscissa and ordinate and why are these two variables plotted and not some other potential candidates What is the state of the DNA in phases I and II of the graph ... 

In addition to the assay described earlier, we have also performed routine qualitative measurements of exonuclease activity using [ H]-P. coli genomic DNA (5.8 jtCi/p,g) (NEN), which has the advantage of being commercially available. A typical exonuclease reaction cocktail consists of 0.4 p,g/ml H-labeled E. coli genomic DNA in 1 x reaction buffer, and assays are carried out as described above. 

Figure 26-3 Integration of an F agent into a bacterial chromosome and transfer of some bacterial genes into another cell. (A) Incorporation of the F agent into E. coli genome and transfer of the "plus" strand of DNA out to a female recipient cell. (B) Genetic recombination between a piece of transferred DNA and the genome of the recipient cell.

For complex samples containing several different DNAs, multiplex PCR has been carried out. For instance, on-chip multiplex PCR was achieved on four DNAs representing regions in the bacteriophage A.DNA (199 and 500 bp), Escherichia coli genomic DNA (346 bp), and E. coli plasmid DNA (410 bp). After PCR, the fluorescent intercalating dye (TO-PRO) was added to the PCR reservoir, and CGE separation was performed downstream [929]. 

Based on total DNA content the E. coli genome could code for 3000 to 4000 individual proteins (14), and a total of perhaps 1500 have been visualized by two-dimensional electrophoresis during different growth conditions (15,16). Further, the proteins produced vary with the growth phase of the cell and the composition of the growth medium (6,15,16). These numbers are larger and the potential distribution more complex for mammalian host cell systems. From this complex mixture a process specific subset will be enriched, and therefore the distribution reference impurities must be refined to represent that population. 

The term chromosome refers to a physical or organizational unit within which part of or all the genome is contained. Thus, the E. coli genome is contained within just one chromosome, comprising a single DNA molecule. It has a size of 2.5 x 109 Da and contains approximately 4.6 x 106 base pairs. The size of DNA molecules is more commonly expressed in kilobase pairs (kb, 1,000 base pairs). The E. coli chromosome is 4,639 kb in size. Another feature of this particular molecule is that it is a closed, or circular, structure, i.e., there are no free ends. 

Information content, (a) How many different 8-mer sequences of DNA are there (Hint There are 16 possible dinucleotides and 64 possible trinucleotides.) (b) How many bits of information are stored in an 8-mer DNA sequence In the E. coli genome In the human genome (c) Compare each of these values with the amount of information that can be stored on a personal computer diskette. A byte is equal to 8 bits. 

DNA replication must be very rapid, given the sizes of the genomes and the rates of cell division. The E. coli genome contains 4.8 million base pairs and is copied in less than 40 minutes. Thus, 2000 bases are incorporated per second. We shall examine some of the properties of the macromolecular machines that replicate DNA with such high accuracy and... 

Inspection of the complete E. coli genome sequence reveals two sites within 500 bp of the primary operator site that approximate the sequence of the operator. Other lac repressor dimers can bind to these sites, particularly when aided by cooperative interactions with the lac repressor dimer at the primary operator site. No other sites that closely match sequence of the lac operator site are present in the rest of the E. coli genome sequence. Thus, the DNA-binding specificity of the lac repressor is sufficient to specify a nearly unique site within the E. coli genome. 

Counting sites. Calculate the expected number of times that a given 8-base-pair DNA site should be present in the E. coli genome. Assume that all four bases are equally probable. Repeat for a 10-base-pair site and a 12-base-pair site. 

The lac repressor does not bind DNA when the repressor is bound to a small molecule (the inducer), whereas pur repressor binds DNA only when the repressor is bound to a small molecule (the corepressor). The E. coli genome contains only a single lac repressor-binding region, whereas it has many sites for the pur repressor. 

In eubacteria and eukaryotes, several types of DNA polymerases have been characterized three in eubacteria (DNA polymerases I, II and III), and five in eukaryotes (DNA polymerases a, 3, 6, e and )). Some of these enzymes, named DNA replicases , are specifically involved in DNA-chain elongation at the replication fork. They have a multi-subunit structure and can prime and perform DNA replication in a processive way when they are associated with the other replicative proteins. In eubacteria, only one DNA replicase has been isolated (DNA polymerase III), whereas several DNA replicases co-exist in eukaryotes DNA polymerases a, 6 and e, which are essential for the replication of nuclear DNA, and DNA polymerase y, which is responsible for the replication of the mitochondrial genome. The other eubacterial and eukaryotic DNA polymerases are monomeric and are preferentially involved in mechanisms which require replication of short DNA fragments, in the course of either DNA repair (DNA polymerases I and II from E. coli, eukaryotic DNA polymerase 3), or DNA replication (maturation of Okasaki fragments by E. coli DNA polymerase I). 

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