The DNA fragments obtained from using endonucleases such as SnaBI produce a distinct pattern of bands useful for physical mapping of the chromosomes. However, there is a kb of DNA unaccounted, which makes it arithmetically impossible since there must be a total SnaBI fragment length of 1, kb [12]. Through the process of narrowing down how the fragments connect, the chromosomes were physically mapped.
In a molecular-basis, this experiment was used to determine that R. In terms of a genetic-basis, the interrupted mating experiments using Hfr strains strengthened the evidence for the presence of multiple chromosomes in R. In , the first evidence of linear chromosomes was discovered, but the limited availability of scientific techniques, and a strong belief of bacterial chromosomal circularity was so convincing that many people did not take in this idea till much later.
By , the pulse-field gel electrophoresis technique was developed, and used in conjunction with restriction digests to verify that spirochete Borrelia burgdorferi has a linear chromosome using a similar process of narrowing down by connecting the separated fragments like the R.
This chromosome organization is comparable to the eukaryotic chromosome organization. Two problems arise with the presence and use of linear chromosomes compared to circular chromosomes in prokaryotes.
Firstly, the free double-stranded DNA ends must have some kind of protection so that intracellular nucleases do not degrade them [14]. Secondly, the telomeres, which are the ends of the linear DNA molecules, will require a different process for DNA replication [14]. For protection, there are two types of telomeres that have been observed.
The first one is called the palindromic hairpin loops, in which there are no free double-stranded ends available [8] , [14]. This can be found in Streptomyces linear chromosomes. Supercoils are both inserted and removed by topoisomerases. DNA topoisomerases are, therefore, essential in the unwinding, replication, and rewinding of the circular, supercoiled bacterial DNA.
For example, a topoisomerase called DNA gyrase catalyzes the negative supercoiling of the circular DNA found in bacteria. Topoisomerase IV, on the other hand, is involved in the relaxation of the supercoiled circular DNA, enabling the separation of the interlinked daughter chromosomes at the end of bacterial DNA replication.
In general, DNA is replicated by uncoiling of the helix, strand separation by breaking of the hydrogen bonds between the complementary strands, and synthesis of two new strands by complementary base pairing. Replication begins at a specific site in the DNA called the origin of replication ori C. DNA replication is bidirectional from the origin of replication. To begin DNA replication, unwinding enzymes called DNA helicases cause short segments of the two parent DNA strands to unwind and separate from one another at the origin of replication to form two "Y"-shaped replication forks.
Single-strand binding proteins bind to the single-stranded regions so the two strands do not rejoin. Unwinding of the double-stranded helix generates positive supercoils ahead of the replication fork.
Enzymes called topoisomerases counteract this by producing breaks in the DNA and then rejoin them to form negative supercoils in order to relieve this stress in the helical molecule during replication.
As the strands continue to unwind and separate in both directions around the entire DNA molecule, new complementary strands are produced by the hydrogen bonding of free DNA nucleotides with those on each parent strand.
As the new nucleotides line up opposite each parent strand by hydrogen bonding, enzymes called DNA polymerases join the nucleotides by way of phosphodiester bonds. Actually, the nucleotides lining up by complementary base pairing are deoxynucleotide triphosphates, composed of a nitrogenous base, deoxyribose, and three phosphates.
In bacteria, Par proteins function to separate bacterial chromosomes to opposite poles of the cell during cell division. They bind to the origin of replication of the DNA and physically pull or push the chromosomes apart, similar to the mitotic apparatus of eukaryotic cells.
Fts proteins, such as FtsK in the divisome, also help in separating the replicated bacterial chromosome. DNA polymerase enzymes are only able to join the phosphate group at the 5' carbon of a new nucleotide to the hydroxyl OH group of the 3' carbon of a nucleotide already in the chain.
As a result, DNA can only be synthesized in a 5' to 3' direction while copying a parent strand running in a 3' to 5' direction. Each DNA strand has two ends. The two strands are antiparallel, that is they run in opposite directions.
However, the other parent strand - the one running 5' to 3' and called the lagging strand - must be copied discontinuously in short fragments Okazaki fragments of around nucleotides each as the DNA unwinds. This occurs, as mentioned above, at the replisome. The lagging DNA strand loops out from the leading strand and this enables the replisome to move along both strands pulling the DNA through as replication occurs. They can only attach new nucleotides onto 3' OH group of a nucleotide in a preexisting strand.
Therefore, to start the synthesis of the leading strand and each DNA fragment of the lagging strand, an RNA polymerase complex called a primase is required. The primase, which is capable of joining RNA nucleotides without requiring a preexisting strand of nucleic acid, first adds several comlementary RNA nucleotides opposite the DNA nucleotides on the parent strand.
Yet even with this complicated procedure, a micrometer-long macromolecule of tightly-packed, supercoiled DNA can make an exact copy of itself in only about 10 minutes time under optimum conditions, inserting nucleotides at a rate of about nucleotides per second! The chromosome is the genetic material of the bacterium.
Messenger RNA is then translated into protein at the ribosomes. In general then, DNA determines what proteins and enzymes an organism can synthesize and, therefore, what chemical reactions it is able to carry out.
The epigenome refers to a variety of chemical compounds that modify the genome typically by adding a methyl CH 3 group to the nucleotide base adenine at specific locations along the DNA molecule. This methylation can, in turn, either repress or activate transcription of specific genes.
By basically turning genes on or off, the epigenome enables the bacterial genome to interact with and respond to the bacterium's environment. The epigenome can be inherited just like the genome. Each deoxynucleotide contains a phosphate, a 5-carbon sugar 2-deoxyribose and one of four nitrogenous bases: adenine, cytosine, thymine or guanine.
The phosphate and sugar make up the backbone of each strand of DNA, while the bases are responsible for holding the two strands together via hydrogen bonds in a structure called the double helix see figure. The order of the bases in a DNA strand contains the coded genetic information. All of the DNA found in an organism is collectively referred to as the genome.
The human genome is comprised of 23 pairs of linear chromosomes, and approximately megabases Mb of DNA, while the genome of the bacterium Escherichia coli consists of a single 4. By studying the genomes of bacteria we are able to better understand their metabolic capabilities, their ability to cause disease and also their capacity to survive in extreme environments.
Many of the well-studied bacterial model organisms, such as E. However, advances in molecular genetics have shown that bacteria possess more complex arrangements of their genetic material than just a single circular chromosome per cell.
The following are a few examples of bacteria with unusual genomes. Deinococcus radiodurans was first discovered in by Arthur W.
While inspecting spoiled meat, he noticed reddish colonies forming despite the fact that the meat had been sterilized with megarads of radiation! This radiation resistant organism was given the name Deinococcus radiodurans - which literally means "strange berry that withstands radiation.
That is 3, times greater than the amount of radiation exposure that would kill a human. Ionizing radiation makes double-strand breaks in the DNA. Somehow, D. The genome of D.
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