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2.6Problems

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.

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.

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?

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)

Some 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.

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.

1)

Draw the structural formulas for DNA and RNA, and point out the structural differences between the two.

See Figs. 2-2 to 2-6. The difference between RNA and DNA is the presence/absence of the hydroxy group at the 2’ position of a pentose.

2)

DNA is more stable than RNA. Explain the reasons for this in terms of the structural differences between them.

As mentioned above, the difference between RNA and DNA is the presence/absence of the hydroxy group at the 2’ position of a pentose. This difference results in a difference in affinity to water molecules, or in other words, a difference in sensitivity to hydrolysis. RNA, which has the hydroxy group, is more highly subject to hydrolysis.

3)

Explain the implications of DNA being more stable than RNA for organisms.

Since DNA retains genetic information, it is very important that the molecules are stable.

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.

The total number of bases in exons is divided by the total number of bases in the genome. The answer is 1.3%.

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.

The total number of bases in exons is divided by the number of genes, and is further divided by 8.8. The answer is 180 bases.

3)

As with 2), assuming that one gene contains 7.8 introns, calculate the average DNA sequence length per intron.

Calculate in accordance with number 2 above. The answer is 5,600 bases.

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.

DNA exists as two strands held together by the formation of complementary base pairs, and performs semi-conservative replication using a template. At the origin of a replication, double-stranded DNA (which will serve as a template) is partially loosened to form single strands, and complementary (daughter) strands are synthesized by adding nucleotides complementary to those of the two template strands (Ts for As and Cs for Gs, and vice versa), thus producing double-stranded DNA chains made of parent and daughter chains.

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?

Heavy DNA : Light DNA : Intermediate DNA

i) 1 : 0 : 0 (only heavy DNA is detected.)

ii) 0 : 0: 1

iii) 0 : 6 : 2 or 0 : 3 : 1

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.

Correct. Although not dealt with in this book, restrictive enzymes are essential in life science research. A range of information has been published elsewhere on the subject.

2)

Restriction enzymes recognize a specific sequence and cut it; the cut sequences are always located within the amino-acid-coding regions of genes.

Incorrect. Cut sequences occur in DNA at a certain frequency irrespective of gene regions.

3)

Some viruses have reverse transcriptase, which transcribes RNA into DNA.

Correct. The winners of the Nobel Prize for Physiology or Medicine in 1975 included H. Temin and D. Baltimore, who discovered reverse transcriptase.

4)

DNA reverse-transcribed from mRNA by reverse transcriptase contains promoter sequences.

Incorrect. mRNA transcription starts downstream of the promoter sequence. The mRNA transcribed does not therefore contain the promoter sequence.

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.

Correct. Apart from a few exceptions, most cells contain the complete sequence of the organism’s genome. However, since the genes expressed differ by tissue, organ and time, cDNA include the genes that happen to be expressed, and does not include those that are not expressed.

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.

The molecule type that only includes the region between two primers, the type that has been elongated from one primer (Column Fig. 2-3: the inside molecules in (3) in the second cycle) and the type with a template DNA strand (Column Fig. 2-3: the outside molecules in (3) in the second cycle). The numbers of molecules are 2N - 2N, 2(n - 1) and 2, respectively. After 10 cycles, they are 1,004, 18 and 2, meaning that the inter-primer molecules dominate.

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