Prokaryotes do not have a nucleus. However, they still must fit DNA that is 1000 times the length of the cell within the cell membrane (Fig. 3.6). The genome of Escherichia coli, a bacterium widely used in molecular biology research, is 4700 kb in size and exists as one double-stranded circular DNA molecule, with no free 5′ or 3′ ends. The chromosomal DNA is organized into a condensed ovoid structure called a nucleoid. A considerable number of nonessential proteins, called histone-like proteins or nucleoid-associated proteins, are
Figure 3.6 The bacterial genome. False-color transmission electron micrograph (TEM) of a lysed bacterial cell (E. coli). The DNA is visible as the gold colored fibrous mass lying around the bacterium. Magnification: ×15,700.
(Credit: G. Murti / Photo Researchers, Inc.)
thought to be involved in DNA compaction and genome organization. These include HU (heat-unstable protein), IHF (integration host factor), HNS (heat-stable nucleoid structuring), and SMC (structural maintenance of chromosomes). HU and HNS are particularly abundant. Further condensation packs the bacterial genome into supercoiled domains of 20–100 kb. Approximately 50% of DNA supercoiling is unrestrained. These domains are dynamic and unlikely to have sequence-specific domain boundaries.
Negative superhelicity is maintained by the action of topoisomerases, in particular by the ability of gyrase to remove the positive supercoils generated during replication and transcription (see Section 2.8).
3.4 Plasmids
Plasmids are small, double-stranded circular or linear DNA molecules carried by bacteria, some fungi, and some higher plants. They are extrachromosomal (meaning separate from the host cell chromosome), independent, and self-replicating. At least one copy of a plasmid is passed on to each daughter cell during cell division. Their relationship with their host cell could be considered as either parasitic or symbiotic.
They range in size from 2 to 100 kb (Fig. 3.7). The majority of plasmids are circular; however, a variety of linear plasmids have been isolated. A notable example is the linear plasmid pC1K1 carried by Claviceps purpurea, a fungus found on rye. The fungus contains poisonous alkaloids that cause ergotism – hallucinations and sometimes death – in humans who eat the infected grain and was a likely contributor to the Salem Witch Trials.
The focus in this textbook will be on the circular plasmids from bacteria. Plasmids are important for our study for two main reasons: they are carriers of resistance to antibiotics, and they provide convenient vehicles for recombinant DNA technology (see Section 8.4).
3.5 Bacteriophages and mammalian DNA viruses
The motivation for most of the early studies on viruses centered on their pathogenicity, but they have also proved extremely useful systems for analysis of fundamental principles of molecular biology. For example, DNA viruses provide a cloned set of genes, organized in a physiologically meaningful array on a single DNA molecule. Before the advent of gene cloning technologies, viruses provided a readily available source of pure
Bacterial chromosome
Plasmid DNA
Figure 3.7 Schematic representation of a bacterium containing plasmid DNA. Plasmids are small, circular molecules of DNA that are extrachromosomal and self-replicating within the host bacterium.
DNA for studies of genomic expression, function, and replication. Bacteriophages and mammalian DNA viruses have DNA genomes that occur in a myriad of forms, ranging from double-stranded to single-stranded DNA and linear to circular forms (Table 3.2).
Bacteriophages
The chromosome of bacterial viruses usually consists of a single DNA molecule, largely devoid of associated proteins. For example, bacteriophage lambda (λ) has a double-stranded linear genome. Upon infection of a host bacterium, the DNA closes to form a circle. This phage is used widely as a tool for molecular biology research (see Chapter 8). In contrast, another phage commonly used by molecular biologists, M13, has a single-stranded circular genome. All bacteriophages have the ability to package an exceedingly long DNA molecule into a relatively small volume. A case in point is bacteriophage λ that uses the enzyme terminase to package 17µm of DNA into a preformed protein “head” (capsid) which is less that 0.1 µm on any side.
After packaging of the genome, a preformed tail is attached to the viral head.
Mammalian DNA viruses
Mammalian DNA viruses infect mammalian cells and make use of the host cell machinery for their replication. For this reason, the papovaviruses (for papilloma, polyoma, and vacuolating), in particular, have been one of the most important model systems for understanding molecular and genetic characteristics of eukaryotes. Their genomes come in a diversity of forms. For example, human papilloma virus (HPV), a causative agent of cervical and other cancers (see Chapter 16), has a double-stranded circular genome.
Likewise, simian virus 40 (SV40) from rhesus monkey, also has a double-stranded circular genome. In contrast, adenovirus, a vector used for human gene therapy (see Section 17.3), has a double-stranded linear genome.
Little is known about how mammalian DNA viruses package their genome into the viral capsid (the protein shell encoded by viral genes). Some viruses encode their own basic proteins, while others usurp the host cell machinery. For example, papovavirus uses the host cell histones, H2A, H2B, H3, and H4 to package its genome. Histone H1 is absent from the nucleosome-like particles. Electron micrographs of SV40 show that the covalently closed, circular, double-stranded DNA is organized in a chromatin-like structure called a minichromosome (Fig. 3.8).
(A) (B) (C) (D)
Figure 3.8 Chromatin formation in simian virus 40 (SV40). (A) Electron micrograph of SV40 viral particles.
(Photograph courtesy of Norm Olson and Timothy Baker, University of California, San Diego.) (B) SV40 DNA. (C) SV40 condensed minichromosome. (D) SV40 extended minichromosome associated with host cell histones. (Parts B-D reproduced with permission from Singer, M. and Berg, P. 1997. Exploring Genetic Mechanisms, University Science Books, Sausalito, CA. Copyright © 1997 by University Science Books.)
3.6 Organelle genomes: chloroplasts and mitochondria
Both mitochondria and chloroplasts contain their own genetic information (Fig. 3.9). The genomes are usually, but not always, circular. In circular form, the mitochondrial and chloroplast genomes look remarkably similar to bacterial genomes. This similarity, along with other observations, led to the
“endosymbiont hypothesis” – the idea that both mitochondria and chloroplasts are derived from primitive organisms that were free-living and much like bacterial organisms. Organelle genomes are inherited independently of the nuclear genome and they exhibit a uniparental mode of inheritance, with traits being passed to offspring only from their mother. The organelles are only contributed from the maternal gamete (e.g. egg cell), and not from the paternal gamete (e.g. sperm cell or pollen grain).
Chloroplast DNA (cpDNA)
Chloroplasts are found in higher plants, some protozoans, and algae. The cpDNA encodes enzymes involved in photosynthesis. The most standard depiction of cpDNA is as a circular, double-stranded DNA molecule, ranging in size from 120 to 160 kb, with 20– 40 copies per organelle. However, this is a subject of debate.
Recent studies suggest that, in fact, most cpDNA is linear and only a minor amount is in a circular form.
(A)
(B)
matrix cpDNA
stroma thylakoids
cristae
mtDNA
Figure 3.9 Organelle DNA.
(A) Chloroplast in the freshwater red alga Compsopogon. Red algal chloroplasts are similar to green algae and land plants in that they are bounded by a typical double membrane envelope. The small, somewhat electron translucent region in the middle of the chloroplast stroma is one of many chloroplast DNA (cpDNA) sites. Thylakoids are typically unstacked and often reveal small attached granules knowns as phycobilisomes, the site of the red and/or blue accessory pigments. (B) Two mitochondria of mouse (Peromyscus) heart tissue. Two possible mitochondrial DNA (mtDNA) regions are evident in the matrix, as well as the double membrane envelope and shelf-like cristae. (Photographs courtesy of Joe Scott, College of William and Mary.)
In 1988, 25 years after the discovery that mitochondria have their own genes, researchers made a link between certain human diseases and mtDNA mutations. Most mtDNA defects lead to degenerative disorders, especially of the brain and muscles, but because of the essential function of mitochondria in cellular ATP production, the effects can be widespread.
One of the first diseases to be linked to a small inherited mutation in a mitochondrial gene was a form of young adult blindness (Leber’s hereditary optic neuropathy, LHON). The most common defects associated with LHON occur in genes coding for protein components of complex I of the electron transport chain. mtDNA mutations such as deletions or duplications that affect many genes at once have also been
identified. One example is Kearns–Sayre syndrome, which involves paralysis of eye muscles, progressive muscle degeneration, heart disease, hearing loss, diabetes, and kidney failure.
Normally, all of the mtDNA within the cells of an individual are identical – a condition called homoplasmy.
However, a mutation occurring in one copy of mtDNA can eventually result in both mutant and normal mtDNA coexisting within the same cell – a condition called heteroplasmy. Consequently, an individual may have some tissues enriched for normal mtDNA and others enriched for mutant mtDNA. This leads to differences in the severity and the kind of symptoms that may be displayed for a particular disease.
D I S E A S E B O X 3 . 1
Mitochondrial DNA and disease
Whatever the form, cpDNA is free of the associated proteins characteristic of eukaryotic DNA. Compared with nuclear DNA of the same organism, it has a different buoyant density and base composition.
Mitochondrial DNA (mtDNA)
Mitochondria are found in plants, animals, fungi, and aerobic protists. The mtDNA encodes essential enzymes involved in ATP production (Disease box 3.1). mtDNA is usually a circular, double-stranded DNA molecule that is not packaged with histones. There are a few exceptions where mtDNA is linear, generally in lower eukaryotes such as yeast and some other fungi. mtDNA differs greatly in size among organisms. In animals, it is typically 16–18 kb, while in plants it ranges in size from 100 kb to 2.5 Mb. There are multiple copies of mtDNA per organelle, with anywhere from several to as many as 30 copies in Euglena protozoans.