BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 29. EUKARYOTIC CHROMOSOMES AND GENE EXPRESSION IN EUKARYOTES

Eukaryotic Cells contain vastly more Genetic information than prokaryotic ones. For instance, a human Cell harbors about 1,000 times more DNA than an E. coli cell and roughly 100,000 times more than a single phage λ virion. This Abundance of DNA endows eukaryotes with immense potential capabilities absent in prokaryotes. Another key distinction is that the DNA of higher organisms is complexed with basic Proteins called Histones, whereas the Chromosomes of lower organisms lack such proteins. The primary function of these basic proteins is to package DNA so that a contour length of many centimeters can fit into a volume just a few micrometers in diameter. The characteristic Morphology of eukaryotic chromosomes, readily observable under a Light Microscope, indicates that they are organized with significantly greater complexity than Prokaryotic Genomes. Furthermore, the shape of eukaryotic chromosomes changes dramatically during the Cell Cycle. Yet another crucial feature distinguishing eukaryotes from prokaryotes is that eukaryotic chromosomes are enclosed within a nuclear membrane. Prokaryotes lack this membrane, as well as any other internal membranes. Consequently, METABOLISM/31.html">Transcription and Translation are separated in both time and space in eukaryotes, whereas they are tightly coupled in prokaryotes. In the nuclei of higher organisms, primary transcripts undergo extensive modification, splicing, and joining of fragments. Only a small fraction of the RNA synthesized in The Nucleus reaches the Cytosol as mRNA.

Clearly, Gene Expression IN eukaryotes is regulated through a much more complex pathway than in prokaryotes. Research in this field is advancing rapidly, driven by our ability to isolate and clone eukaryotic genes, determine their nucleotide sequences, and express them in well-characterized systems. All indications suggest that we are on the threshold of unraveling one of biology's most fundamental problems: The Mechanism of cellular differentiation.

Class="center">Fig. 29.1. Phase-contrast micrograph of a lampbrush-type chromosome from an oocyte

29.1. A Eukaryotic Chromosome Contains a Single Double-Stranded DNA Molecule

Is it valid to assert that a chromosome consists of a single long DNA molecule? For many years, this question was difficult to answer because extremely long DNA molecules are exceptionally fragile and prone to shearing by hydrodynamic forces. Bruno Zimm resolved this problem using viscoelastic elasmometry, a technique that permits the measurement of the length of very large DNA molecules in solution. DNA molecules are stretched in a liquid flow and then allowed to relax into their random-coil conformation. The time required for half of the molecules to recoil depends on their molecular weight. Fruit fly cells were lysed directly within the measurement chamber to prevent DNA breakage during sample transfer. Nucleases were inactivated by incubating the samples in the presence of detergent at 65°C. In addition, pronase was added to hydrolyze DNA-associated proteins.

The Molecular Weight of the largest DNA molecules in the resulting mixture was determined to be 41 × 10⁶ kDa. This value aligns remarkably well with the established DNA content of the largest Drosophila melanogaster chromosome, which is 43 × 10⁶ kDa. An equally striking agreement was observed in a translocation mutant involving the largest chromosome, which carries an additional segment of DNA, raising its expected DNA content to 59 × 10⁶ kDa. The measured amount of DNA in this molecule was 58 × 10⁶ kDa. Autoradiographs of D. melanogaster DNA (Fig. 29.2) confirm the existence of these exceptionally long DNA molecules. These studies demonstrate that the Drosophila chromosome contains a single continuous DNA molecule. Moreover, this DNA molecule is linear and unbranched.

Fig. 29.2. Autoradiograph of a Drosophila melanogaster DNA molecule. The contour length of this DNA is 1.2 cm

29.2. Eukaryotic DNA Is Tightly Complexed with Basic Proteins Called Histones

DNA in eukaryotic chromosomes does not exist in a naked state; rather, it is tightly bound to a group of small, basic proteins known as histones. Histones account for roughly half of the mass of eukaryotic chromosomes, with DNA comprising the other half. This nucleoprotein material of the chromosome is termed Chromatin. Treating chromatin with salt or dilute acid allows the histones and DNA to dissociate. The resulting mixture can then be resolved using Ion-exchange Chromatography. Histones are classified into five major types, designated H1, H2A, H2B, H3, and H4. They range in molecular mass from 11 to 21 kDa (Table 29.1). A remarkable feature of histones is their high proportion of positively charged side chains: approximately every fourth residue is either Lysine or Arginine.

Through post-translational modifications of specific side chains, each histone exists in multiple molecular variants. For instance, the lysine-16 residue of histone H4 can be acetylated. Furthermore, histones can undergo methylation, ADP-ribosylation, and phosphorylation. The resulting alterations in charge, hydrogen-bonding capacity, and molecular conformation driven by these covalent modifications likely play a vital role in regulating DNA accessibility for Replication and transcription.

Table 29.1. Histones