Introduction to Molecular Genetics

(Draft)

(from Concepts of Genetics, 2nd Edition,

by W. Klug and M. Cummings, Merrill Publishing Company, 1986)

INTRODUCTION

Expression of the information stored in the genetic material is a complex process

and is the basis for the concept of information flow within the cell. The initial event

is the transcription of genetic information stored in DNA. Transcription results in

the synthesis of three types of RNA molecules: messenger RNA (mRNA), transfer

RNA (tRNA) and ribosomal RNA (rRNA). Of these, mRNAs are translated into

proteins. Each type of mRNA is the product of a specific gene and leads to the

synthesis of a different protein.

Translation, or protein synthesis, involves many molecular components, a supply

of energy, and the cellular organelle, the ribosome. The ribosome consists of several

types of rRNA plus a variety of individual proteins. The role of tRNA is to adapt

the information present in mRNA to the correct amino acids during translation.

Amino acids are the building blocks of proteins. In eukaryotic dells, transcription

occurs in the nucleus and translation occurs in the cytoplasm.

The genetic material is also responsible for newly arising variability among the

organisms through the process of mutation. If a change in the chemical composition

of DNA occurs, the alteration will be reflected during transcription and translation,

perhaps affecting the specified protein. If a mutation is present in gametes, it

will be passed to future generations and, with time, may become distributed in

the population. Genetic variation, which also includes rearrangements within and

between chromosomes, provides the raw material for the process of evolution.

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PROTEIN AS THE GENETIC MATERIAL

First, proteins are abundant in cells. Although the protein content may vary

considerably, these molecules compose over 50 percent of the dry weight of cells.

Since cells contain such a large amount and variety of proteins, it is not surprising

that early geneticists believed that some of this protein could function as the genetic

material.

DNA was first studied in 1868 by Friedrick Miescher, a Swiss chemist. He was

able to separate nuclei from the cytoplasm of cells and then isolate from them an

acid substance that he called nuclein, Miescher shoed that nuclein contained large

amounts of phosphorous and no sulfur, characteristics that differentiate it from

proteins.

As analytical techniques were improved, nucleic acids, including DNA, were

shown to be composed of four similar molecules called nucleotides. Around 1910,

Phoebius A. Levene proposed the tetranucleotide hypothesis to explain the chemical

arrangement of these nucleotides in nucleic acids. He proposed a very simple four-

nucleotide unit as shown in Figure 1. Levene based his proposal on studies of the

composition of the four types of nucleotides. Although his actual data revealed

proportions of the four that varied considerably, he assumed a 1 : 1 : 1 : 1 ratio.

The discrepancy was ascribed to inadequate analytical technique.

Between 1910 and 1930, other proposals for the structure of nucleic acids were

advance, but they were generally overturned in favor of the tetranucleotide hy-

pothesis. It was not until the 1940s that the work of Erwin Chargriff led to the

realization that Levene’s hypothesis was incorrect. Chargaff showed that, for most

organisms, the 1 : 1 : 1 : 1 ratio was indeed inaccurate, thus discrediting Levene’s

hypothesis.

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Figure 1. Levene’s proposed structure of a DNA tetranucleotide.

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THE NUCLEOTIDE: THE BASIC UNIT

Nucleotides are the building blocks of all nucleic acid molecules. Sometimes

called mononucleotides, these structural units consist of three essential components:

a nitrogenous base, a pentose sugar (5 carbons), and phosphoroic acid (a phosphate

group). There are two kinds of nitrogenous bases: the nine-membered double-

ringed purines and the six membered single ringed pyrimidines. Two types of

purines and three types of pyrimidines are found commonly in nucleic acids. The

two purines are adenine and guanine, abbreviated A and G. The three pyrimidines

are cytosine, thymine, and uracil, abbreviated C, T, and U. The chemical structures

of A, G, C, T, and U are as shown here in Figure 2. Both DNA and RNA contain

A, C, and G; only DNA contains the base T, whereas only RNA contains the base

U.

The pentose sugars found in nucleic acids give them their names. Ribonucleic

acids (RNA) contain ribose, while deoxyribonucleic acids (DNA) contain deoxyri-

bose. See Figure 3.

If a molecule is composed of a purine or pyrimidine base and a ribose or de-

oxyribose sugar, the chemical unit is called a nucleoside. If a phosphate group is

added to the nucleoside, the molecule is now called a nucleotide. Nucleosides and

nucleotides are named according to the specific nitrogenous base (A, T, G, C, or

U) that is part of the building block. The nomenclature and general structure are

as given in Figure 4.

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Figure 2. Chemical structures if the pyrimidines and purines.

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Figure 3. Chemical structures of ribose and 2-deoxyribose.

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Figure 4. The structure and names of the nucleosides and

nucleotides of RNA and DNA.

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POLYNUCLEOTIDES

The linkage between mononucleotides consists of a phosphate group linked to

two sugars. A phosphodiester bond is formed, because phosphoric acid has been

joined to two alcohols (the hydroxyl groups on the two sugars) by an ester linkage

on both sides. See Figure 5. The joining of two nucleotides forms a dinucleotide;

of three nucleotides, a trinucleotide; and so forth. When long chains of nucleotides

are formed, the structure is called a polynucleotide.

Long polynucleotide chains would account for the observed molecular weight

and would explain the most important property of DNA — genetic variation. If

each nucleotide position in this long chain may be occupied by any one of four

nucleotides, extraordinary variation is possible/ for example, a polynucleotide that

is 1000 nucleotides in length may be arranged 41000 different ways, each one different

from all other possible sequences. This potential variation in molecular structure

is essential if DNA is to serve the function of storing the vast amounts of chemical

information necessary to direct cellular activities.

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Figure 5. The linkage of nucleotides by the formation of C-3′ – C-5′

phosphodiester bonds, producing a polynucleotides chain.

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THE STRUCTURE OF DNA

In 1953, James Watson and Francis Crick proposed that the structure of DNA

is in the form of a double helix. Their proposal was published in a short paper in

Nature, volume 171, no. 4356, pp. 737–38. In a sense, this publication constituted

the finish line in a highly competitive scientific race to obtain what some consider to

be the most significant finding in the history of biology. This “race,” as recounted

in Watson’s book The Double Helix, demonstrates the human interaction, genius,

frailty, and intensity involved in the scientific effort that eventually led to the

elucidation of DNA structure.

Watson and Crick published their analysis of DNA structure in 1953. By build-

ing models under the known constraints, they proposed the double-helical form of

DNA as shown Figures 5, 6, and 7. This model has the following major features:

1. Two right-handed helical polynucleotide chains are coiled around a central axis;

the coiling is plectonic, meaning that the two coils can only be separated by

completely unwinding them.

2. The two chains are antiparallel; that is, one is upside down with respect to the

other (their C-5′-to-C-3′ orientations are in opposite directions).

3. The bases of both chains are flat structures, lying perpendicular to the axis;

they are “stacked” on one another, 0.34 nm (3.4 Angstrom) apart.

4. Each complete turn of the helix is 3.4 nm long; thus 10 bases exist in each chain

per turn.

5. The nitrogenous bases of opposite chains are electrostatically attracted to one

another as the result of the formation of hydrogen bonds: specifically only A-T

and G-C pairs are allowed.

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6. In any segment of the molecule, alternating large major grooves and smaller

minor grooves are apparent along the axis.

7. The double helix measures 2.0 nm in diameter.

The specific A-T and G-C base pairing is the basis for the concept of complemen-

tarity. This term is used to describe the chemical affinity provided by the hydrogen

bonds between the bases.

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Figure 6. A schematic representation of the DNA double helix

as proposed by Watson and Crick.

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Figure 7. A representation of the antiparallel nature

of the two strands of the helix.

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Figure 8. The hydrogen bonds between cytosine and guanine

and between thymine and adenine.

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THE MODE OF DNA REPLICATION

It was apparent to Watson and Crick that because of the arrangement and

nature of the nitrogenous bases. each strand of a DNA double helix could serve as

a template for the synthesis of its complement. They proposed that if the helix were

unwound, each nucleotide along the two parent strands would have an affinity for its

complementary nucleotide. The complementarity is due to the potential hydrogen

bonds that can be formed. If thymidylic acid were present, it would “attract”

adenylic acid; if guanidylic acid were present, it would “attract” cytidylic acid;

and so on. If these nucleotides were then covalently linked into polynucleotide

chains along both templates, the result would be the production of two new but

identical double strands of DNA. See Figure 9. Each replicated DNA molecule

would consist of an “old” and a “new” strand. Therefore, this mechanism is called

semiconservative replication.

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Figure 9. General model of semiconservative replication of DNA.

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EUKARYOTIC CHROMOSOMES: GROSS STRUCTURE

The structure and organization of the genetic material in eukaryotic cells is much

more intricate than in viruses and bacteria. This complexity is due to the greater

amount of DNA per chromosome and the presence of large numbers of proteins

associated with DNA in eukaryotes.

By the metaphase of mitosis it becomes apparent that each chromosome is really

a double structure consisting of two sister chromatids. Sister chromotids are held

together at a single point, the centromere, which is the area of attachment to the

spindle fibers. Chromosomes for any given species are classified by the location of

the centromere and the overall size of the chromosome. The karyotype consists of

a micrograph of the chromosome pairs in the metaphase arranged by size and cen-

tromere location. The number of chromosome pairs in the karyotype is equal to the

haploid number. In species with a low haploid number, each pair of chromosomes

may be distinct in gross morphology from all other pairs. Humans have 23 pairs of

chromosomes.

EUKARYOTIC CHROMOSOMES: MOLECULAR

ORGANIZATION

Thus, the eukaryotic genetic material is composed of nucleoprotein; such mate-

rial is generally referred to as chromatin, particularly during interphase, when it

is uncoiled. The associated proteins are divided into basic, positively charged his-

tones and less positively charged nonhistones. The histones seem to be intimately

associated with chromatin structure, while the nonhistone proteins are thought to

play other roles, including genetic regulation. Despite the presence of protein in

the chromatin, the DNA component is universally believed to be in the part that

stores genetic information.

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Research in the past several years has made it possible to develop a general model

for chromatin structure. This model is based on the assumption that chromatin

fibers, composed of DNA and protein, must undergo extensive coiling and folding

in order to fit into the cell nucleus.

Of the proteins associated with DNA, the histones are now believed to be es-

sential to the structural integrity of chromatin. Histones contain large amounts of

the positively charges amino acids lysine and arginine. Thus histones can bond

electrostatically to the negatively charge phosphate groups of nucleotides.

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