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Almost all research in molecular biology until the 1960s had centered on bacteria or viruses. Now scientists turned their attention to multicellular organisms. These are all called eukaryotes because they package their DNA in a nucleus. Nuclei are examples of organelles, intracellular compartments for special functions. One of the first results of the new approaches was a reorganization of our classification of organisms. Whereas before two great kingdoms were recognized—bacteria and eukaryotes—there were now found to be three distinct groups, with two classes of bacteria.

Here we summarize the entire pathway of information transmission from gene to protein. The key discovery was messenger RNA, a transient RNA transcribed from one strand of duplex DNA, and then attached to ribosomes, where it could be translated into protein sequences. This represented a whole revision of earlier thought, which held that ribosomes contained the messages. The transfer of amino acids to the ribosome was found to be accomplished by transfer RNA molecules, each of which contained an anticodon complementary to the RNA codon for a specific amino acid. With these matters at least partly explained, attention turned to regulation of information transfer. The model system used in the pioneering studies by Jacob and Monod was the lac operon. This is a group of genes coding for proteins involved in lactose metabolism in bacteria. It was found that transcription of the genes coding for the enzymes needed was controlled by another protein, the repressor, which interacted with the DNA at the operator locus. This binding was in turn modulated by presence of lactose in the medium, which they termed an allosteric effect. This has proved a paradigm for much regulation of gene expression.

The cells of most eukaryotes are of a variety of types and thus must undergo selective differentiation during development from the fertilized egg. This process has long fascinated scientists and philosophers but was buried in superstition until the 19th century. Then, the growth of embryology as a science led to the recognition that different kinds of cells exhibit distinct levels of potency for further differentiation in form and function. The levels range from many somatic cell, incapable of further development, to the fertilized egg, which can yield all cell types of the organism. Intermediate are the embryonic stem cells, which can be induced to produce a variety of cell types.

Molecular biology and its attendant techniques have had a bewildering variety of applications in government, industry, and medicine. We mention only a few here. For example, the observation that human genomes carry different numbers of short variable repeats has led to a powerful tool for forensics. The ability to clone proteins in bacteria or yeast has allowed the production of hundreds of protein reagents essential for therapeutics or diagnosis that were difficult to obtain directly from their natural source. Gene therapy seems more and more to be practical and of immense potential. The production of genetically modified plant species, resistant to insects or to insecticides, has become an enormous industry. Whole animals can be cloned, and their genetics modified at will. The possibilities seem endless.

This chapter is all about the code. By 1955, it was clear that acode must exist, connecting the four-letter alphabet of nucleotides on nucleic acid sequence to the 20-letter amino-acid alphabet on proteins, but its nature was a matter of speculation. Then, in a matter of a few years, its structure was deduced. The code was comprised of three-letter (nucleotide residue), nonoverlapping codons, without any punctuation between them. This meant that a defined start site was necessary to define reading frame. Deducing the specific codons corresponding to specific amino acids was more difficult. Initial success with homopolynucleotides used as synthetic messenger RNA molecules provided a breakthrough, and within a few years all codons were assigned. The fact that there are 64 (4 × 4 × 4) possible combinations was taken care of by the fact that most amino acids had multiple codons, and some were used for start and stop signals in translation. The curious fact that the third residue in each codon was often undiscriminating was explained by Crick's “wobble” hypothesis.

This chapter describes what many would consider the origin of molecular biology, with the pioneering studies by Delbruck and Ellis on bacteriophage genetics. The fundamentals of the phage infection cycle were elucidated. Methods for transferring genetic information between bacteria were developed as genetic tools. The Hershey-Chase experiment used such transfer to prove the new belief that DNA was indeed the genetic material. The monumental discovery of the structure of DNA as a duplex helical structure of two antiparallel complementary strands provided the final step into a new era of biology. The success of working with bacteria and viruses shows the importance of choosing the appropriate creatures for fundamental biological studies.

Toward the end of the renaissance (c. 1700), both chemistry and biology were beginning to emerge as established sciences, and a few chemists were beginning to study biological systems. Proteins were early recognized as a distinct type of substance, filling many roles, including structure (as in fibers). The functioning of some proteins as catalysts (enzymes) was recognized. The role of amino acids as components of proteins was also understood although the mode of their incorporation was still unclear as late as 1900.

This chapter provides some examples of the impact molecular biological insights and techniques have had on other branches of biology. Here we center on evolution, beginning with Darwin, with special emphasis on the concept of natural selection. The chapter begins with a brief summary of the classical development of evolution, emphasizing the importance of mutations in that process. Molecular biology has not only clarified the basic mechanisms of evolution but also provided a new, more rational approach to phylogenetic classification. The study of human genomes, and the genomes of related species, has given us a clearer picture of human evolution, and the migrations of our ancestors over the ages.

This is a brief description of how DNA is replicated. Although the Watson-Crick structure provided immediate suggestions, full elucidation required answering some difficult questions over many years. First, the mode of replication was proved to be semiconservative (each strand serving as template for the complementary strand) by the elegant Meselson-Stahl experiment. Then a number of DNA-polymerase enzymes were discovered which could catalyze the synthesis of a complementary strand on a single-strand template. It was found that whereas one strand (leading strand) could be synthesized continuously, the other (lagging strand) had to be made in discontinuous pieces, which were then spliced together. The whole process has been more recently found to require a very complex molecular apparatus.

At the beginning of the 20th century, Edwin Fischer and Franz Hofmeister independently proposed that proteins were long-chain polymers formed by the condensation of amino acids into polypeptides. This model was for some time challenged by theories which held that proteins were simply colloidal aggregates. Several lines of experimentation refuted colloid models, including Theodor Svedberg's use of his ultracentrifuge to show that hemoglobin was a defined, high-molecular-weight molecule. The remaining question, as to whether protein sequences were repetitive or unique, was decided by Fredrick Sanger, who in 1951 determined the unique sequence of insulin.

Although understanding the one-dimensional structure of proteins via sequence analysis told a great deal, the question of how this chain was structured in three dimensions (3D) long remained unsolved. To be sure, certain special proteins, like those in hair and silk, were understood to exist as regular fibers. However, it was clear that the vast majority of “globular” proteins must be folded more compactly. Linus Pauling and his collaborators using simple rules derived from the structures of amino acids and their possible hydrogen bonding deduced a number of possible ways to fold a polypeptide chain into either helical or sheet structures. These were only speculation until an actual structure could be determined. This was accomplished by Max Perutz and John Kendrew who completed X-ray diffraction studies of hemoglobin and its smaller counterpart myoglobin in the early 1950s. Since then, similar methods have been used to determine the 3D structures of thousands of proteins.

In the early 1970s, molecular biologists suddenly devised a set of techniques that were to revolutionize the field and produce whole new industries. These go under the name of “DNA cloning” and allow the isolation, copying, altering, and amplifying almost any desired sequence of DNA. The methods depended on the discovery of restriction endonucleases (enzymes that cleave DNA at selected sites), and ligases which seal the cut DNA pieces back together. With the development of vectors to insert the newly constructed DNA into cells, it then was possible to clone the DNA—make many copies of it by growing it in bacteria, for example. A quite different way to amplify DNA, discovered more recently, is via the polymerization chain reaction (PCR). This utilizes repeated copying of the template DNA in a temperature-cycling regime, using a temperature-tolerant polymerase.

The origin of genetics is usually attributed to Gregor Mendel, who carried out thousands of breeding experiments with garden peas around 1860. From these, he established the first laws of genetics, which, with certain exceptions and extensions, are regarded as valid today. Unfortunately, Mendel's work was completely neglected for over 30 years. After 1900, the major fig. in genetics is Thomas Hunt Morgan, who established the fruit fly as a model for genetic studies. Morgan's laboratory was the center of a number of advances, including the mapping of genes on chromosomes and the artificial production of mutations via high-energy radiation.

Since its initial development in the 1970s, DNA sequencing has increased enormously in both rapidity and scope, starting with viruses and progressing to the whole genomes of mammals. The culmination of this work was the Human Genome Project, a massive international effort only completed after the turn of the millennium. It was followed by the ENCODE project, to extract structural and functional data from the genome sequence. This has been enormously successful, especially in pinpointing interactions of regulatory and transcription start sites with proteins. A major surprise was the finding that whereas a large fraction of the genome is transcribed into RNA, only a tiny fraction of that is translated into proteins. These, and other findings from ENCODE, raise once more the old question “What is a gene?”

Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.

Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism.

Kayoko Hayashihara, Susumu Uchiyama, Shigeru Shimamoto, Shouhei Kobayashi, Miroslav Tomschik, Hidekazu Wakamatsu, Daisuke No, Hiroki Sugahara, Naoto Hori, Masanori Noda, Tadayasu Ohkubo, Jordanka Zlatanova, Sachihiro Matsunaga, Kiichi Fukui. The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA. Journal of Biological Chemistry, Volume 285, Issue 9, 2010, Pages 6498-6507. https://doi.org/10.1074/jbc.M109.092833., In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 M NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys⁹⁷-Lys²⁷⁴), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by ∼25 bp. The solution structure determined byNMRrevealed that the globular domain (Met¹⁵³-Thr²³⁷) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.

BRCA1, the protein product of the Breast Cancer Susceptibility Gene (BRCA1) has been implicated in multiple pathways that preserve genome stability, including cell cycle control, DNA repair, transcription, and chromatin remodeling. BRCA1, in complex with another RING-domain protein BARD1, possesses ubiquitin-ligase activity. Only a few targets for this activity have been identified in vivo. Nucleosomal histones may also be targets in vivo since they can be modified by the BRCA1/BARD1 complex in vitro. Here we demonstrate that the BRCA1/BARD1 complex can ubiquitylate both free H2A and H2B histones and histones in the context of nucleosomal particles. These results raise the possibility that BRCA1/BARD1 can directly affect nucleosomal structure, dynamics, and function through its ability to modify nucleosomal histones.

Chromatin structure is a powerful tool to regulate eukaryotic transcription. Moreover, nucleosomes are constantly remodeled, disassembled, and reassembled in the body of transcribed genes. Here we propose a general model that explains, in quantitative terms, how transcription elongation affects nucleosome structure at a distance as a result of the positive torque the polymerases create as they translocate along DNA templates.