Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
How the Present Concepts Evolved
DNA as the Genetic Material

The discovery of deoxyribonucleic acid dates back to 1869, when Friedrich Miescher isolated a new chemical compound from leukocytes (from pus) and subsequently from spermatozoa. This substance was named nucleic acid. Sometime later, it was found to occur in both plants and animals, with the Thymus and Yeast Cells proving to be the best sources of Nucleic Acids. Chemical research soon established that the nucleic acids isolated from the thymus and from yeast are different. As we now know, thymus nucleic acids are predominantly represented by DNA, while yeast nucleic acids are represented by RNA. For some time, it was believed that animal cells contained only DNA, while plant cells contained only RNA; this view prevailed until the early 1940s, when it became clear that both compounds are present in All living organisms [5, 6].

a. Bacterial "Transformation"

Important experiments performed on Diplococcus pneumoniae cells in 1928 showed that the Genetic information controlling The properties of capsular Polysaccharides (Ch. 5, Sec. D) can be transferred from one bacterial strain to another. According to these experiments, some substance present in killed cells and Cell-free extracts stably alters the properties of capsules exposed to this substance. This phenomenon, termed bacterial "transformation," remained a mystery for many years. At the time these experiments were carried out, there was not even a hint of the genetic role of nucleic acids, which were universally perceived as rather peculiar material. Moreover, the covalent nature of bonds in nucleic acids had not yet been proven at that time. METABOLISM/2.html">THE CONCEPT OF the tetranucleotide as the repeating unit of some regular polymer was widely accepted. It was generally believed that genes were proteinaceous in nature.

In 1944, Avery¹⁾ and his coworkers demonstrated that purified pneumococcal DNA extracts could induce Bacterial Transformation [7, 8]. The purified transforming agent contained only a small amount of protein. Proteolytic Enzymes did not inactivate it, whereas deoxyribonuclease did.

Thus, bacterial transformation experiments conclusively demonstrated that DNA is the genetic material. This was also indicated by the results of several other experiments. For instance, it was found that DNA is localized in the nuclei of Eukaryotic cells. The absolute amount of DNA per cell for a given species Organism turned out to be constant. The fact that DNA constitutes the genetic material of certain Viruses was proven in 1952 by D. Hershey and M. Chase [8a], who discovered that upon infection of a bacterial cell (bacteriophage) by a virus, the viral DNA penetrates inside the bacterium, while the protein "coat" remains outside. This was successfully demonstrated by preparing Two Types of isotopically labeled T2 Bacteriophages (Supplement 4-D). In one of them, the DNA was labeled with the ³²P isotope, whereas in the other, the ³⁵S isotope was incorporated into the protein. E. coli cells were infected with preparations of labeled phages and then vigorously blended in a Waring blendor to remove the phage particles. As a result, the following occurred: about 80% of the ³⁵S was detached from the bacterium, whereas the greater part of ³²P penetrated inside the Bacteria and could even be detected in subsequent generations of bacteriophages [3].

¹⁾ Chargaff points out that by making this discovery at the age of 67, Avery refuted the widespread view that scientific discoveries are made exclusively by young people.

b. The Double Helix [9]

With The Development of new Methods for Investigating the Chemical composition of Nucleic Acids, it was established (by Chargaff) that, despite very substantial differences in the relative Abundance of various bases in different DNAs, the molar ratio of adenine to thymine, as well as that of cytosine to guanine, in all examined DNAs is approximately 1 : 1 [10]. Based on these data, the concept of base pairing in DNA was put forward. Definitive results were obtained from X-Ray Diffraction studies of stretched DNA fibers. These studies implied that DNA molecules almost certainly possess a helical Structure consisting of more than one strand. The decisive experiments were performed by Franklin [11] and Wilkins, whose data were utilized by Watson and Crick in 1953 to construct their double helix model [12, 13] (Fig. 2-21). Once The structure of DNA was elucidated, it immediately suggested Conclusions regarding the coding Properties of the DNA molecule and The Mechanism of its natural Replication. It seemed obvious that The sequence of NUCLEOTIDES must serve as The basis of The Genetic Code and that base pairing represents the mechanism that enables the Separation of two mutually complementary strands and the subsequent Biosynthesis of a new complementary strand (along each of the parental strands). In this manner, accurate Gene copying can occur. Analogously, RNA molecules can be synthesized along a DNA "template" and subsequently emerge into the Cytoplasm.

The Link Between the presence of RNA in the cytoplasm and Protein Synthesis was established through a series of experiments conducted in the early 1940s [i.e., before the structure of DNA was deciphered — Trans.]. Immediately following the Discovery of the double helix, a concept was proposed stating that DNA plays The Role of the primary "template" from which secondary RNA templates can be copied. These RNA copies, subsequently termed messenger RNAs (mRNAs; Ch. 1, Sec. A, 4), carry the genetic information that determines the Amino Acid Sequence in a protein. The flow of information from DNA to RNA and to protein can be symbolically represented as follows:

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Proteins control all metabolic processes in one way or another, including the reactions leading to The formation of nucleic acid nucleotide precursors and the reactions resulting in the polymerization of Amino Acids and nucleotides. Thus, the flow of information from DNA to proteins represents only a part of a larger loop of metabolic processes, with the DNA replication process itself proceeding with a high degree of fidelity. The flow of genetic information is always directed from DNA to The Cell, and copies of the primary template are transmitted from generation to generation in an almost unchanged form. The simple concept expressed by equation (15-1) rapidly attracted the attention of the scientific community and led to the explosive development of biochemical genetics.

c. Chromosome Map of E. coli

Let us now consider the most important aspect of Introduction/20.html">DNA Structure, namely The nucleotide sequence in which the genetic information is embodied. The circular DNA molecule comprising the E. coli chromosome contains 3.8 million nucleotides. In essence, we have only just begun the detailed Study of the nucleotide sequence of certain regions of this chromosome. However, speaking in broad terms rather than details, we know quite a lot about the chromosome. Specifically, it is precisely known that individual genes are arranged linearly on this chromosome. By 1972, the locations of 460 genes had been mapped on the chromosome map (Fig. 15-1; see also Table 15-1).

FIG. 15-1. Chromosome map of E. coli strain K-12 (data taken from Bachman B. J., Low K. B., Taylor A. L., Bacteriol. Rev., 40, 116–167 (1976)). The time scale is constructed based on interrupted conjugation experiments. The thr locus is arbitrarily chosen as the starting point. A list of genetic symbols and explanations for them are given in Table 15-1. In any given region of The Genome, typically only one of the DNA strands is transcribed. Since DNA strands are antiparallel, Transcription proceeds clockwise for one strand and in the opposite direction for the other (it is conventionally assumed that, given the chromosome orientation shown in the figure, the r-strand is transcribed clockwise). The directions of transcription of various operons are indicated by arrows. Maps for Salmonella typhimurium have been obtained using Similar Methods [Sanderson K. E., Bacteriol. Rev., 36, 558–586 (1972)].

To understand how the chromosome map shown in Fig. 15-1 was obtained, it is necessary to briefly review some of the genetic Research Methods. (We shall return to this issue in Sec. B.) Early work on genetic mapping dates back to the time when it was discovered that mutants exist whose growth depends on the presence of specific factors in the medium. Normal "wild-type" E. coli cells can grow on a minimal medium containing a carbon compound as an energy source and inorganic nutrients. Ultraviolet irradiation or Treatment with Chemical Mutagens leads to the appearance of A large number of mutant cells unable to grow on such a minimal medium. However, upon The addition of one or more specific compounds such as amino acids or Vitamins, the growth of mutant bacteria is usually restored. The Selection of auxotrophs based on nutritional requirements (as these mutants are called) is most often carried out by plating a large number of irradiated or chemically treated cells onto petri dishes containing a solid, nutrient-rich medium. Then, after colonies (clones) develop from individual bacteria, replica plating is performed by transferring replicas of the resulting colonies onto petri dishes containing minimal medium with specific supplements¹⁾.

Table 15-1 Some E. coli Genes^a

^a This list includes only 125 out of the 650 genes whose positions on the genetic map have been established.

^b Gene locations are schematically shown in Fig. 15-1.

¹⁾ To replicate patterns onto a petri dish containing nutrient Agar on which small bacterial colonies are growing, a sterile velvet pad is pressed against the plate and then used to "print" replicas onto petri dishes containing minimal medium. The original colonies and the colonies formed on the replica plates (with minimal medium) are compared, after which auxotrophic colonies (which failed to grow on the minimal medium) are selected. In the second stage, auxotrophs can be transferred by the same replica-plating method to petri dishes containing minimal medium supplemented with various nutrients (amino acids, Purines, Pyrimidines, vitamins, etc.). Selection is facilitated by preincubating irradiated cells in minimal medium treated with penicillin (Supplement 7-G). Penicillin kills growing cells, whereas auxotrophs, which do not grow on minimal medium, survive. Subsequently, penicillin is destroyed by adding penicillinase (Supplement 7-G). As a result of these Procedures, the percentage of auxotrophic mutants in the suspension is significantly increased [3].

Nutritional auxotrophs typically possess a defective gene that determines a protein without which The biosynthesis of the nutrient required by the given auxotroph cannot proceed. Based on this, the mutated gene is identified and assigned an appropriate genetic symbol. For example, the gene determining the synthesis of one of the protein subunits of tryptophan synthase is designated trpA. The replica-plating method can also be used to identify other Types of mutations, such as those disrupting motility or other cellular properties; in these cases as well, the mutant genes are assigned corresponding symbols. Some of these genetic symbols are indicated on the genetic map shown in Fig. 15-1.

Normally, bacteria reproduce by simple Cell Division; that is, The amount of DNA in the chromosome doubles, the cells divide, and the daughter cells receive identical Chromosomes. However, as Lederberg and Tatum demonstrated in 1946 [13a], bacteria can also reproduce sexually. Direct evidence for mating in bacteria was initially lacking, but it was shown that if cells of two different mutant strains of E. coli K-12 are mixed and cocultivated for several generations, some bacteria regain The ability to grow on minimal medium. Since each of these strains contained a single defective gene, the formation of an individual lacking either defect could only occur as a result of genetic material recombination between the two strains. These very experiments served as the basis for concluding that conjugation exists in bacteria. It was subsequently shown that true genetic recombination can occur during conjugation. This means that genes from two mating cells can be integrated to form a single continuous strand of bacterial DNA.

d. Sex Factors in Bacteria

We now know that certain E. coli K-12 cells contain small additional DNA fragments that act as a sex factor (F factor, fertility factor). The presence of the F factor in a bacterial cell essentially determines its male mating type. Among other elements, the F factor carries genes necessary for the synthesis of F-pili (sex hairs). These thin appendages, 8.5 nm in diameter, grow rapidly—within 4–5 min they reach a length of approximately 1.1 µm (see also Ch. 1, Sec. A, 6; Fig. 4-7). The end of an F-pilus attaches to a female cell. Brinton [14] suggested that DNA might pass from the male cell to the female cell through the pili. Although some researchers have indeed managed to observe cytoplasmic bridges between closely apposed cells, the true mechanism of DNA transfer remains to be elucidated.

The high Significance of the F factor for chromosome mapping is due to the fact that it occasionally integrates with the bacterial chromosome. Direct Electron Microscopy has shown that both the chromosome and the F factor are circular in structure. Their integration requires the Enzymatic Cleavage of both the chromosomal DNA and the F factor DNA, followed by the rejoining of the ends to form a continuous circle (Fig. 15-2). Specific enzymes catalyzing these reactions are discussed later in Section G. Different F factors can integrate into the chromosome at various sites. Bacterial strains containing an integrated F factor are designated as Hfr strains (derived from high frequency of recombination).

¹⁾ It remains unclear, however, whether F-pili form tubular structures with an inner diameter of about 2.5 nm (Fig. 4-7) or whether they possess a more open structure.

FIG. 15-2. Integration of the F factor with the bacterial chromosome and transfer of certain bacterial genes to another cell. A. Incorporation of the F factor into the E. coli genome and transfer of the "plus" DNA strand to a female recipient cell. B. Genetic recombination between the transferred DNA fragment and the genome of the recipient cell.

During the conjugation of an Hfr strain bacterium with an F^- (female) bacterium, the following occurs: at a point located close to the end of the integrated F factor, the chromosome begins to replicate, and the bacterial genes, followed by the F factor genes, are transferred into the female cell. According to current models, only one of the DNA strands—usually referred to as the "plus" strand—is transferred from the donor cell to the recipient cell, likely via the F-pilus (Fig. 15-2). A complementary "minus" strand is synthesized within the recipient cell, resulting in a double-stranded DNA molecule carrying genes from the Hfr cell. Only in rare cases does the "plus" strand of the donor cell transfer entirely to the female cell. More frequently, the DNA strand or the pilus itself breaks, and only a portion of the chromosome is transferred.

The partial transfer of the chromosome from the male cell results in the F^- cell becoming partially diploid (a merozygote), meaning it contains a duplicate set of many genes. Genetic information is exchanged (genetic recombination) between the two chromosomes within such a partially diploid cell (Fig. 15-2). The Chemical Reactions underlying this process, which is of vital importance for all sexually reproducing organisms, will be examined in Section G. Ultimately, the recombination process leads to daughter cells formed during subsequent divisions containing only a single chromosome with the standard gene Complement. However, certain genes in this chromosome originate from each of the parental strains. Thus, an F^- cell of a mutant strain, unable to grow on media lacking specific nutritional supplements, may acquire a gene from the male cell that allows it to grow on minimal medium. Although the number of such recombinant bacteria is small, they can easily be selected from a very large population of the initially mixed mutant bacteria.

d. Chromosome mapping by interrupted conjugation

The E. coli chromosome map can be constructed by mixing Hfr and F^- cells, allowing conjugation to proceed for a defined time interval, and then vigorously agitating the cells, for example, in a Waring blender. This Procedure disrupts all conjugation bridges and interrupts the bacterial mating process. Mating is interrupted at various time intervals, and the recipient bacteria are assayed for the presence of genes transferred from the donor strain cells. Using this method, it was demonstrated that complete chromosomal transfer at 37 °C requires approximately 100 min, and the approximate chromosomal Location of any gene can be determined by the time required for that gene to be transferred to the recipient cell. In reality, however, the situation is somewhat more complex. Since complete transfer of the entire chromosome is a rare event, experiments typically utilize various E. coli K-12 substrains in which the F factor is located at different sites; in all cases, genes located clockwise¹⁾ immediately downstream of the integration point (Fig. 15-1) are transferred rapidly and at high frequency.

In constructing the map shown in Fig. 15-1, not only the interrupted conjugation data were used, but also data obtained from bacteriophage P1 Transduction studies [15]. Phage transduction, discussed in more detail in Section F, makes it possible to transfer short DNA fragments approximately 2 min in length (see the map

of E. coli). Cotransduction—for example, the simultaneous incorporation of two genes into the recipient cell chromosome—occurs at a frequency that correlates with the distance between these two genes on the map. This approach has allowed refined mapping of many Regions of the E. coli chromosome.

It should be noted that although the map in Fig. 15-1 is calibrated in minutes, in the near future it will likely be possible to represent the genetic map directly in micrometers of DNA length (its total length is approximately 1100 µM) or in thousands of nucleotide units, frequently called kilobases (kb). The total DNA length is approximately 3800 kb₁₎.

¹⁾ For an F factor of a single type. Other types integrate in the opposite direction.