Cellular Roles of the Nucleic Acids (With Diagram)

The Discovery of DNA:

Friedrich Miescher (Swiss, 1844-1895) is credited with the discovery of nucleic acid in 1869.

He isolated nuclei from white blood cells present in pus, using di­lute solutions of hydrochloric acid to dissolve away other cell structures and then adding the protein- digesting enzyme pepsin to further degrade residual cell protein adhering to the nuclei.

Nuclei isolated in this manner were then extracted with alkali, and the chemical composition of the extract was analyzed.

The chemical composition of the extract differed markedly from that of protein, so Miescher called this material “nuclein.” (The term nucleic acid was introduced 20 years following Miescher’s discovery by another bio­chemist, Richard Altmann.)

By comparing the chemi­cal analyses reported by Miescher with those carried out more recently, it is clear that Miescher’s nuclein was, in fact, the nucleic acid deoxyribonucleic acid (DNA).

Miescher also worked with the sperm cells of salmon, which contain particularly large nuclei (more than 90% of the cell mass is accounted for by the nu­cleus). In addition to isolating nuclein from the sperm nuclei, he used acid to extract an organic material having an unusually high nitrogen content. He called this substance “protamine.” The protamines are pro­teins containing an unusually rich content of lysine and arginine residues. We now recognize that extracts of cell nuclei also contain proteins called histories and that these are intimately associated with the nuclear DNA.

The function of the cell nucleus was unknown in Miescher’s time, and while Miescher was convinced of the fundamental importance of nu­clein, especially during fertilization, it was not until 60 years after his discovery that a series of experiments was carried out that established the genetic role of the nucleic acids.

Also during this period, it was shown that two types of nucleic acid occur in cells: deoxy­ribonucleic acid and ribonucleic acid (RNA).

Most no­table among the experiments that established the ge­netic role of the nucleic acids were those on:

(1) The transformation of bacterial types and

(2) Virus repro­duction.

Transformation of Bacterial Types:

Two types of pneumonia bacteria (Diplococcus pneumoniae) exist and are readily distinguished by the appearance of their colonies when cultured on agar plates; they are called “smooth” (S) and “rough” (R) types. The S type of pneumonia bacterium (which is the normal, virulent kind) is enclosed within a poly­saccharide capsule and gives rise to smooth, shiny col­onies.

In contrast, the R type is non-infective (i.e., non- virulent), is unable to synthesize the polysaccharide capsule, and gives rise to granular (and therefore rough-appearing) colonies. In 1928, F. Griffith showed that it was possible to transform the rough bacteria into the smooth type. He simultaneously injected small numbers of live R bacteria and large numbers of heat-killed S bacteria into mice, many of which subse­quently died.

When these mice were examined, they were found to contain live S bacteria. Because the S bacteria originally injected into the mice were heat- killed and thus incapable of reproducing, Griffith con­cluded that some of the R bacteria must have been transformed into the S type in the presence of the dead S cells. These observations were subsequently confirmed by a number of investigators who also ruled out the possibility of contamination of the inocu­lum by a few live S cells or of the mutation of some of the R cells into the S type following injection.

In 1932, J. L. Alloway showed that the same trans­formation was possible in vitro, for when an extract of S cells was added to a culture of R cells, some of the latter were permanently transformed into the viable S type. Therefore, there appeared to be a substance in S cells that was capable of bringing about an inheritable change in the R cells.

The substance responsible for the transformations observed by Griffith and Alloway (called “transform­ing principle”) was finally identified in 1944 by 0. T. Avery, C. M. MacLeod, and M. McCarty. Although crude extracts of heat-killed S cells were found to con­tain protein, polysaccharide, lipid, and nucleic acid, the removal of the protein, polysaccharide, and lipid by a combination of chemical and enzymatic proce­dures, including enzymatic hydrolysis, chloroform extraction, and alcohol fractionation, resulted in a product that retained the transforming activity.

How­ever, when the product was treated with the enzyme deoxyribonuclease (which degrades DNA), the capac­ity to transform R cells was lost. This evidence, to­gether with chemical analyses, showed that the trans­forming principle was DNA.

Virus Reproduction:

Viruses are composed princi­pally of protein and nucleic acid. Depending on the type of virus, the nucleic acid is either DNA or RNA. The protein forms a coat around the virus and en­closes the core of nucleic acid. In 1952, A. D. Hershey and M. W. Chase conducted a series of experiments to determine whether it was the viral protein coat or the viral nucleic acid that was required for virus reproduc­tion. Hershey and Chase used the bacterium Esche­richia coli and the T2 virus (a DNA-containing virus that infects E. coli) in two sets of experiments. In one set of experiments, E. coli was cultured in a medium containing the radioactive isotope of sulfur, ³⁵S.

During the growth of the bac­terial population, ³⁵S was incorporated into the cells. The culture was then infected with the T2 bacterio­phage, and during the reproduction of the bacterio­phage within the host cells, bacterial ³⁵S was used in the synthesis of phage protein (i.e., it was incorpo­rated into cysteine and methionine residues). Nucleic acids do not contain sulfur.

Following lysis of the bac­terial cells, the ³⁵S-labeled viruses were collected and used to infect E. coli cultured on media devoid of ³⁵S. A Waring blender was then used to separate by physi­cal agitation what was left of the attached viruses from the surfaces of the bacterial hosts.

Centrifugation was then used to separate the bacteria from unattached virus and from viruses that were detached by the action of the blender. A comparison of the ra­dioactive sulfur content of the sedimented bacteria and unsedimented viruses revealed that nearly all the sulfur remained with the viruses and had not entered the bacterial cytoplasm.

In other experiments, E. coli was cultured in media containing the radioisotope ³²P prior to infection with the phage. The nucleic acids contain large quantities of phosphorus, and therefore ³²P was incorporated into newly synthesized viral DNA. When labeled vi­ruses were used to infect additional bacteria and the blender again was employed to shake off the attached viruses, it was found that the ³²P radioactivity was within the infected cells.

The two sets of experiments are depicted in Figure 7-1. Hershey and Chase con­cluded that it was the DNA of the virus that entered the host cell during infection and that DNA was re­quired for the reproduction of genetically identical virus particles by the metabolic machinery of the host cell.

The observations of Hershey and Chase also explained earlier findings by T. F. Anderson and R. M. Herriott that the T2 phage loses its capability to re­produce when distilled water is added to a suspension of the viral particles prior to their addition to a bac­terial culture.

Even though such viruses are still able to attach to the bacterial host, the sudden osmotic shock created by exposure to distilled water causes them to empty their nucleic acid content into the sus­pending medium. Therefore, it must be the nucleic acid injected into the bacterium that allows for viral replication.

Tobacco mosaic virus (TMV), the virus that infects tobacco leaves, contains RNA instead of DNA. In the early 1950s, H. Fraenkel-Conrat separated the RNA and protein components of TMV and found that the RNA separately injected into the tobacco leaves could infect the plant and produce new virus particles con­taining both RNA and protein.

Fraenkel-Conrat also found that when protein isolated from one strain of TMV was mixed with RNA isolated from another strain, a reconstituted “hybrid” virus was produced that retained the capability to infect the tobacco leaves. When progeny viruses produced following in­fection by the hybrid were isolated and their RNA and protein components separated and analyzed, it was found that the type of protein in the virus coat was identical to that of the strain used as the source of RNA (Fig. 7-2).

It was not the same as the protein of the hybrid viruses causing the infection. These obser­vations support earlier conclusions concerning the ge­netic role of the nucleic acids and also prove that these molecules alone contain information that determines the specific nature of newly synthesized protein.

Although the most direct evidence supporting the notion that DNA (or in the case of some viruses, RNA) is the genetic material was obtained using microbial systems, observations supporting this idea were also made with higher organisms. Based on numerous studies of the mechanism of fertilization, including those by 0. Hertwig (1865), H. Fol (1877), E. Stras- burger (1884), A. Weismann (1892), and E. B. Wilson (1895), it was generally acknowledged by the turn of the century that the chromosomes contained within the cell nucleus were concerned with the transmission of heredity.

Therefore, the development of staining re­actions (such as the Feulgen reaction) that are specific for DNA and the subsequent microscopic localization of the DNA in the chromosomes implicated DNA as the genetic material. Further evidence was provided during the 1940s from quantitative chemical analyses of DNA present in measured quantities of cells (i.e., numbers, dry weight, etc.).

These analyses revealed that the amount of DNA per cell was more or less con­stant within the various tissues of an organism. More­over, it was also found that the total quantity of DNA present in the cell nucleus was related to its ploidy (i.e., the number of complete sets of chromosomes). The gametes (sperm and egg cells) of an organism were shown to have one-half as much DNA as the dip­loid somatic cells. This is precisely what would be ex­pected if DNA served as the genetic material.

Although RNA appears to be the genetic material of some virus particles, there is no evidence that it plays a similar role in cells. Instead, RNA serves as an intermediary between the genetic information of DNA and the expression of this information as the synthesis of the cell’s enzymes and other proteins.

In eukaryotic cells, most of the DNA is present in the nucleus, although certain organelles (mitochondria and chloroplasts) also contain DNA. Before we explore just how the relationship between DNA and RNA can be effected and the mechanism by which DNA serves as the genetic material, it is first necessary to con­sider the chemistry of these two macromolecules.