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What is the diameter of each type of deoxyribonucleotide (if the types are significantly different in diameter, if they are fairly similar one value would suffice)? I.e., if a hole was to be created through which only a single deoxyribonucleotide could pass at a time, how small would it have to be?
DNA is about 20 angstroms wide, so each of the two strand is about 10 angstroms wide. That width is the average between a larger purine unit and smaller pyrimidine unit, so let's round up to 12 or 13 angstroms for the larger unit.
DNA is a helically twisted double-chain poly deoxyribonucleotide macromolecule that constitutes the genetic material of all the organisms with the exception of ribovirus. In prokaryotes, it occurs in nucleoid and plasmid. The DNA is usually circular. Unlike, in eukaryotes, it occurs in chromatin of the nucleus and this is linear. Single-stranded DNA occurs as genetic material in some viruses. In addition, smaller quantities of DNA are also present in mitochondria and plastids. In this case, it can either be circular or linear.
DNA was discovered in 1869 by J. Friedrich Miescher, a Swiss researcher. He named the phosphorus-containing substance as “nuclein”. Erwin Chargoff and his colleagues (in the late 1940s) found that the four nucleotide bases of the DNA occur in different ratios in different organisms. Furthermore, the demonstration that DNA contains genetic information made in 1944 by Avery, Macleod, and MacCary .
Mitochondrial Machineries for Protein Import and Assembly
Nils Wiedemann and Nikolaus Pfanner
Vol. 86, 2017
Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport . Read More
Figure 1: Overview of the five major protein import pathways of mitochondria. Presequence-carrying preproteins are imported by the translocase of the outer mitochondrial membrane (TOM) and the presequ.
Figure 2: The presequence pathway into the mitochondrial inner membrane (IM) and matrix. The translocase of the outer membrane (TOM) consists of three receptor proteins, the channel-forming protein To.
Figure 3: Role of the oxidase assembly (OXA) translocase in protein sorting. Proteins synthesized by mitochondrial ribosomes are exported into the inner membrane (IM) by the OXA translocase the ribos.
Figure 4: Carrier pathway into the inner membrane. The precursors of the hydrophobic metabolite carriers are synthesized without a cleavable presequence. The precursors are bound to cytosolic chaperon.
Figure 5: Mitochondrial intermembrane space import and assembly (MIA) machinery. Many intermembrane space (IMS) proteins contain characteristic cysteine motifs. The precursors are kept in a reduced an.
Figure 6: Biogenesis of β-barrel proteins of the outer mitochondrial membrane. The precursors of β-barrel proteins are initially imported by the translocase of the outer membrane (TOM), bind to small .
Figure 7: The dual role of mitochondrial distribution and morphology protein 10 (Mdm10) in protein assembly and organelle contact sites. Mdm10 associates with the sorting and assembly machinery (SAM) .
Figure 8: Multiple import pathways for integral α-helical proteins of the mitochondrial outer membrane. The precursors of proteins with an N-terminal signal anchor sequence are typically inserted into.
Figure 9: The mitochondrial contact site and cristae organizing system (MICOS) interacts with protein translocases. MICOS consists of two core subunits, Mic10 and Mic60. Mic10 forms large oligomers th.
Discovering the Double Helix
By the early 1950s, considerable evidence had accumulated indicating that DNA was the genetic material of cells, and now the race was on to discover its three-dimensional structure. Around this time, Austrian biochemist Erwin Chargaff  (1905–2002) examined the content of DNA in different species and discovered that adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine was very close to equaling the amount of thymine, and the amount of cytosine was very close to equaling the amount of guanine, or A = T and G = C. These relationships are also known as Chargaff’s rules.
Figure 4. The X-ray diffraction pattern of DNA shows its helical nature. (credit: National Institutes of Health)
Other scientists were also actively exploring this field during the mid-20th century. In 1952, American scientist Linus Pauling (1901–1994) was the world’s leading structural chemist and odds-on favorite to solve the structure of DNA. Pauling had earlier discovered the structure of protein α helices, using X-ray diffraction, and, based upon X-ray diffraction images of DNA made in his laboratory, he proposed a triple-stranded model of DNA.  At the same time, British researchers Rosalind Franklin (1920–1958) and her graduate student R.G. Gosling were also using X-ray diffraction to understand the structure of DNA (Figure 4). It was Franklin’s scientific expertise that resulted in the production of more well-defined X-ray diffraction images of DNA that would clearly show the overall double-helix structure of DNA.
James Watson (1928–), an American scientist, and Francis Crick (1916–2004), a British scientist, were working together in the 1950s to discover DNA’s structure. They used Chargaff’s rules and Franklin and Wilkins’ X-ray diffraction images of DNA fibers to piece together the purine-pyrimidine pairing of the double helical DNA molecule (Figure 5). In April 1953, Watson and Crick published their model of the DNA double helix in Nature.  The same issue additionally included papers by Wilkins and colleagues,   each describing different aspects of the molecular structure of DNA. In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Physiology and Medicine. Unfortunately, by then Franklin had died, and Nobel prizes at the time were not awarded posthumously. Work continued, however, on learning about the structure of DNA. In 1973, Alexander Rich (1924–2015) and colleagues were able to analyze DNA crystals to confirm and further elucidate DNA structure. 
Figure 5. In 1953, James Watson and Francis Crick built this model of the structure of DNA, shown here on display at the Science Museum in London.
Think about It
Question: 21. During Gel Electrophoresis, The DNA Fragments That Are __________________ Will Move Fastest Through The Gel Matrix. Group Of Answer Choices Smallest In Size (number Of Nucleotides) Largest In Size (number Of Nucleotides) Simplest In Base Sequence Most Complex In Base Sequence 22. True/false. When A Deoxyribonucleotide Is Incorporated Into A Growing .
21. During gel electrophoresis, the DNA fragments that are __________________ will move fastest through the gel matrix.
Smallest in size (number of nucleotides)
Largest in size (number of nucleotides)
Simplest in base sequence
Most complex in base sequence
When a deoxyribonucleotide is incorporated into a growing DNA strand, the strand cannot elongate.
23. In sequencing by chain termination (or "Sanger" sequencing), dideoxynucleotides (ddNTPs) are used because:
ddNTPs are in fact RNA nucleotides
ddNTPs lack the hydroxyl group at the 3' carbon
ddNTPs lack phosphate groups at the 5' carbon
ddNTPs have an unusual base composition that prevents normal base-pairing
24. After you run sequencing by chain termination ("Sanger" sequencing), you use gel electrophoresis to resolve the product sizes, as shown in the gel below.
The DNA chain synthesized in your reactions is read as:
25. Which of the following sequences could be a palindromic restriction endonuclease site?
Figure 2: Phenolphthalein trial
Afterwards, the nucleus DNA should be isolated before executing the Deoxyribonucleic acid analysis. The basic stairss in the Deoxyribonucleic acid extraction are shown as follows:
Cytolysis to interrupt the cell and expose the Deoxyribonucleic acid by adding the lysis buffer. The common lysis buffers are tris-HCl, EDTA in Na chloride solution. Incubate the sample with lysis buffer for 30 proceedingss on ice and extractor for 10 proceedingss at 4oC to acquire the DNA pellet.
Removal of cell membrane by the add-on of detergent, such as Na dodecyl sulphate ( SDS )
Removal of protein by the add-on of peptidase K and incubate it at 37oC in the H2O bath overnight.
Perform the phenol-chloroform extraction by the add-on of same volume of phenol: trichloromethane mixture and aqueous buffer solution and centrifugate the mixture at 4oC. After the stage separation, the aqueous bed is on the top while the organic bed is at the underside. Most of the Deoxyribonucleic acid is in the aqueous bed so that it can be isolated.
Precipitation of DNA in ethyl alcohol or iso-propanol and re-dissolve in Tris EDTA buffer at pH 8.0.
Chelating agent can be used to adhere with metal ions, such as Mg and Ca ions that inhibit the Polymerase concatenation reaction ( PCR ) .
Two common techniques are used for the Deoxyribonucleic acid analysis. They are restriction fragment length polymorphism ( RFLP ) and polymerase concatenation reaction – short tandem repetitions ( PCR-STR ) . RFLP is the traditional DNA profiling technique which uses the variable figure of tandem repetitions ( VNTR ) in the non-coding parts. The limitation enzyme should be used which cut the long Deoxyribonucleic acid strand into a fragment at a precise sequences of 4 – 8 base brace called acknowledgment sites. The common limitation enzymes are shown as follows:
Bacillus amyloliquefaciens H
Haemophilus influenzae Rd
Agarose and Acrylamide Gels
The polymerization of acrylamide is inhibited by oxygen. These gels must therefore be poured vertically between two glass plates that are held apart by two plastic spacers. This process is time-consuming and technically more demanding than horizontal agarose gel electrophoresis. However, acrylamide gels have excellent resolving power. They are used when a high degree of separation (i.e. a size difference of a single base pair) is required, such as when sequencing DNA .
Gels can be stained either before or after an electrophoresis run. To stain a gel before an electrophoresis run, ethidium bromide is added to the melted agarose before it is poured on to the gel tray. This staining method is the quickest and offers the advantage that the position of the DNA can be monitored during the separation. Alternatively, the gel may be placed in a dilute solution of ethidium bromide for 10–30 min after the electrophoresis run.
Factors influencing mobility
Department of Health and Human Services (DHHS), Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, 27709, North Carolina, USA
Jessica S. Williams, Scott A. Lujan & Thomas A. Kunkel
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You can also search for this author in PubMed Google Scholar
You can also search for this author in PubMed Google Scholar
Chapter 16 - The Molecular Basis of Inheritance
- The specific pairing of nitrogenous bases in DNA was the flash of inspiration that led Watson and Crick to the correct double helix.
- The possible mechanism for the next step, the accurate replication of DNA, was clear to Watson and Crick from their double helix model.
During DNA replication, base pairing enables existing DNA strands to serve as templates for new complementary strands.
- In a second paper, Watson and Crick published their hypothesis for how DNA replicates.
- Essentially, because each strand is complementary to the other, each can form a template when separated.
- The order of bases on one strand can be used to add complementary bases and therefore duplicate the pairs of bases exactly.
- One at a time, nucleotides line up along the template strand according to the base-pairing rules.
- The nucleotides are linked to form new strands.
- In their experiments, they labeled the nucleotides of the old strands with a heavy isotope of nitrogen (15N), while any new nucleotides were indicated by a lighter isotope (14N).
- Replicated strands could be separated by density in a centrifuge.
- Each model—the semiconservative model, the conservative model, and the dispersive model—made specific predictions about the density of replicated DNA strands.
- The first replication in the 14N medium produced a band of hybrid (15N-14N) DNA, eliminating the conservative model.
- A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model.
A large team of enzymes and other proteins carries out DNA replication.
- It takes E. coli 25 minutes to copy each of the 5 million base pairs in its single chromosome and divide to form two identical daughter cells.
- A human cell can copy its 6 billion base pairs and divide into daughter cells in only a few hours.
- This process is remarkably accurate, with only one error per ten billion nucleotides.
- More than a dozen enzymes and other proteins participate in DNA replication.
- Much more is known about replication in bacteria than in eukaryotes.
- The process appears to be fundamentally similar for prokaryotes and eukaryotes.
- These enzymes separate the strands, forming a replication “bubble.”
- Replication proceeds in both directions until the entire molecule is copied.
- At the origin sites, the DNA strands separate, forming a replication “bubble” with replication forks at each end.
- The replication bubbles elongate as the DNA is replicated, and eventually fuse.
- The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells.
- Each has a nitrogenous base, deoxyribose, and a triphosphate tail.
- ATP is a nucleoside triphosphate with ribose instead of deoxyribose.
- The exergonic hydrolysis of pyrophosphate to two inorganic phosphate molecules drives the polymerization of the nucleotide to the new strand.
- Each DNA strand has a 3’ end with a free hydroxyl group attached to deoxyribose and a 5’ end with a free phosphate group attached to deoxyribose.
- The 5’ --> 3’ direction of one strand runs counter to the 3’ --> 5’ direction of the other strand.
- A new DNA strand can only elongate in the 5’ --> 3’ direction.
- The DNA strand made by this mechanism is called the leading strand.
- Unlike the leading strand, which elongates continuously, the lagging stand is synthesized as a series of short segments called Okazaki fragments.
- They can only add nucleotides to the 3’ end of an existing chain that is base-paired with the template strand.
- The primer is 5–10 nucleotides long in eukaryotes.
- RNA polymerases can start an RNA chain from a single template strand.
- Another DNA polymerase, DNA polymerase I, replaces the RNA nucleotides of the primers with DNA versions, adding them one by one onto the 3’ end of the adjacent Okazaki fragment.
- This untwisting causes tighter twisting ahead of the replication fork, and topoisomerase helps relieve this strain.
- The lagging strand is copied away from the fork in short segments, each requiring a new primer.
- For example, helicase works much more rapidly when it is in contact with primase.
Enzymes proofread DNA during its replication and repair damage in existing DNA.
- Mistakes during the initial pairing of template nucleotides and complementary nucleotides occur at a rate of one error per 100,000 base pairs.
- DNA polymerase proofreads each new nucleotide against the template nucleotide as soon as it is added.
- If there is an incorrect pairing, the enzyme removes the wrong nucleotide and then resumes synthesis.
- The final error rate is only one per ten billion nucleotides.
- DNA molecules are constantly subject to potentially harmful chemical and physical agents.
- Reactive chemicals, radioactive emissions, X-rays, and ultraviolet light can change nucleotides in ways that can affect encoded genetic information.
- DNA bases may undergo spontaneous chemical changes under normal cellular conditions.
- Each cell continually monitors and repairs its genetic material, with 100 repair enzymes known in E. coli and more than 130 repair enzymes identified in humans.
- A hereditary defect in one of these enzymes is associated with a form of colon cancer.
- DNA polymerase and ligase fill in the gap.
- These individuals are hypersensitive to sunlight.
- Ultraviolet light can produce thymine dimers between adjacent thymine nucleotides.
- This buckles the DNA double helix and interferes with DNA replication.
- In individuals with this disorder, mutations in their skin cells are left uncorrected and cause skin cancer.
The ends of DNA molecules are replicated by a special mechanism.
- Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes.
- The usual replication machinery provides no way to complete the 5’ ends of daughter DNA strands.
- Repeated rounds of replication produce shorter and shorter DNA molecules.
- In human telomeres, this sequence is typically TTAGGG, repeated between 100 and 1,000 times.
- Telomeric DNA tends to be shorter in dividing somatic cells of older individuals and in cultured cells that have divided many times.
- If the chromosomes of germ cells became shorter with every cell cycle, essential genes would eventually be lost.
- There is now room for primase and DNA polymerase to extend the 5’ end.
- It does not repair the 3’-end “overhang,” but it does lengthen the telomere.
- Telomere length may be a limiting factor in the life span of certain tissues and of the organism.
- Cells from large tumors often have unusually short telomeres, because they have gone through many cell divisions.
- This overcomes the progressive shortening that would eventually lead to self-destruction of the cancer.
- Immortal strains of cultured cells are capable of unlimited cell division.
Lecture Outline for Campbell/Reece Biology, 7th Edition, © Pearson Education, Inc. 16-1
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