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Modern Genetic Analysis Chapter 1. Genetics and the Organism Experimental Genetics
Genetics In Process 1-2: Deducing the nature of genes

The present understanding of gene structure and function arose out of approximately 100 years of research, so it is important to trace some of the landmark experiments that contributed to our understanding of the gene as a concept and as a reality. All the discoveries mentioned below will be revisited throughout the book; they are listed here as a time line in order to give an initial overview of the sequence by which genetics has evolved. The overview also gives the names of the scientists who made the discoveries, a reminder that it is people who advance scientific understanding, through their genius and their dedication. More detailed treatments of selected historical discoveries will appear throughout the book in boxes.

Although it is likely that the human speciesand even its progenitor specieshas always been interested in heredity, the oldest surviving records of ideas on the mechanisms of heredity are from the ancient Greeks. For example, Aristotle speculated that since blood perfused and nourished the organs of the body, procreation too must be via blood. He proposed that male semen was purified blood and that the female genetic contribution to the next generation was menstrual blood. However, these and other ideas made little or no contribution to the eventual development of our present understanding of heredity. After the Greeks, there was a 2000-year silence on the mechanisms of heredity. Genetics as a scientific discipline did not exist before the work of Gregor Mendel, in the middle of the nineteenth century. The reason is that Mendel was the first person to conceive of the idea of a gene, and although at the time he knew nothing of the physical or chemical nature of genes, genes became the basis for the development of our present understanding of heredity. All present research in genetics can be traced back to Mendel's discovery. Follow the discussion of the evolution of genetic understanding below using the accompanying figure, which shows diagrammatic representations of some of the key steps discussed.

Before Mendel, hereditary thought was based mainly on logic and speculation, not on experimentation. However, Mendel set the tone for genetics as an experimental discipline. In his monastery garden, over several years he carried out a large number of experimental cross-pollinations between pea plants that showed contrasting expression of certain characters, such as yellow versus green seeds and tall versus dwarf stature. In certain types of crosses he observed precise and repeatable mathematical ratios in the progeny, and from these ratios in a set of brilliant deductions he concluded not only that genes (what he called "factors") exist, but also that they are present in pairs in the pea plant and that the pairs separated during the formation of ovules and pollen (see part 1 of the figure, showing gene pair A and a and a second gene pair B and b). Contrasting properties such as tall and dwarf were caused by different forms of a single type of gene (which we now call alleles). Mendel also analyzed plants that differed in two characters, and from progeny ratios in these crosses he deduced that different gene pairs are packaged independently into ovules and pollen. Note that in the figure above alleles are given letter symbols (for example, pair A and a); this is the convention in genetics, as we shall see later.

Mendel's genes were only hypothetical entities, factors that could be inferred to exist in order to explain his results. The twentieth century saw tremendous strides in the development of the understanding of the nature of genes and how they function. Mendel's publications lay unmentioned in the research literature until 1900, when the same conclusions were reached independently by several other investigators. Then there followed hundreds of publications showing "Mendelian" inheritance in a wide array of plants and animals including humans. It seemed that Mendel's ideas on genes and their inheritance patterns were of general validity. In the early years of this century biologists noted that the inheritance of genes closely parallels the inheritance of chromosomes during the nuclear divisions called meiosis, which occur in the cell divisions during the formation of eggs and sperm. It seemed that genes must be parts of chromosomes (see part 2 of the figure), and genes must be in pairs because in most plants and animals chromosomes are in pairs. This idea was strengthened through the demonstration by Thomas Hunt Morgan (1910) of parallel inheritance of certain fruit fly genes with the sex-determining chromosomes; that is, the pattern of inheritance of certain genes seemed to follow (parallel) the pattern of inheritance for the sex chromosomes, again suggesting that the genes were on the chromosomes. Further experiments by Morgan (1911) and one of his students, Alfred Sturtevant (1913), also showed parallel inheritance between genes themselves, suggesting that these genes were on the same chromo-somesthe concept of linkage. In part 3 of the figure, for example, allele A might be inherited with allele C more often than chance would predict, suggesting they were linked on the same chromosome. The same would be true for alleles a and c. The researchers developed a method by which the distance between genes on the same chromo-some could be mapped by measuring the frequency at which the parallel inheritance of two genes was disrupted, yielding new combinations of alleles (for example, Ac and aC). These new combinations were thought to be caused by a process of regulated chromosomal breakage and reunion called crossing-over. The farther apart two linked genes are (that is, the greater their map distance), the more likely crossing-over will occur between them, disrupting their parallel inheritance. In 1916 another student of Morgan, Calvin Bridges, proved beyond reasonable doubt that genes are on chromosomes. Using fruit flies with an extra chromosome, he showed that the abnormal inheritance of certain genes could be explained only if they were part of the extra chromosome. Eventually in 1931 Harriet Creighton and Barbara McClintock demonstrated that the production of new combinations of genes on the same chromosome was indeed correlated with physically exchanged chromo-some parts.

In 1927 Hermann Muller showed that mutations (part 4) can be produced at high frequencies by treating cells with X rays, the first demonstration of an environmental mutagenic agent. (Mutations also arise spontaneously but at a lower frequency.) Muller's work on mutation induction opened the door to the genetic technique of using mutations to dissect biological processes, which is still used extensively today. In 1943 Salvador Luria and Max Delbruck used bacteria to show that mutation is a random process that can occur in any cell at any time.

Molecular genetics began in 1941 when George Beadle and Edward Tatum used the fungus Neurospora crassa to show that certain genes concerned with cellular chemistry acted by coding for catalytic proteins called enzymes. Subsequent studies in other organisms extended this idea to show that most genes code for some type of protein, either active proteins such as enzymes or proteins that play a structural role in cells. Soon afterwards, Oswald Avery, C. M. McLeod, and M. McCarthy (1944) found that the genetic makeup of a bacterial cell could be changed by adding exogenous DNA (the first example of transgenesis), showing that bacterial genes are made of DNA, a finding that was later extended to all organisms. A major landmark was attained in 1953 when James Watson and Francis Crick proposed a double-helix model for DNA structure. This model showed that DNA could replicate by progressive unwinding of the two intertwined strands of the double helix and new synthesis using the exposed strands as templates. Each of the two intertwined strands of DNA was proposed to be a chain of chemical groups called nucleotides, of which there were known to be four types. Because proteins are strings of amino acids, it was suggested that a specific nucleotide sequence of DNA (a gene) could contain coded information for specifying amino acid sequence and hence protein structure (part 5).

In 1955 Seymour Benzer, extending earlier studies in fruit flies, showed that mutant alleles in bacterial viruses are produced by changes of only localized regions within the gene (mutant sitesrefer back to part 4) and that the mutant sites within a gene could be mapped in relation to one another, forming a linear map. Hence it was likely that indeed the gene itself is a linear structure as in the Watson and Crick proposal. It also seemed likely that the DNA of genes was part of a continuous DNA molecule, the chromosome (see part 6), and indeed this was proved later. The strand separation method for DNA replication (called the semiconservative methodpart 7) was demonstrated experimentally for the first time by Matthew Meselson and Frank Stahl in 1958. At the end of replication of a DNA molecule, two molecules result. Each of these is a hybrid consisting of one of the parent strands intertwined with one newly synthesized strand, hence the term semiconservative. In 1961 Francis Crick and Sydney Brenner showed that the coded information in the nucleotide sequence must be translated in groups of three nucleotides for each amino acid. Charles Yanofsky (1966) showed that the positions of mutant sites within a gene matched perfectly the positions of altered amino acids in the polypeptide sequence of the corresponding protein. The complete genetic "dictionary" of all 64 possible triplet coding units (codons), and the specific amino acids they stand for, was deduced in 1966 by Marshall Nirenberg and Gobind Khorana. Subsequent studies in many organisms showed that the double-helical structure of DNA, the mode of its replication, and the codon dictionary are the same in virtually all organisms, whether plants, animals, fungi, or bacteria.

In 1961 Francois Jacob and Jacques Monod established the prototypic model for gene regulation by showing that bacterial genes can be turned on (to produce RNA transcripts) and off through the binding action of regulatory proteins to a region just adjacent the site from which RNA synthesis begins (part 8).

Technical advances have played an important role in the advance of genetic understanding. Daniel Nathans and Hamilton Smith in 1970 discovered a specialized class of enzymes called restriction enzymes that cut DNA at specific nucleotide target sequences. This discovery allowed Paul Berg in 1972 to make the first artificial recombinant DNA molecule, composed of DNA from two different organisms. He isolated DNA molecules from the different sources, cut them, and joined them in a test tube. These advances allowed Herbert Boyer and Stanley Cohen in 1973 to isolate individual genes and amplify them to a high copy number. They spliced the genes into self-replicating DNA molecules (small chromosomes of various types) and inserted these into living cells. From these methodologies arose the whole field of recombinant DNA technology that currently dominates genetics. In 1977 two different methods were invented for determining the nucleotide sequence of DNA, one by Allan Maxam and Walter Gilbert and the other by Fred Sanger. It was then possible to examine the structure of genes directly by nucleotide sequencing, thereby confirming many of the inferences about genes originally made indirectly. In 1985 Kary Mullis invented a technique called the polymerase chain reaction that made it possible to find and amplify specific segments of DNA from complex mixtures. In 1985 Michael Smith invented site-directed mutagenesis, a technique that makes it possible to introduce specific nucleotide changes into a gene of choice.

The late 1980s and early 1990s saw the initiation of projects to completely sequence the genomes of several organisms important in genetic research. In 1990 the human genome sequencing project was begun. In 1995 the first complete organismal genome sequence was announced, that of a bacterium, Haemophilus influenzae. The first complete eukaryotic sequence was for baker's yeast, Saccharomyces cerevisiae, published in 1996. Sequencing of several other eukaryotic genomes is nearing completion, and the complete human sequence is expected in 2005.

© 1999 by W. H. Freeman and Company. All rights reserved.