BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 29. EUKARYOTIC CHROMOSOMES AND GENE EXPRESSION IN EUKARYOTES

29.9. Mitochondria and Chloroplasts Contain Their Own DNA

Not all the Genetic information of Eukaryotic Cells is contained in nuclear chromosomal DNA. Genetic studies of Yeast led to the discovery of a Mitochondrial Genome distinct from the nuclear genome. In 1949, Boris Ephrussi discovered that certain mutants of baker's yeast are incapable of Oxidative Phosphorylation. These Respiration-deficient mutants grow slowly via Fermentation. They are called petites (French for "small") because they form very small colonies. Genetic analysis led to the surprising discovery that petite Mutations segregate independently of The Nucleus, suggesting that Cell/35.html">Mitochondria possess their own genome. Indeed, DNA was discovered in mitochondria a few years later. Moreover, the Mitochondrial DNA from a petite strain differed in buoyant density from wild-type yeast mitochondrial DNA, implying that a significant portion of the mitochondrial genome is altered in the mutant. Subsequently, it was shown that the METABOLISM/14.html">Chloroplasts of photosynthetic eukaryotes also contain DNA and that it is replicated, transcribed, and translated.

Animal cell mitochondrial DNA is a circular double-stranded molecule with a contour length of about 5 µm, corresponding to 15 kb. Yeast mitochondrial DNA is typically about 5 times longer, and chloroplast DNA about 10 times longer. The DNA molecules in mitochondria and chloroplasts are not associated with Histones. They are relatively small, comparable in size to viral genomes. The yeast mitochondrial genome is the best studied; it encodes approximately ten Proteins, two ribosomal RNA molecules, and about 26 species of Transfer RNA. The molecules encoded by mitochondrial DNA and synthesized within the organelle account for only about 5% of the mitochondrial protein. Thus, the majority of mitochondrial proteins are encoded by the nuclear genome. However,

the genetic contribution of mitochondrial DNA is essential. For example, three of the seven subunits of cytochrome oxidase and three of the ten subunits of inner mitochondrial membrane ATP ase are encoded by the mitochondrial genome. The existence of separate genomes raises A number of questions. How is the Replication of mitochondrial DNA coordinated with chromosome duplication and Cell Division? How do proteins synthesized in the Cytosol enter mitochondria and interact with mitochondrial Gene products? But the most puzzling question is: why do mitochondria need their own genomes if 95% of their proteins are encoded by the nuclear genome? Answers to these intriguing questions are not yet available.

Class="center">Fig. 29.16. Electron micrograph of a mitochondrial DNA molecule containing two genomes joined HEAD-to-tail to form a ring. Replication of this DNA molecule has just begun. The arrows point to two loops located on opposite sides of the ring. These displacement loops (D-loops, from English displacement) contain newly synthesized DNA. The thinner line in each loop is the displaced single-stranded region of parental DNA

29.10. Eukaryotic DNA Contains Many Repeated Base Sequences

Roy Britten and his coworkers investigated the reassociation kinetics of heat-denatured DNA and found that eukaryotic DNA, unlike prokaryotic DNA, contains many repeated base sequences. In these experiments, DNA was sheared into short fragments and then denatured by heating the solution above the melting Temperature of the DNA (T_m). The resulting single-stranded DNA solution was then cooled to a temperature approximately 25°C below T_m, which is optimal for the reassociation of complementary strands to form double-helical DNA. The reassociation kinetics can be monitored in various ways. One method involves measuring the absorbance of the solution at 260 nm (sec. 24.9). At this wavelength, the absorbance coefficient of double-stranded DNA is about 40% lower than the corresponding value for single-stranded DNA; this phenomenon is called hypochromism. Another experimental approach relies on the fact that double-stranded DNA binds to columns of hydroxyapatite (calcium phosphate), whereas single-stranded DNA passes right through. The appeal of this method is that it allows the fractionation of large amounts of DNA based on its reassociation rate following thermal Denaturation.

The observed reassociation kinetics of E. coli or phage T4 DNA conform to the expected bimolecular reaction kinetics

where S and S' are complementary single-stranded molecules, DS is the reassociated double helix, and k is the association rate constant. In such a reaction, the fraction of single-stranded molecules f decreases with time According to the equation

where C₀ is the initial DNA concentration (expressed in moles of NUCLEOTIDES per liter) and t is time in seconds. For a given DNA and specified experimental conditions (i.e., Ionic strength, temperature, DNA fragment size), f depends solely on C_0t, the product of DNA concentration and time. It is convenient to plot reassociation kinetics graphically by showing f as a function of the common logarithm of C_0t. Such a C_0t curve has a sigmoidal shape (Fig. 29.17). A characteristic feature of any given DNA preparation is the C_0t0.5 value, which is readily determined from this curve. C₀t_0.5 is the C₀t value at which half of the DNA has reassociated (f = 0.5). For E. coli DNA, the C₀t_0.5 value is approximately 9 M • s; for phage T4, C₀t_0.5 = 0.3 M • s. These figures show that E. coli DNA reassociates about 30 times more slowly than phage T4 DNA. This is because E. coli DNA is longer, and the number of Different types of fragments contained in the sheared DNA preparation is greater than in the T4 DNA preparation. Thus, the concentration of complementary fragments in a solution of fragmented E. coli DNA is lower than in a phage T4 DNA solution (containing the same amount of nucleotides), and consequently, the reassociation rate is lower. Studies of a number of prokaryotic DNAs have shown that the C₀t_0.5 value is directly proportional to Genome Size.

Fig. 29.17. Plots of f versus C₀t ("C₀t curves") illustrating the reassociation kinetics of several heat-denatured DNAs. The ordinate represents the fraction of single-stranded molecules, and the abscissa represents C₀t. The rapid reassociation of mouse satellite DNA indicates that it contains a vast number of repeated sequences

When researchers began exploring mouse DNA using this method, they obtained an unexpected result. Mammalian genomes are approximately three orders of magnitude larger than the E. coli genome, and it was expected that a C₀t_0.5 value on the order of 10⁴ M • s would be obtained. A DNA solution with a concentration of 10^-4 M and such a C₀t_0.5 value should be half-reassociated in 10⁸ seconds (about 3 years). To the researchers' surprise, they found that 10% of the mouse DNA is half-reassociated in a few seconds. This fraction of mouse DNA reassociates faster than even the smallest viral DNAs and, therefore, contains many repeated sequences. Analysis of the f versus C₀t curve revealed that this fraction of mouse DNA contains on the order of a million copies of a repeated sequence about 300 Base Pairs long. Approximately 20% of the mouse DNA renatures at an intermediate rate. According to the authors' interpretation, this fact indicated that this fraction contains 10³–10⁴ copies of certain sequences. The remaining 70% of the mouse DNA renatured very slowly. The C₀t_0.5 value for this fraction indicated that it consists of unique or nearly unique base sequences.

All Eukaryotic Genomes studied to date, except perhaps yeast, contain repeated DNA sequences, whereas prokaryotes do not. For example, human DNA consists of 30% sequences repeated at least 20 times. The relative Abundance of highly repetitive, moderately repetitive, and unique DNA varies among different species.

29.11. Highly Repetitive DNA (Satellite DNA) is Localized in Centromeres

Many highly repetitive DNAs can be isolated by density gradient centrifugation because their buoyant density differs from that of the main DNA. For instance, Drosophila virilis DNA yields a main peak and three satellite peaks of lower density (Fig. 29.18). These satellite peaks consist exclusively of repetitive DNA. Each of them represents a repeating heptanucleotide sequence:

Fig. 29.18. Three distinct peaks of satellite DNA (ρ = 1.692, 1.688, and 1.671) are visible on this sedimentation curve. The result of equilibrium centrifugation of D. virilis DNA in a neutral CsCl gradient is shown

The Role of highly repetitive DNA remains unknown. However, the chromosomal localization of this fraction was determined using in situ Hybridization, a method developed by Joseph Gall and Mary Lou Pardue. Cells were immobilized under a thin layer of Agar and treated with alkali to denature the DNA. This preparation was then incubated with tritium-labeled RNA, transcribed in vitro using purified satellite DNA AS A template. Hybrids formed between the radioactive RNA and chromosomal regions containing satellite DNA were detected by autoradiography (Fig. 29.19). A very clear result was obtained: mouse satellite DNA is found exclusively in the centromeric regions. The localization of these sequences and the apparent absence of complementary RNA in The Cell suggest that satellite sequences are involved in chromosome movements during Mitosis and Meiosis.

Fig. 29.19. Autoradiogram of mouse cells showing the localization of satellite DNA

Fig. 29.20. Micrograph of a nucleus isolated from a *Xenopus* oocyte. The nucleus is stained to reveal hundreds of nucleoli formed As a result of ribosomal RNA gene Amplification