14: DNA Structure and Function Flashcards
Historical Basis of Modern Understanding, DNA Structure and Sequencing, Basics of DNA Replication, DNA Replication in Prokaryotes, DNA Replication in Eukaryotes, DNA Repair
How large is the human genome?
Each human cell has 23 pairs of chromosomes, one from each parent, and a mitochondrial genome, inherited exclusively from the mother. The human haploid genome contains 3 billion base pairs and has between 20,000 and 25,000 functional genes.
What is transformation?
The process in which external DNA is taken up by a cell.
Who discovered DNA?
In the 1860s, Friedrich Miescher, a physician by profession, was the first person to isolate phosphate-rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.
When was bacterial transformation discovered?
The first experiment, reported in 1928 by Frederick Griffith, a British bacteriologist, suggesting that bacteria are capable of transferring genetic information through a process known as transformation. He used Streptococcus pneumoniae, injecting mice with combinations of the R (non-pathogenic) and S (pathogenic) strains. The strains are so named depending on whether they have a Rough or Smooth appearance, which is caused be the absence or presence of a capsule. They survived with live R strains and heat-killed S strains, but died with a mixture of live R strain and heat-killed S strain. Upon isolating the live bacteria from the dead mouse, only the S strain was recovered. Injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain, and he called this the transforming principle. These experiments are now famously known as Griffith’s transformation experiments.
When was DNA identified as the transforming principle?
An experimental demonstration, reported in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, that DNA is the substance that causes bacterial transformation, in an era when it had been widely believed that it was proteins that served the function of carrying genetic information (with the very word “protein” itself coined to indicate a belief that its function was primary). They isolated the S strain from the dead mice in Griffith’s experiment and isolated the proteins and nucleic acids (namely RNA and DNA), and conducted a systematic elimination study. They used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain. They found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria. This led them to conclude that DNA was the transforming principle.
When was the first application of DNA analysis in forensics?
DNA evidence was used for the first time to solve an immigration case. The story started with a teenage boy returning to London from Ghana to be with his mother. Immigration authorities at the airport were suspicious of him, thinking that he was traveling on a forged passport. After much persuasion, he was allowed to go live with his mother, but the immigration authorities did not drop the case against him. All types of evidence, including photographs, were provided to the authorities, but deportation proceedings were started nevertheless. Around the same time, Dr. Alec Jeffreys of Leicester University in the United Kingdom had invented a technique known as DNA fingerprinting. The immigration authorities approached Dr. Jeffreys for help. He took DNA samples from the mother and three of her children, plus an unrelated mother, and compared the samples with the boy’s DNA. Because the biological father was not in the picture, DNA from the three children was compared with the boy’s DNA. He found a match in the boy’s DNA for both the mother and his three siblings. He concluded that the boy was indeed the mother’s son.
What do forensic scientists analyze?
Forensic scientists analyze many items, including documents, handwriting, firearms, and biological samples. They analyze the DNA content of hair, semen, saliva, and blood, and compare it with a database of DNA profiles of known criminals.
What does forensic DNA analysis include?
Analysis includes DNA isolation, sequencing, and sequence analysis; most forensic DNA analysis involves polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci and electrophoresis to determine the length of the PCR-amplified fragment. Only mitochondrial DNA is sequenced for forensics.
Where do forensic scientists work?
Forensic scientists are expected to appear at court hearings to present their findings. They are usually employed in crime labs of city and state government agencies. Geneticists experimenting with DNA techniques also work for scientific and research organizations, pharmaceutical industries, and college and university labs.
What education is required to be a forensic scientist?
Students wishing to pursue a career as a forensic scientist should have at least a bachelor’s degree in chemistry, biology, or physics, and preferably some experience working in a laboratory.
When was DNA identified as genetic material?
A series of experiments conducted in 1952 by Alfred Hershey and Martha Chase that helped to confirm that DNA is genetic material. They worked with bacteriophages, labeling the protein coat in one batch of phage with radioactive sulfur, 35S, and labeling their DNA in another batch with radioactive phosphorus, 32P. After infection, the phage-bacterial suspensions were put in blenders, causing the phage coat to be detached from the host cell. The suspensions were then spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant. In the 35S-labeled tube, the supernatant contained the radioactivity. In the 32P-labeled tube, the pellet contained the radioactivity. This suggested that it was DNA and not protein that was injected into the bacterial cells, providing evidence that DNA was the genetic material.
What is a bacteriophage?
A virus that infects and replicates within bacteria and archaea. It infects the host cell by attaching to its surface, and injecting its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages.
How are viruses structured?
Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA.
What are Chargaff’s rules?
Austrian biochemist Erwin Chargaff examined the content of DNA in different species and found that the amounts of 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 equals the amount of thymine, and the amount of cytosine equals the amount of guanine, or A = T and G = C. This is also known as Chargaff’s rules.
What is electrophoresis?
A technique used to separate DNA fragments according to size.
What are nucleotides?
The building blocks of DNA are nucleotides. The important components of the nucleotide are a nitrogenous base, deoxyribose (5-carbon sugar), and a phosphate group. The nucleotide is named depending on the nitrogenous base. The nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T).
What is a phosphodiester bond?
The nucleotides combine with each other by covalent bonds known as phosphodiester bonds or linkages. The carbon atoms of the five-carbon sugar are numbered 1’, 2’, 3’, 4’, and 5’ (1’ is read as “one prime”). The phosphate residue is attached to the hydroxyl group of the 5’ carbon of one sugar of one nucleotide and the hydroxyl group of the 3’ carbon of the sugar of the next nucleotide, thereby forming a 5’-3’ phosphodiester bond.
What are purines?
The purines have a double ring structure with a six-membered ring fused to a five-membered ring.
What are pyrimidines?
Pyrimidines are smaller in size than purines; they have a single six-membered ring structure.
Who was involved in the discovery of the structure of DNA?
In the 1950s, Francis Crick and James Watson worked together to determine the structure of DNA at the University of Cambridge, England. Other scientists like Linus Pauling and Maurice Wilkins were also actively exploring this field. Pauling had discovered the secondary structure of proteins using X-ray crystallography. In Wilkins’ lab, researcher Rosalind Franklin was using X-ray diffraction methods to understand the structure of DNA. Watson and Crick were able to piece together the puzzle of the DNA molecule on the basis of Franklin’s data because Crick had also studied X-ray diffraction. In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Medicine. Unfortunately, by then Franklin had died, and Nobel prizes are not awarded posthumously.
What is the DNA double helix?
In this model, base-pairing occurs between a purine and pyrimidine (A-T and G-C), which are called complementary base pairs. The base pairs are stabilized by hydrogen bonds; adenine and thymine form two hydrogen bonds, and cytosine and guanine form three. The two strands are anti-parallel, where the 3’ end of one strand faces the 5’ end of the other. The sugar and phosphate of the nucleotides form the backbone of the structure, and the nitrogenous bases are inside. Each base pair is separated from the other by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm. Ten base pairs are present per turn of the helix. The diameter of the DNA double helix is 2 nm, and is uniform throughout. The twisting of the two strands around each other results in the formation of uniformly spaced major and minor grooves.
How have DNA sequencing techniques improved over time?
Until the 1990s, the sequencing of DNA (reading the sequence of DNA) was a relatively expensive and long process. Using radiolabeled nucleotides also compounded the problem through safety concerns. With currently available technology and automated machines, the process is cheap, safer, and can be completed in a matter of hours. Fred Sanger developed the sequencing method used for the human genome sequencing project, which is widely used today.
What is a dideoxynucleotide?
A chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing. They are known as 2’,3’ because both the 2’ and 3’ positions on the ribose lack hydroxyl groups (hence they differ from deoxyribonucleotides by the lack of a 3’ hydroxyl group), and are abbreviated as ddNTPs. If a ddNTP is added to a growing DNA strand, the chain is not extended any further because the free 3’ OH group needed to add another nucleotide is not available.
How does Sanger sequencing work?
A method of DNA sequencing developed by Fred Sanger based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication. By using a predetermined ratio of deoxyribonucleotides to dideoxynucleotides, it is possible to generate DNA fragments of different sizes. It uses dye-labeled ddNTPs to generate DNA fragments that terminate at different points. The DNA is separated by capillary electrophoresis on the basis of size, and from the order of fragments formed, the DNA sequence can be read. The DNA sequence readout is shown on an electropherogram that is generated by a laser scanner. The DNA sample to be sequenced is denatured (separated into two strands) by heating it to high temperatures. The DNA is divided into four tubes in which a primer, DNA polymerase, and all four nucleotides (A, T, G, and C) are added. In addition to each of the four tubes, limited quantities of one of the four ddNTPs are added to each tube respectively. The tubes are labeled as A, T, G, and C according to the ddNTP added. For detection purposes, each of the four ddNTPs carries a different fluorescent label. Chain elongation continues until a fluorescent dideoxy nucleotide is incorporated, after which no further elongation takes place. After the reaction is over, electrophoresis is performed. Even a difference in length of a single base can be detected. The sequence is read from a laser scanner. For his work on DNA sequencing, Sanger received a Nobel Prize in chemistry in 1980.
How does gel electrophoresis work?
Gel electrophoresis is a technique used to separate DNA fragments of different sizes. Usually the gel is made of a chemical called agarose. Agarose powder is added to a buffer and heated. After cooling, the gel solution is poured into a casting tray. Once the gel has solidified, the DNA is loaded on the gel and electric current is applied. The DNA has a net negative charge and moves from the negative electrode toward the positive electrode. The electric current is applied for sufficient time to let the DNA separate according to size; the smallest fragments will be farthest from the well (where the DNA was loaded), and the heavier molecular weight fragments will be closest to the well. Once the DNA is separated, the gel is stained with a DNA-specific dye for viewing it.
What are Neanderthals?
Neanderthals are the closest ancestors of present-day humans. They were known to have lived in Europe and Western Asia before they disappeared from fossil records approximately 30,000 years ago.
How was the Neanderthal genome sequenced?
The first draft sequence of the Neanderthal genome was published by Richard E. Green et al. in 2010. Green’s team studied almost 40,000-year-old fossil remains that were selected from across the world. Extremely sophisticated means of sample preparation and DNA sequencing were employed because of the fragile nature of the bones and heavy microbial contamination. In their study, the scientists were able to sequence some four billion base pairs.
How closely related are Neanderthal and human genomes?
The Neanderthal genome has 2 to 3 percent greater similarity to present-day humans living outside Africa than to people in Africa. The data from the Neanderthal genome thus contradicts current theories that suggest that all present-day humans can be traced to a small ancestral population in Africa. Green and his colleagues also discovered DNA segments among people in Europe and Asia that are more similar to Neanderthal sequences than to other contemporary human sequences. Another interesting observation was that Neanderthals are as closely related to people from Papua New Guinea as to those from China or France. This is surprising because Neanderthal fossil remains have been located only in Europe and West Asia. Most likely, genetic exchange took place between Neanderthals and modern humans as modern humans emerged out of Africa, before the divergence of Europeans, East Asians, and Papua New Guineans.
Which genes are different between Neanderthals and modern humans?
Several genes seem to have undergone changes from Neanderthals during the evolution of present-day humans. These genes are involved in cranial structure, metabolism, skin morphology, and cognitive development. One of the genes that is of particular interest is RUNX2, which is different in modern day humans and Neanderthals. This gene is responsible for the prominent frontal bone, bell-shaped rib cage, and dental differences seen in Neanderthals. It is speculated that an evolutionary change in RUNX2 was important in the origin of modern-day humans, and this affected the cranium and the upper body.
How large is the E. coli genome?
The genome of E. coli, which is comprised of 4.6 million base pairs (approximately 1.1 mm, if cut and stretched out).
How is DNA packed in prokaryotic cells?
The DNA is twisted by what is known as supercoiling. Supercoiling means that DNA is either under-wound (less than one turn of the helix per 10 base pairs) or over-wound (more than 1 turn per 10 base pairs) from its normal relaxed state. Some proteins are known to be involved in the supercoiling; other proteins and enzymes such as DNA gyrase help in maintaining the supercoiled structure.
How is DNA packed in eukaryotic cells?
At the most basic level, DNA is wrapped around proteins known as histones to form structures called nucleosomes. The histones are evolutionarily conserved proteins that are rich in basic amino acids and form an octamer. The DNA (which is negatively charged because of the phosphate groups) is wrapped tightly around the histone core. This nucleosome is linked to the next one with the help of a linker DNA. This is also known as the “beads on a string” structure. This is further compacted into a 30 nm fiber, which is the diameter of the structure. At the metaphase stage, the chromosomes are at their most compact, are approximately 700 nm in width, and are found in association with scaffold proteins.
What are heterochromatin and euchromatin?
In interphase, eukaryotic chromosomes have two distinct regions that can be distinguished by staining. The tightly packaged region is known as heterochromatin, and the less dense region is known as euchromatin. Heterochromatin usually contains genes that are not expressed, and is found in the regions of the centromere and telomeres. The euchromatin usually contains genes that are transcribed, with DNA packaged around nucleosomes but not further compacted.
What were the three original models for DNA replication?
The double helix model of DNA suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested: conservative, semi-conservative, and dispersive.
What was the conservative model of DNA replication?
A model that suggests that parental DNA remains together and newly-formed daughter strands are also together.
What was the semi-conservative model of DNA replication?
A model that suggests that two parental DNA strands serve as a template for new DNA and after replication, each double-stranded DNA contains one strand from the parental DNA and one new (daughter) strand.
What was the dispersive model of DNA replication?
A model that suggests that, after replication, the two daughter DNAs have alternating segments of both parental and newly-synthesized DNA interspersed on both strands.
How was the mechanism of DNA replication discovered?
Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N) that gets incorporated into nitrogenous bases, and eventually into the DNA.
The E. coli culture was then shifted into medium containing 14N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in 14N and spun again. During the density gradient centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in 15N will band at a higher density position than that grown in 14N. Meselson and Stahl noted that after one generation of growth in 14N after they had been shifted from 15N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15N and 14N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N, and the other corresponded to the band of 14N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.
What is helicase?
During replication, this enzyme helps to open up the DNA helix by breaking the hydrogen bonds.
