Introduction and Goals
Deoxyribonucleic acid (DNA) is the genetic material in cells. In eukaryotic cells, the bulk of DNA resides in the nucleus. This tutorial describes the organization of nuclear DNA in a typical eukaryotic cell, how it is packaged into chromosomes, and the structure and features of eukaryotic chromosomes.
By the end of the tutorial you should know:
- The experimental evidence supporting that DNA is the genetic material
- The properties of DNA that allow denaturation and hybridization
- The features of typical chromosomes, including a centromere, telomeres and origins of replication
- The organization of the genome
- The packaging of DNA into chromatin and chromosomes
- The structure of a typical nucleus
DNA is the Genetic Material
The knowledge that DNA is the genetic material is so well established that it has become part of the lexicon of popular culture, as seen in the many police, courtroom and forensic television dramas in which DNA is used to solve a crime (in under an hour!). However, this common knowledge was not widely accepted by the scientific community until the early 1950s. It was known that chromosomes carried the genes, but the chemical nature of genes was disputed because chromosomes are composed of DNA and proteins. Several seminal experiments demonstrated that the heritable genetic material is DNA. In the 1920s, Fred Griffith established the principle of transformation - a permanent and heritable change (or transformation) in a cell or organism is caused by the acquisition of foreign DNA. Griffith injected mice with either a pathogenic form of the Streptococcus pneumoniae bacteria (S strain), which was lethal to the mice, or a nonpathogenic form of the bacteria (R strain), which was not lethal. Griffith found he could kill the S strain by heating it (heat-kill) and it would no longer be lethal to the mice; however, if he mixed heat-killed S strain bacteria with the live noninfectious R strain and injected this mixture into mice, they died. Furthermore, when live bacteria were isolated from the dead mice, they were infectious. Therefore, some component of the heat-killed S strain was capable of transforming the live R strain to an infectious and lethal strain of bacteria. In the 1940's Oswald Avery, Colin Macleod and Maclyn McCarty purified the transforming component of the S strain bacteria and demonstrated it was DNA. However, many in the scientific community were still not convinced. They countered that the DNA might have been contaminated with protein. Furthermore, it was hard to imagine that such a relatively "simple" molecule with only four repeating nucleotides was capable of encoding all of the genetic complexity because proteins are more varied and complex. In 1952, Alfred Hershey and Martha Chase performed the infamous "blender experiment" (Figure 1), which ended this controversy. They examined the genetic material of the T2 virus, which infects bacteria, replicates and causes the bacteria to produce many more copies of the virus, eventually killing the bacteria. They knew that the T2 virus was composed of DNA and proteins, so they designed an experiment to determine whether it was the DNA or the protein that was injected into the bacteria that directed the synthesis of new virus. Hershey and Chase grew viruses in the presence of radioactive sulfur (S35) to label the protein, and radioactive phosphate (P32) to label the DNA. The labeled viruses were mixed with bacteria. Then, the mixture was subjected to agitation in a blender, thereby shearing off any viral particles adhering to the outside of the bacteria. The mixture was subjected to centrifugation, then the radioactivity in the pellet (containing the bacteria) and in the supernatant was measured. Hershey and Chase found that the infected bacteria contained the P32-labeled DNA but not the S35-labeled protein, demonstrating conclusively that DNA was injected into the bacteria by the virus. Furthermore, they were able to detect P32-labeled virus particles. The conclusion was undeniable - DNA is the genetic material.
The Structure of DNA is Suited For Its Role as the Genetic Material
In 1953, James Watson and Francis Crick proposed a physical model for the structure of double-stranded DNA based on X-ray crystallography data. The model they proposed is a double-stranded, right-handed helix (illustrated in Figure 2). The key features of this model are that the two strands of DNA are wrapped around each other so that the sugar phosphate backbones are on the outside and the nitrogenous bases are pointing toward each other and hold the double helix together via hydrogen bonds. Furthermore, the pairing of the bases has a precise pattern; adenine base pairs with thymine (A-T), and guanine base pairs with cytosine (G-C). Review the tutorial titled Properties of Macromolecules II: Nucleic Acids, Polysaccharides and Lipids, which introduced this subject earlier in the course.
In what seems an incredible understatement today, the authors stated: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material" (Watson and Crick, 1953, Nature 171, pp 737-738). The complementary nature of DNA allows it to serve as a template during replication, to make two exact copies of the DNA when a cell divides so that each daughter cell receives the same genetic material. (DNA replication will be described in detail in a future tutorial entitled DNA Replication and Repair.)
Nucleic Acid Denaturation and Renaturation
The two strands of a DNA double helix are held together by hydrogen bonds. The denaturation of DNA occurs when these bonds are disrupted by the application of heat; the two strands become structurally separated. The melting temperature (Tm) of DNA is the temperature at which this occurs for half of the DNA molecules in a solution, and it is a function of the nucleotide number and composition. Remember that the A-T base pairs are held together by double hydrogen bonds, whereas the G-C base pairs are held together by triple hydrogen bonds. If the DNA is composed mostly of G-C base pairs, it will be more stable and have a higher Tm than DNA composed mostly of A-T base pairs. Furthermore, the Tm is higher for longer molecules of double-stranded DNA because there are more base pairs to disrupt. After denaturation and as the temperature is reduced, renaturation occurs; the two strands will come together again in a complementary fashion to reform the double helix. Denatured DNA (as well as RNA) from different sources can be mixed and they will anneal to each other in the regions that contain complementary sequences. This is the basis of an important application of DNA technology referred to as nucleic acid hybridization, which is used to detect specific DNA or RNA sequences based on their ability to anneal to a short, specific, single-stranded DNA fragment called a probe. Denatured DNA or single-stranded RNA can hybridize with a radioactive probe, allowing one to detect the presence of the nucleic acid that is complementary to the probe. Under the correct conditions of temperature and salinity, the probe can hybridize with a nucleic acid that is not completely complementary but that has some mismatched bases. This can be useful when using a probe from one species to detect related sequences from another species. The application of nucleic acid hybridization will be described in greater detail in the tutorial entitled Recombinant DNA Technology and Cloning.
Eukaryotic DNA is Distributed Among Chromosomes
The genome refers to the complete genetic information (the complete DNA content) of an organism. The nuclear genome is the DNA sequence of the DNA in the nucleus, which is where the bulk of the DNA is found in a eukaryotic cell. Genome sizes vary greatly, depending on the species. For instance, the size of the genome of the bacteria Haemophilus influenzaeis 1.8 million bp, whereas the size of the human nuclear genome is 3200 million bp. In general, the entire genome of a bacterium is found in one circular DNA molecule. In eukaryotes, the nuclear DNA is distributed across several chromosomes. Each chromosome is composed of a very long linear DNA molecule associated with proteins, which greatly compresses the DNA. The number of chromosomes varies from species to species (e.g. a fruitfly has four pairs of chromosomes, whereas a normal human has 23 pairs of chromosomes). The size of a chromosome can also vary between species and within a single species. (For example, a human chromosome can vary in size from 50 to 245 million bp!) Each chromosome carries a linear array of genes. A geneis defined as the segment of DNA that contains all the instructions necessary for making a particular protein. This includes the sequence that encodes the protein, as well as other sequences that regulate when the gene is turned "on" or "off." There are approximately 30,000 genes in the human genome.
Diploid organisms contain a pair of each chromosome, referred to as homologous chromosomes(one inherited from the father and one inherited from the mother), with the same organization of genes but not necessarily the identical DNA sequences. In humans, there are 22 pairs of homologous chromosomes (two copies each of chromosomes 1 - 22) and a pair of the sex chromosomes (XX in a normal female and XY in a normal male). A karyotype is a display of the full set of 46 human chromosomes (see Figure 3). The karyotype represents the mitotic chromosomes, which are the duplicated condensed chromosomes undergoing mitosis(nuclear division). All eukaryotic chromosomes have several structural features in common that are required for replication and stability: a centromere(a specific region of DNA, and its associated proteins, which allows the duplicated chromosomes to be separated during cell division); telomeres (the DNA sequences at the ends of the chromosomes); and several origins of replication (ORI; sequences scattered along the chromosomes, which initiate DNA replication). These are illustrated schematically in Figure 4.
Organization of the Genome
The majority of the human genome (greater than 50%) is composed of repetitive DNA sequences that do not encode for any genes and that have no known function. Interspersed among the repetitive DNA sequences are the genes (which are, on average, 27,000 bp long). Approximately, 10% of the genome is short tandem repeats consisting of short DNA sequences (less than 14 bp) repeated many times (100-100,000 times) in tandem for long stretches. For example, the sequence at the telomeres is TTAGG repeated 250-1500 times. Most of the repetitive DNA in the nuclear genome is composed of longer stretches of sequences (100s or 1000s bp), repeated fewer times in the genome, interspersed with other sequences and distributed throughout the genome. Some of the interspersed repeated sequences arose from mobile genetic elements that could copy themselves and insert the new copies at random into the genome. The function of the repetitive DNA is not well understood.
DNA is Packaged into Chromatin and Chromosomes
Nuclear DNA in chromosomes is highly compacted and associated with a variety of proteins. This association of DNA and protein is referred to as chromatin, and it is assembled in an ordered fashion. The first level of DNA packaging is the nucleosome, the DNA helix wrapped around a core particle of histone proteins (see Figure 5). The core particle is composed of a pair of each of the four core histones (H2A, H2B, H3 and H4). Precisely, 146 bp of DNA are tightly wrapped around the core histones, and the nucleosomes are separated by a region of DNA (termed the linker DNA) that is not associated with the core histones. Arrays of nucleosomes along the length of a DNA molecule appear as "beads on a string" (see Figure 6). This arrangement is further compressed into the so-called 30-nm chromatin fiberthrough the action of an additional histone (H1) that binds the linker DNA and pulls the nucleosomes closer together. The 30-nm fiber is further condensed into loops, and then, finally, into the dense, visible mitotic chromosome (Figure 6).
The association of DNA with the histones is not just structural. The DNA:histone interactions are regulated and can change to allow the DNA to become more or less accessible to the other proteins involved in DNA replication and transcription. The histones can be modified by the addition of a phosphate, an acetyl group or a methyl group, thus altering their interactions with the DNA and the accessibility of the DNA, as well as recruiting other proteins to the DNA. During the lifetime of a cell, the structure of chromatin is very dynamic and constantly changing in response to the needs of the cell.
The packaging of DNA in chromosomes between each round of mitosis (interphase chromosomes) is a looser configuration than the chromatin in mitotic chromosomes. However, not all the regions of an interphase chromosome are uniform. Some portions of the chromosome (heterochromatin) are in a much more compact configuration. Regions of the chromosome that contain heterochromatin generally do not contain many genes, and the genes in these regions are not highly expressed. These regions also include the centromere and the telomeres. The rest of the interphase chromosome (the euchromatin) is more extended.
Structure of a Typical Nucleus
The bulk of the DNA in a eukaryotic cell is found in interphase chromosomes in the nucleus (illustrated in Figure 7). The nucleus is surrounded by a double membrane termed the nuclear envelope, which is composed of an outer and inner membrane. Immediately below the inner membrane is a network of proteins termed the nuclear lamina. The outer membrane is part of the endomembrane system. The nuclear envelope is interrupted by nuclear pores, small channels created by the proteins in the pore complexthat allow for the regulated movement of material in and out of the nucleus. The chromatin is distributed throughout the interior of the nucleus such that each interphase chromosome has a distinct location. The nucleus also contains the nucleolus. This structure forms when the regions of chromatin encoding ribosomal genes cluster, and is the site of the synthesis of ribosomal RNA. In addition to the nucleus, eukaryotic DNA is found in mitochondria and chloroplasts. The genomes of these organelles are small circular DNA molecules, and they encode only a small number of genes. Interestingly, the inheritance of the mitochondrial genome is strictly maternal. (You only inherited your mother's mitochondria.)
Experimental data have demonstrated that nucleic acid in the form of DNA is the genetic material of cells. The structure of the DNA double helix and the complementary base pairing make double-stranded DNA uniquely suited as a template during replication. The two strands of double-stranded DNA can be denatured by heat and then renatured in a complementary fashion. This property of DNA is exploited in nucleic acid hybridization, where a short single-stranded probe can hybridize to the complementary sequence in denatured DNA or RNA. The melting temperature (Tm) of double-stranded DNA is a function of nucleotide length and composition. The DNA in the nucleus is found in the chromosomes, which are composed of linear DNA associated with proteins. Each chromosome has a centromere, two telomeres, and several origins of replication (ORI). Mitotic chromosomes are highly condensed and easiest to visualize in a karyotype. The genome of a typical eukaryote is composed of unique DNA sequences, interspersed repeated DNA, and short tandem repeats. The DNA in chromatin is highly packed and compressed. The first level of packaging is the nucleosome, DNA wrapped around the core histones. The nucleosomes (so-called "beads on a string") are further packaged into 30-nm chromatin fibers, looped domains of chromatin, and finally, the condensed mitotic chromosome. The interphase chromosomes are less condensed in the regions of the euchromatin, and more condensed in the regions of the heterochromatin. The interphase chromosomes reside in the nucleus, whose nuclear envelope has a double membrane. Transport of molecules, in and out of the nucleus, occurs through the nuclear pores.