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Chap t e r 2 R ep l i c a t i o n o f Gene t i c I n f o r ma t i o n

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CSLS / THE UN IVERS ITY OF TOKYO 27

Part I

Relationship between Cells and Genetic Information

Chapter 2Replication of Genetic Information

As the smallest units of organisms, cells share the single basic property/function

of dividing and multiplying to create progeny cells. All cellular components must

be doubled in number before a cell can divide, but DNA, a genetic material, has

the distinctive characteristic of existing as a single molecule in each cell. Since

DNA molecules carry the full genetic information of the organisms to which they

belong, a DNA molecule from the parent cell must be accurately replicated (i.e.,

doubled), and the two molecules must be distributed equally to the two daughter

cells without fail. In this chapter, we will first learn how genetic-information-

carrying DNA consists of two long polymers of small low-molecular units called

nucleotides (i.e., double-stranded DNA). We will then learn how DNA has an

ingenious mechanism to accurately replicate itself using a strand from the parent

DNA as a template – a mechanism not used in the synthesis of other polymers.

I . Cell Growth and DNA Replication

Cell Growth – the Most Basic Cell Function

All organisms on earth have one thing in common: they are made of cells.

Surrounded by a phospholipid bilayer, cells contain proteins as functional polymers

and have DNA as genetic information that dictates cell structures and functions. As

the smallest units of organisms, all cells divide and multiply to create progeny cells.

Although multicellular organisms produce offspring as a function on the level of

individual organisms, this function is supported by the multiplication of component

cells, and cell multiplication is a basic function of the process by which a fertilized

egg develops into an individual organism. Cell multiplication is the most basic and

common of cell functions, and has survived a long process of evolution.

Special Characteristics of DNA Replication

Cells multiply by binary fission. Before cell division, all cellular components must



be doubled (Fig. 2-1). However, cellular components consist of a great number

of molecules, and the concept of “doubling” here is applied loosely; components

are not necessarily equally divided and distributed precisely into two cells (i.e.,

daughter cells).

DNA, which consists of genetic information, is quite different. Prokaryotes have

only one DNA molecule per cell. Although eukaryotes have a structure slightly

more complex than that of prokaryotes, they also have only one molecule of the

same DNA type per cell. Since DNA molecules contain all the genetic information

of the organism to which they belong, a DNA molecule identical to that of the

parent cell must be replicated during cell multiplication, and the two resulting

identical copies of the DNA must be equally distributed to the two daughter cells.

This phenomenon, which involves individual molecules, presents a rigorous

condition not found in other cellular molecules. First, let’s start by looking at

exactly what DNA is.

I I . What Kind of Molecule is DNA?

Nucleic Acids as a Unit

DNA is a type of nucleic acid. A nucleic acid is a compound consisting of a

base, a pentose and a phosphate (Fig. 2-2A). As shown in Figure 2-2B, bases

are aromatic ring (heterocyclic) compounds containing nitrogen, and are roughly

divided into purines and pyrimidines. There are five main bases in nucleic acids

(Fig. 2-2B). Their names and one-letter abbreviations are frequently referred to

throughout this book, and should therefore be remembered. There are two types

of pentose: ribose and 2-deoxyribose (Fig. 2-2C). Compounds consisting of a

base and a pentose are collectively called nucleosides (Fig. 2-3). In nucleosides,

the carbon numbers of a sugar are expressed as “number’.” Compounds in

which a phosphate or phosphates are linked to the hydroxyl group of their sugar

in nucleosides are called nucleotides or nucleic acids (Fig. 2-4). The number of

phosphates added is not necessarily one, and in fact many nucleotides have

three phosphates (Fig. 2-5). The position of a phosphate is not limited to 5’, but

many of the phosphates found in living bodies are 5’-phosphates. There are

many types of functional nucleotides. Typical examples include ATP (adenosine

5'-triphosphate), which supplies energy to enzymatic reactions that require it,

and cAMP (adenosine 3', 5'-cyclic monophosphate), which works in the signal

Figure 2-1 Schematic diagram of cell division


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transduction pathway. When bases are not specified, they are called NMP

(ribonucleoside monophosphate) or dNTP (deoxyribonucleoside triphosphate).

Nucleic acids are roughly classified into DNA (deoxyribonucleic acid) and RNA

(ribonucleic acid). The difference between the two lies in whether the pentose is

2-deoxyribose (in this case, DNA) or ribose (in this case, RNA). There is also a

base-level difference between DNA and RNA: A, C and G are common, but T

is found only in DNA and U is found only in RNA (Fig. 2-5).

High-molecular Nucleic Acids

DNA is polydeoxyribonucleotide in which nucleotides are polymerized. 5’ and

3’ of 2-deoxyribose are joined by phosphodiester linkage (Fig. 2-6). High-

molecular RNA is required when genes are expressed, and as shown in Figure

2-6, high-molecular DNA and high-molecular RNA have very similar structures.

Both are long strings of molecules with the linkage of pentoses in a certain

direction (in Fig. 2-6, the upward part is the 5’ direction or the 5’ end, and the

downward part is the 3’ direction or the 3’ end).

From the specification of whether a high-molecular nucleic acid is DNA or RNA,

its structure can be simply expressed as a sequence of single letters (a base

(A)

(C)

Figure 2-2 Nucleic acids, bases and pentoses

(B)



Figure 2-3 Nucleosides Figure 2-4 Nucleotides


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sequence). By convention, the 5’ end is written on the left -hand side and the 3’

end on the right-hand side, unless otherwise specified.

DNA – a Double Strand

All high-molecular DNA found in nature (excluding that of viruses) is double-

stranded (Fig. 2-7). DNA takes a shape called B-form, and as shown in Figure

2-8, two bases – A and T – are linked by two hydrogen bonds while C and G

are linked by three hydrogen bonds. These respectively form base pairs, and

create a right-handed helix with a diameter of approximately 2 nm (Fig. 2-7).

Figure 2-6 Structure of high-molecular nucleic acids

Figure 2-5 Nucleotide types



Since a particular pairing rule exists, once the base sequence of one DNA strand

is known, that of the other strand is automatically determined; these two are

called complementary strands. The length of DNA is often expressed as the

number of base pairs (bp).

The directions of the two strands (5’ → 3’) run opposite to each other in an

orientation referred to as antiparallel. The “spiral stairs” formed by the bases are

not situated in the center of the helix structure; rather, they are slightly deviated

from the center, creating wider grooves (major grooves) and narrower grooves

(minor grooves) (Fig. 2-7). These grooves play important roles when proteins that

control the expression of genes recognize base sequences and attach to them.

RNA – a Single Strand

All high-molecular RNA found in nature (except that of viruses) is single-stranded. In

many cases, however, RNA is partially double-stranded due to the pairing of bases

within the strand. This structure is called A-form, and is characterized by a minimal

difference between wider and narrower grooves. Like DNA-DNA and RNA-RNA

pairings, DNA and RNA form pairs by creating antiparallel double strands.

Figure 2-7 Double-stranded structure of DNA (B-form)

Figure 2-8 Hydrogen bonds forming Watson-Crick base pairs


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Circular Strands for Prokaryotes and Linear Double Strands for Eukaryotes

Many prokaryotes have closed-circular double-stranded DNA. In other words,

their DNA has no ends. Its structure forms a twisted shape (Type I) (Fig. 2-9). Type

II is a form in which the twist of Type I is uncoiled as a result of nicking in the

DNA strands, but this form is rare in nature. On the other hand, all nuclear DNA

in eukaryotes is linear double-stranded (Type III) and has ends. This difference in

form between prokaryotes and eukaryotes is a key characteristic of DNA.

Denaturation and Renaturation of DNA

Double strands of DNA are separated into single strands in an alkali

environment with a pH value of 12 or more or by heating to 90˚C or higher.

This is called the denaturation of DNA. The phenomenon of proteins losing

their higher-order structure is also called denaturation; however, organic

media and acids that denature proteins precipitate but do not denature

DNA. The transformation of single-stranded DNA back to double strands is

called renaturation or annealing. During this process, base pairs are formed

between two single strands. Annealing between heterogeneous DNA, or

between DNA and RNA, is called hybridization. These techniques are

frequently used in genetic engineering.

Column

Figure 2-9 Circular and straight-chain structures of DNA



DNA is the thread of life. E. coli has circular double-stranded DNA with an

approximate length of 2 mm. A human somatic cell contains linear double-

stranded DNA of approximately 2 m, consisting of 6 x 109 base pairs. To

help visualize this, if they were magnified to 500,000 times, the diameter

of the DNA would be 1 mm and its length would be 1,000 km. Since DNA

in human somatic cells is distributed to 46 threads, the length per thread

would be approximately 22 km. This 1,000-km DNA would be housed in

a nucleus with a diameter of 5 m. The number of DNA threads is doubled

to 96 before cell division, and these are distributed equally to the two

daughter cells without fail.

I I I . Genes and DNA

Definitions of Genes

It is often said that genes are DNA, or conversely, that DNA consists of genes;

however, genes are not spread throughout high-molecular DNA from one end to

the other. A gene is defined as a region of high-molecular DNA containing

information that determines the primary structure of proteins (amino acid sequence)

or the structure of non-coding RNA (base sequence) (explained in Chapter 3).

Generally in prokaryotes, genes are densely located with very narrow intervals

between them. In eukaryotes, they are sparsely located throughout the DNA with

wide intervals between them.

Genomes

The overall DNA contained in one cell is called a genome. In prokaryotes, cells

contain one thread (i.e., one molecule) of DNA, whereas human somatic cells

have 46 threads of DNA per cell (i.e., per nucleus), of which 23 are derived

from the mother (the ovum) and 23 from father (the sperm). The somatic cells of

eukaryotes commonly have two sets of genes derived from both parents, and

these cells are called diploids. Cells that have only one set of genes are called

Column DNA — a Long, Thin Thread


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haploids. Most prokaryotes are haploids, as are the germ cells of eukaryotes.

The whole DNA of diploid cells is called a genome; however, in some cases,

from a functional viewpoint, the DNA of haploid cells is called a genome and

that of diploid cells is referred to as two sets (copies) of the genome.

DNA Content of Organisms

DNA content greatly varies among organisms. Figure 2-10 shows the amount of

DNA per haploid in several organisms. Generally, DNA content per cell is larger

in eukaryotes than in prokaryotes. Human somatic cells contain approximately

1,000 times as much DNA as those of E. coli (per haploid). For diploid cells, the

amount per cell is 6 pg. Generally among eukaryotes, the higher an organism,

the more DNA content there is, although there can be great variations among

organisms of the same group. Among vertebrates, such variations can be very

distinctive in fish and amphibians, with some species having more DNA content

than humans. Some higher plant species also have more DNA than humans, so

it does not necessarily hold true that higher DNA content represents a higher

organism. In other words, humans are not the highest species with the largest

amount of DNA.

Figure 2-10 Distribution of DNA content per cell



The Number of Genes in Organisms

The Human Genome Project, which aims to determine the sequence of all the

chemical base pairs in human DNA, is now almost complete, and the genome

sequences of many other organisms are also being increasingly identified.

Contrary to predictions, the number of genes in humans is now estimated to be

only six times as many as that in E. coli (approx. 26,000 in humans and 4,300

in E. coli ). The number of genes also does not differ greatly among fruit flies,

Arabidopsis thaliana and humans.

Despite the less-than-significant difference in the number of genes between

humans and E. coli, in eukaryotes (including humans), one gene can synthesize

multiple types of protein with different amino acid sequences, and the number of

protein types produced in humans is estimated to be around 100,000. This

mechanism is discussed later (Chapter 3).

Eukaryotes – Characterized by Many Non-gene DNA Regions

The DNA of eukaryotes has a much larger proportion of regions that are not

genes (amino-acid-coding sequences) than prokaryotes. As an example, humans

have a much higher DNA content than E. coli, but have only slightly more genes.

As shown in Figure 2-11, in mammals, only 3% of the whole DNA sequence

codes for amino acids. One of the characteristics of eukaryotic DNA is that –

unlike DNA in prokaryotes – it has a large number of repetitive sequences, which

represent over half the total DNA in some species. Short repetitive sequences

may be located at the same sites or be scattered throughout the genome, and

lit t le is known about the meaning and function of their existence.

Only a small part of the gene that determines the structure of a protein in

eukaryotes has an amino-acid-coding sequence for protein synthesis. Figure 2-12

shows a schematic diagram of a eukaryotic gene. Introns do not contain an

amino-acid-coding sequence. Based on the classical definition, therefore, introns

are not genes, but by convention in eukaryotes, genes include introns and exons.

Some genes have introns that are 10 to 100 times as long as exons.

In prokaryotes, regions that regulate gene expression are short (from tens of bp

to a hundred bp), whereas in eukaryotes they are much longer (dozens of kbp).

This is another major difference between prokaryotes and eukaryotes.


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IV. Replication of DNA

Outline of DNA Replication

The replication of DNA involves the production of high-molecular DNA by

polymerizing deoxyribonucleotides, which are units of DNA.

Generally, this process is expressed as follows:

[dNMP]n + dNTP → [dNMP]n+1 + PPi

(PPi: pyrophosphate)

dNMP – a compound generated as a result of the detachment of pyrophosphate*1

from dNTP – is added to the 3’-OH of [dNMP]n. This indicates that DNA synthesis

occurs in the direction from 5’ to 3’. This is also the case for RNA synthesis, and high-

molecular nucleic acids are always synthesized from 5’ toward 3’.

Deoxyribonucleotides are linked by DNA polymerase. In E. coli, DNA polymerase I,

II, III, etc. are known, and III is the main replicative enzyme. In mammals, several types,

including α, β, γ, δ and ε, have been identified. Of these, α, δ and ε are the

main replicative enzymes, while the others serve mainly to repair damage to DNA.

*1

Figure 2-11 What types of DNA do mammals have? Figure 2-12 Exons and introns



DNA is continually subjected to damage. There are many natural or artificial

chemicals that bond with DNA bases, form base-base bonds, or cut DNA

strands. Radiation such as ultraviolet rays and cosmic rays also modifies

bases or cuts strands. DNA faces such threats from the birth of the organism

that carries it, and all organisms are equipped with a range of functions to

detect and repair DNA damage. As an example, damage to bases caused

by ultraviolet rays or certain chemicals is fixed by a mechanism called

excision repair, in which the damaged sites (including the surrounding

regions) are cut out and removed, and the resulting gaps are filled with

DNA polymerase (Column Fig. 2-1). Defects in the genes coding for the

enzymes involved in this mechanism result in a hereditary disease called

xeroderma pigmentosum, which makes sufferers prone to developing cancer.

Many other hereditary diseases associated with repair-enzyme defects are

known; these often cause cancer and, in rare cases, accelerated senescence

(progeria). In short, many repair-enzyme systems are in place to continually

repair gene damage, thus minimizing the accumulation of defects.

The Need for a Template in Replication

During the process of replication, the original double strands are unwound, and

new nucleotides are added to each single strand in a way that forms base pairs

(Fig. 2-13). This diagram shows the DNA double-helix structure published in

1953 by James Watson and Francis Crick, which suggested the possibility that

DNA was replicated using templates. In fact, during the replication process, base

pairs (C-G and A-T) are formed using each original strand as a template. As a

result, once the process is complete, two new double strands of DNA with the

same base sequence as the original are created. One strand of the new double

strand is the original template (i.e., the parent strand), and the other is a new

strand (i.e., the daughter strand). This method of replication is called

semiconservative replication. While many high molecules such as proteins and

sugar chains exist in the living body, this template-based semiconservative

synthetic method is unique to DNA.

Column DNA Damage and Repair

Column Figure 2-1 Excision repair of DNA damages


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Accuracy of Replication

DNA replication must be accurate. If DNA were synthesized using

nucleotides that do not follow the pairing rule, changes in the sequence

would occur in the newly created strand to form what is known as a mutation.

A mutation that occurs within the region of an important gene, if the effect is

significant, results in the death of the cell. Alternatively, in some cases, the

cell may become cancerous. In humans, one cell contains 6 x 109 base

pairs, and the division of 1011 – 1012 cells is thought to occur each day.

When DNA polymerase extends a new strand, the possibility of its inserting

incorrect nucleotides is said to be 10-6 – 10-4. DNA polymerase has a

proofreading function by which incorrectly inserted nucleotides are removed

and replaced with the correct ones. Additionally, errors missed by the

proofreading function are detected and replaced with the correct nucleotides

by a mismatch repair mechanism. There are multiple mismatch repair

systems, and the final frequency of errors is in the range of 10-11 – 10-10. To

artificially build a reaction system that achieves such low error rates is

difficult, even in the field of precision engineering.

Column

Figure 2-13 Semiconservative replication using a template



Replication – a Discontinuous Process

Double strands of DNA always run in opposite directions. This is the case for

completed DNA as well as for DNA during the process of replication. When

DNA synthesis is considered based on the Watson-Crick model (Fig. 2-14), one

of the daughter strands must be synthesized in the 3’→5’ direction. However,

DNA polymerase always synthesizes in the 5’→3’ direction. Let’s look at this in

more detail.

During the synthesis of the daughter strands following the uncoiling of the parent

strand, three double strands of DNA appear in a structure called the replication

fork (Fig. 2-14). Looking closely at this fork structure, at the point where DNA

synthesis occurs (i.e., the replication point), one of the daughter strands (i.e., the

leading strand) is synthesized in the same direction as that in which the replication

fork runs. The other daughter strand (i.e., the lagging strand) is synthesized in the

direction opposite to that of the replication fork, because DNA is synthesized in

the 5’→3’ direction (Fig. 2-14). Along the lagging strand, short DNA fragments

of approximately 100 nucleotides are continually synthesized, and are

Figure 2-14 Discontinuous replication of the lagging strand


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subsequently linked with each other. These short strands are called Okazaki

fragments after Reiji Okazaki, the molecular biologist who discovered them. This

type of replication is called discontinuous replication.

DNA polymerase follows the reaction below:

[dNMP]n + dNTP → [dNMP]n+1 + PPi

However, this reaction does not occur when n = 1. At least a fragment of two or

more nucleotides (called a primer) is needed so that new nucleotides can be

added there. On the other hand, RNA polymerase can synthesize RNA from n = 1

using DNA as a template. RNA primers are synthesized by RNA polymerase

prior to DNA synthesis, and DNA synthesis starts from there using DNA polymerase

(Fig. 2-15). This mechanism was also discovered by Okazaki et al.

DNA synthesis proceeds along the lagging strand – removing RNA primers that

have performed their roles on the way – and the gaps between the short DNA

fragments are subsequently bonded by DNA ligase*2.

Figure 2-15 DNA synthesis – the need for RNA primers

*2 DNA ligase: An enzyme that links together breaks between 3’-OH and 5’-phosphate on one of the double strands of DNA. It cannot link a break if even one base is missing.



In fact, replication reactions are even more complicated than replication itself (Column

Fig. 2-2). At the tip of the replication fork, helicase unwinds the parental double

strand. There are single-strand binding proteins that stabilize the single strands

exposed by the helicase. RNA primers are synthesized by primase, and DNA

polymerase extends the primers to form new DNA strands. Along the lagging strand,

as previously mentioned, DNA is synthesized while RNA primers are being removed,

and DNA ligase subsequently joins the deoxyribonucleotides together. Ahead of the

replication point of the fork, topoisomerase (DNA gyrase) cuts the DNA strand to

release the tension held by the parental strand and links it again. Various enzymes

and proteins with such a function form a large replication complex; similar mechanisms

are essentially at work in organisms from bacteria through to humans.

Replication Origin and Endpoint

In prokaryotes, the long DNA strand has a single replication origin from which replication

forks proceed in both directions. Since prokaryotic DNA is circular, the two replication

points meet at the opposite side of the circle at a location known as the replication

endpoint. The replication origin and endpoint have characteristic base sequences, and

Column The Many Enzymes Involved in DNA Replication

Column Figure 2-2 Overview of DNA replication


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particular proteins lead the start and the end of DNA synthesis. A DNA unit that replicates

from a single origin of replication is called a replicon. Prokaryotic DNA consists of one

replicon, and in E. coli the replication of the replicon takes approximately 40 minutes.

Eukaryotes have more DNA content than prokaryotes, and have multiple replication

origins on their DNA. In other words, eukaryotic DNA consists of multireplicons. In this

case, replication forks run in both directions from each replication origin, and the

replication of each replicon takes approximately one hour.

PCR is a technique by which a particular fragment of DNA with a known base

sequence is amplified in tubes. Template DNA, DNA primers (fragments of 10 – 20

nucleotides are chemically synthesized in advance) and DNA polymerase are needed

for this process. As shown in Column Figure 2-3, a particular segment of DNA can

be amplified without limit for analysis. In theory, DNA can be amplified even from a

single cell. Fragmented DNA can also be amplified as long as the target segment is

not fragmented. RNA can also be used as a template, and PCR has a wide range of

application areas such as criminal investigations and court evidence as well as gene

cloning and the amplification and cloning of particular DNA segments.

Column PCR (Polymerase Chain Reaction)

Column Figure 2-3 Schematic diagram of PCR



• All organisms have high-molecular DNA that carries genetic information. The use of this

information involves high-molecular RNA.

• DNA and RNA are both organic compounds called nucleic acids (nucleotides).

• A nucleotide (a structural unit of DNA and RNA) consists of three components – a base, a

pentose and a phosphate. High-molecular DNA and RNA are polynucleotides composed of

nucleotides.

• DNA consists of four base types – adenine (A), cytosine (C), guanine (G) and thymine (T) – and

RNA consists of four base types – adenine (A), cytosine (C), guanine (G) and uracil (U).

• The pentose that constitutes DNA is 2-deoxyribose, while that in RNA is ribose.

• In high-molecular DNA, two long polynucleotide strands form a right-handed double helix.

• Bases are connected between the two strands of high-molecular DNA by a hydrogen bond,

creating base pairs. These are formed between adenine (A) and thymine (T) and between

cytosine (C) and guanine (G).

• Although high-molecular RNA is single-stranded, partial double strands are often formed within

the molecule.

• The structure of high-molecular nucleic acids has a particular nucleotide order characterized by

its base sequence. This is called the base sequence of nucleic acids.

• Although one human cell contains approximately 1,000 times as much DNA as E. coli, humans

have approximately 26,000 genes – only six times as many as E. coli ’s 4,300.

• Each DNA molecule in a cell has a unique base sequence.

• When a cell multiplies, a DNA molecule identical to that of the parent cell is replicated, and the

two molecules are equally distributed to the two daughter cells.

• DNA synthesis is also called DNA replication because genetic information is replicated during

the process.

• When DNA is replicated using the parent strand as a template, four nucleotide types are

individually added to form base pairs, thus synthesizing daughter strands with a sequence

complimentary to that of the parent strand. This method is called semiconservative replication.

• Newly synthesized double-stranded DNA consists of one parent and one daughter strand.

• Although the main agent in the DNA replication reaction is DNA polymerase, the reaction is

complex and involves many types of enzymes and proteins.

Summary Chapter 2


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[1] 1) Draw the structural formulas for DNA and RNA, and point

out the structural differences between the two.

2) DNA is more stable than RNA. Explain the reasons for this

in terms of the structural differences between them.

3) Explain the implications of DNA being more stable than

RNA for organisms.

[2] The DNA sequence that constitutes human chromosomes has

3 billion base pairs, of which 40 million are within exons and

1.1 billion are within introns.

*Answer 1) – 3) to two significant digits.

1) Calculate the percentage of all chromosomes expressed

as mature mRNA.

2) The total number of genes in human chromosomes is

taken as 25,000 here. Assuming that one gene contains

8.8 exons, calculate the average DNA sequence length

per exon.

3) As with 2), assuming that one gene contains 7.8 introns,

calculate the average DNA sequence length per intron.

[3] Respond to the following tasks on DNA replication:

1) DNA is a huge molecule with a molecular weight of 1010

– 1011, and during the process of cell growth must

replicate a molecule that has a structure identical to its

own. Describe the characteristics of the DNA replication

mechanism that are not found in the synthesis of polymers

such as proteins and polysaccharides in terms of their

relationship with DNA structure.

2) E. coli cultured for many hours in a medium containing 15N as a nitrogen source was transferred to a medium

containing normal nitrogen (14N) and divided three times.

Double-stranded DNA was then extracted from the E.

coli, and the specific gravity (buoyant density) was

measured using equilibrium density gradient centrifugation

in cesium chloride.

i) What is the abundance ratio of DNA with heavy, light and

medium specific gravity in E. coli cultured in a medium

containing 15N as a nitrogen source?

ii) What is the abundance ratio of DNA with heavy, light and

medium specific gravity in E. coli transferred to a medium

containing normal nitrogen (14N) and divided once?

iii) What is the abundance ratio of DNA with heavy, light and

medium specific gravity in E. coli transferred to a medium

containing normal nitrogen (14N) and divided three times?

[4] Consider whether the following statements are true or false

and explain your decision:

1) Many DNA cleavage enzymes – called restriction enzymes

– recognize a palindromic sequence, that is, the sequence

on one strand reads the same in the reverse direction on

the complementary strand (e.g., GAATTC/CTTAAG) and

cut it.

2) Restriction enzymes recognize a specific sequence and cut

it; the cut sequences are always located within the amino-

acid-coding regions of genes.

3) S ome viruses have reverse transcriptase, which transcribes

RNA into DNA.

4) DNA reverse-transcribed from mRNA by reverse

transcriptase contains promoter sequences.

5) Even if genome DNA extracted from a cell contains the

coding regions of a gene, DNA reverse-transcribed from

mRNA extracted from the same cell (complementary DNA

or cDNA) does not necessarily contain the gene.

[5] With regard to a PCR reaction using one molecule of

genome DNA as a template, list the types of molecules

generated as the reaction cycles proceed and the number of

molecules for each type using the number of reaction cycles

(N). Also, calculate the number of molecules for each type

when N is 10, and predict the main molecule when N

becomes large.

Problems

(Answers on p.251)

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