graduate school of frontier sciences, the university of

英語版 English version

東京大学大学院新領域創成科学研究科 Graduate School of Frontier Sciences, The University of Tokyo

メディカル情報生命専攻 Department of Computational Biology and Medical Sciences

平成 29（2017）年度 2017 School Year

大学院入学試験問題 Graduate School Entrance Examination Question Booklet

専門科目 Specialties

平成 28 年 8 月 2 日（火） Monday, August 2, 2016

9：30～11：30

注意事項 Instructions

１．試験開始の合図があるまで、この冊子を開いてはいけません。 Do not open this booklet until the start of examination is announced.

２．本冊子の総ページ数は 39 ページです。落丁、乱丁、印刷不鮮明な箇所などがあった場合には申し出ること。 This booklet consists of 39 pages. If you find missing, misplaced, and/or unclearly printed pages, notify it to the staff.

３．解答には必ず黒色鉛筆（または黒色シャープペンシル）を使用しなさい。 Only black pencils (or mechanical pencils) are allowed to answer the questions.

４．問題は 12題出題されます。問題 1～12から選択した合計４問に解答しなさい。ただし、問題 1～12 は同配点です。 There are 12 exam questions (Question 1 to 12). Answer 4 questions out of the 12 questions. Note that Question 1 to 12 are equally weighted.

５．解答用紙は計４枚配られます。各問題に必ず１枚の解答用紙を使用しなさい。解答用紙に書ききれない場合は、裏面にわたってもよい。 You are given 4 answer sheets. You must use one answer sheet for each question. You may continue to write your answer on the back of the answer sheet if you cannot conclude it on the front. But you must not proceed to write on the second sheet.

６．解答は日本語または英語で記入しなさい。 Answers should be given in Japanese or in English.

７．解答用紙の指定された箇所に、受験番号と選択した問題番号を記入しなさい。問題冊子にも受験番号を記入しなさい。 Fill the designated blanks at the top of each answer sheet with your examinee number and the question number you are to answer. Fill the designated blanks at the top of this page with your examinee number.

８．草稿用紙は本冊子から切り離さないこと。 The blank pages are provided for making draft. Do not detach them from this booklet.

９．解答に関係ない記号、符号などを記入した答案は無効とします。 An answer sheet is regarded as invalid if you write marks and/or symbols unrelated to the answer on it.

10．解答できない場合でも、解答用紙すべてに受験番号を記入して提出しなさい。 Turn in the answer sheet with your examinee number, even if you cannot solve the question.

11．解答用紙・問題冊子は持ち帰ってはいけません。 Do not take the answer sheets and this booklet out of the examination room.

受験番号 Examinee number

1

Problem 1 A. Read the following questions (1)-(10), and choose the single most appropriate

answer from A – E. (1) Which amino acids do not have a chiral carbon atom?

A. Ala B. Ile C. Gly D. Met E. Pro

(2) Which combination of amino acids has the maximum absorption spectrum of

ultraviolet light at 280 nm?

A. Leu, Ile, Val B. Trp, Pro, Gly C. Trp, Tyr, Phe D. Asp, Glu, Lys E. Tyr, Phe, Thr

(3) What is the isoelectric point (pI) of the tripeptide alanyl-glutamyl-glycine (NH2-Ala-Glu-Gly-COOH)?

A. approximately 1.9 B. approximately 3.3 C. approximately 6.0 D. approximately 7.4 E. approximately 9.7

(4) Which of the following amino acid combinations could be the targets for phosphorylation?

A. Asn, Ser, Thr B. Cys, Met, Ser C. Arg, Lys, His D. Tyr, Trp, Phe E. Ser, Tyr, Thr

(5) A given nuclease protein has eight cysteine residues forming four disulfide bonds within the molecule. If all of the disulfide bonds are once reduced to thiol groups and then reoxidized to form four disulfide bonds, how many different combinations of disulfide bonds are possible inside the molecule? A. up to 16 combinations B. up to 64 combinations C. up to 105 combinations D. up to 192 combinations E. up to 336 combinations

2

(6) Which pair of methods is inappropriate for protein analysis? A. Circular dichroism and nuclear magnetic resonance B. Ion-exchange chromatography and poly-acrylamide gel electrophoresis C. Mass spectrometry and small angle X-ray scattering D. Western blotting and immunoprecipitation E. Southern blotting and flow cytometry

(7) Which of the following descriptions of the protein is incorrect?

A. Hemoglobin forms a tetramer. B. Lysozyme unfolds in 8.0 M urea. C. Actin and keratin are component proteins of the cytoskeleton. D. Pepsin is an enzyme that digests carbohydrates. E. Antibody is a protein called immunoglobulin.

(8) Which functional group does not exist in a typical natural protein? A. Isopropyl group B. Methoxy group C. Sulfhydryl group D. Guanidino group E. Aryl group

(9) Shown below are short descriptions of the relationship between the substrate

concentration and the reaction rate in the Michaelis-Menten equation. Which one is incorrect? A. When the substrate concentration is 1/9 of Km, the reaction rate is equal to 0.1

times of Vmax. B. When the substrate concentration is 1/4 of Km, the reaction rate is equal to 0.25

times of Vmax. C. When the substrate concentration is equal to Km, the reaction rate is equal to 1/2

of Vmax. D. When the substrate concentration is 9 times of Km, the reaction rate is equal to

0.9 times of Vmax. E. When the substrate concentration is infinite, the reaction rate is equal to Vmax.

(10) Which description of the melting temperature of DNA is incorrect? A. The strong absorbance of ultraviolet light by DNA arises from the bases in

DNA. B. DNA has a strong absorbance of ultraviolet at 260 nm. C. As the temperature of DNA solution is raised, the absorbance at 260 nm of the

DNA solution decreases. D. The GC content of DNA is higher, the melting temperature of the DNA is

higher. E. The melting temperature of double-stranded DNA in the presence of 0.2 M

NaCl is higher than that in the absence of NaCl.

(Problem 1: Continued on the following page.)

3

B. Read the following, and answer the questions (1)-(6). A given protein (protein A) was treated with trypsin to generate a series of fragments of the following sequences:

Gly-Gly-Ile-Arg Ser-Phe-Leu-Gly Trp-Ala-Ala-Pro-Lys Ala-Glu-Glu-Gly-Leu-Arg

And the same protein was treated with chymotrypsin to generate the following fragments:

Leu-Gly Ala-Glu-Glu-Gly-Leu-Arg-Trp Ala-Ala-Pro-Lys-Gly-Gly-Ile-Arg-Ser-Phe

(1) Which is the most appropriate for the analysis of peptide fragments?

A. 1H-NMR B. ELISA C. RT-PCR D. MALDI-TOF MS/MS E. SDS-PAGE

(2) Deduce the amino acid sequence of the protein A from the experimental results.

(3) Calculate the theoretical molecular weight of the protein A using the table below. Cut off after the decimal point.

amino acid molecular weight amino acid molecular weight Alanine 89.0 Leucine 131.2 Arginine 174.2 Lysine 146.2 Asparagine 132.1 Methionine 149.2 Aspartate 133.1 Phenylalanine 165.2 Cysteine 121.2 Proline 115.1 Glutamine 146.2 Serine 105.1 Glutamate 147.1 Threonine 119.1 Glycine 75.1 Tryptophan 204.2 Histidine 155.2 Tyrosine 181.2 Isoleucine 131.2 Valine 117.2

(4) The absorbance of the protein A solution in a cuvette (light path length: 0.50 cm),

was measured to be 2.85. Determine the mass w (mg) of the protein A dissolved in 10 mL of the solution. Answer the value with two significant digits (round the third digit). Molar absorption coefficient ε280 of protein A is 5700 (M-1･cm-1).

4

(5) The apparent molecular weight of the protein A estimated from the gel filtration chromatography was proved to be twice the theoretical value in (3). Why the apparent molecular weight is different from the theoretical molecular weight? Write a probable reason in less than 20 words.


5

C. Read the following, and answer the questions (1)-(3). A given protein (protein-B) binds a short RNA (RNA-X) with high specificity, and there is a single binding site in the protein-B for the RNA-X.

(1) Fill the following blanks, (A) ~ (C), with appropriate equations or words. The dissociation constant (Kd) for the protein-B binding to the RNA-X to form the protein-B: RNA-X complex is expressed as follows;

Kd = ( A )

where [P]: concentration of free protein-B [R]: concentration of free RNA-X [P-R]: concentration of the protein-B: RNA-X complex

In general, the smaller Kd values imply ( B ) binding.

Total RNA-X concentration [R0] is equal to [R] + [P-R], thus, fraction of the RNA-X bound to the protein-B, [P-R] / [R0], can be converted to the following equation, using Kd and [P]

Fraction bound = [P-R] / [R0 ] = ( C )

(2) Fill appropriate values from following (a) ~ (j) in the blanks, (D) ～ (F). When free protein-B concentration [P] is equal to Kd value, the fraction of the RNA-X bound to the protein-B is ( D ) %. When the [P]s are 10 folds Kd and 2 folds Kd, the fractions of RNA-X bound to protein-B are ( E ) % and ( F ) %, respectively.

a. 100 b. 91 c. 83 d. 77 e. 67

f. 50 g. 33 h. 17 i. 9 j. 0

6

(3) Answer the following questions, (G) and (H) RNA-X (final concentration: 10 -11 M) and various amounts of the protein-B (final concentrations: 1 x 10 -9 ～ 4 x 10 -6 M) were mixed, and the fraction of the RNA-X

bound to protein-B were determined (● in graph). Binding of the mutant RNA

(RNA-Y) to the protein-B was also analyzed (▲ in graph).

(G) Estimate the Kd value of the protein-B for the RNA-X.

Kd = ( G )

RNA-XX is an RNA in which two RNA-Xs are linked. Then, the Kd value of the

protein-B for the RNA-XX was analyzed. The overall dissociation constant, K2, can

be expressed as follows

K2 = [P]2 [ R] / [P2-R]

where,

[P]: concentration of free protein-B

[R]: concentration of free RNA-XX

[P2-R]: concentration of the protein-B: RNA-XX complex

7

The overall dissociation constant, K2, of the protein-B and the RNA-XX was

estimated to be 6 x 10-16. The Kd values of the protein-B for the RNA-XY or the

RNA-YX (in which one of the tandem RNA-X sequences in RNA-XX is replaced

with that of the RNA-Y, respectively) are comparable to that of the protein-B for

the RNA-X.

(H) Is the protein-B binding to the RNA-XX cooperative or non-cooperative binding to

two sites in RNA-XX? Answer this question, and describe the reason (within 70

words).

(End of Problem 1)

8

(Blank page for draft)

9

Problem 2 A. Read the following questions (1) - (5), and choose ONE most appropriate

answer from A to D. For each question, write your answer as “(1)-A” in the answer sheet. Choose TWO most adequate terms from the list below for the following questions (6) – (10), write your answer as “(6)-C,D.”

(1) Which of the following statements is correct about replication of genomes?

A. Okazaki fragment is a short RNA, which plays a role in starting the elongation of DNA at the replication fork.

B. Newly synthesized strand and template strand can be distinguished by the existence of methylation sites or the nicks present in the lagging strand.

C. Under optimal growth condition, an Escherichia coli cell divides in every 20 to 30 min, which is approximately twice that for the time required for genomic replication.

D. There is only one replication initiation site for both bacterial and eukaryotic chromosomes.

(2) Which of the following statements is false about topoisomerase in bacteria?

A. Topoisomerase catalyzes the interconversion among A-DNA, B-DNA, and Z-DNA.

B. Topoisomerase I converts supercoiled DNA to relaxed DNA. This reaction does not require ATP.

C. Topoisomerase II is called DNA gyrase, which negatively supercoils DNA in an ATP-dependent manner.

D. During the topoisomerase reaction, there is no insertion or deletion of base sequences.

(3) Which of the following statements is correct about RNA polymerase in bacteria?

A. Sigma subunit determines the specificity in the promoter binding site. B. Bacterial RNA polymerase binds to the 10 to 35 bases upstream from the

translational initiation site in the promoter region. C. Bacterial RNA polymerase releases the messenger RNA upon recognition of

the translational termination codon. D. Kanamycin and hygromycin are the antibiotics that inhibit bacterial RNA

polymerases.

10

(4) Which of the following statements is false about translation?

A. Ribosomes comprise the large and the small subunits in both bacteria and eukaryotes.

B. In bacteria, transcriptional and translational processes are coupled and translation starts before completion of transcription.

C. Translational speed in bacteria is, in general, faster than that in eukaryotes. D. Peptidyl transferase reaction is catalyzed by the side chain of amino acid

residues contained in the large subunit. (5) Which of the following statements is false about bacteria?

A. Gram-negative bacteria contain periplasmic space between the inner and outer membranes.

B. All the proteins localize in periplasmic space carry a signal peptide. C. Lysozyme is a kind of peptidase, which hydrolyzes bacterial cell wall,

peptidoglycan. D. Beta-lactam antibiotics, which are represented by penicillin and ampicillin,

inhibit the biosynthesis of peptidoglycan. (6) Select two enzymes required for replication of circular DNA genomes.

A. Primase B. Helicase C. Protease D. Telomerase

(7) Select two biological events giving rise to diversity of antibodies.

A. Anergy B. Somatic hypermutation C. Genetic recombination D. MHC (Major histocompatibility complex) restriction

(8) Select two regulatory elements required for the transcriptional regulation of

tryptophan synthetase genes in E. coli.

A. Promoter B. Operator C. Silencer D. Enhancer

11

(9) Select two organelles where protein synthesis from mRNAs takes place.

A. Mitochondria B. Golgi apparatus C. Smooth (smooth-surfaced) endoplasmic reticulum D. Rough (rough-surfaced) endoplasmic reticulum

(10) Select a pair of continuous membrane structures.

A. Nuclear membrane B. Endoplasmic reticulum membrane C. Lysosome membrane D. Mitochondria inner membrane


12

B. Following is an explanation for the DNA sequencing method developed by Dr. Frederick Sanger. Read the sentences and answer to the following questions (1) to (3).

In the Sanger DNA sequencing method, a single-stranded DNA is used as a template. A radiolabeled primer is annealed to the template and subjected to primer extension reaction using DNA polymerase. In the elongation reaction, dideoxynucleotides (ddNTPs) are added to the reaction as well as the standard deoxynucleotides (dNTPs). Because DNA polymerase is capable of incorporating ddNTPs instead of dNTPs at certain frequency, the elongation reaction ceases upon incorporation of ddNTPs, generating fragments with various lengths depending on the incorporated ddNTP positions. In the experiment, four sets of reactions are prepared, each of which includes one of the ddNTPs (ddATP, ddGTP, ddCTP, or ddTTP) in addition to dNTPs. Then, each of the reaction mixtures is separated by polyacrylamide gel electrophoresis under denaturing conditions. The gel is exposed to an X-ray film. Now, the sequence information is inferred by reading the order of the ladder bands in the lanes on the film, each of which corresponds to a radiolabeled DNA fragment terminated at each base position by ddNTPs.

(1) Following is the experimental procedure to prepare a radiolabeled oligonucleotide

primer. Select appropriate words for each bracket from the word groups.

Oligonucleotide, (A) that is 32P-radiolabelled at the (B) phosphate group, and (C) are mixed in the reaction buffer. The (D) radiolabeled oligonucleotide is then be purified by using (E). A: ATP; AMP; NAD B: alpha; beta; gamma C: polynucleotide kinase; DNA ligase; terminal transferase D: 5’-end; 3’-end E: gel filtration; agarose gel electrophoresis

(2) Describe one of the procedures to prepare a single stranded DNA template. (3) Explain the reason why the denaturing agent is added to the polyacrylamide gel

electrophoresis to separate DNA fragments.


13

C. Read the following sentence and answer questions in (1)-(3). Eukaryotic mRNAs are processed from pre-mRNAs, which are newly transcribed from chromosomal DNAs via (A) to exclude introns, and also via modification of 5’ and 3’ termini with (B) and (C), respectively. For excision of any given intron via (A), （a）one nucleotide residue in the intron and two nucleotide sequences around exon-intron boundaries are required in general, and these three sequences are recognized by (D) such as U1 and U2. U1 forms base pairs with the 5’ exon-intron boundary sequence and U2 forms base pairs with the intronic sequence, （b）which triggers subsequent events that eventually form a lariat structure to complete excision of the intron. (1) Fill the blanks (A) to (D) with the most appropriate words. (2) What is the (a) one nucleotide residue, which directly takes part in the chemical

reaction to form the structure in (b). Answer a letter for (a). (3) The (a) one nucleotide residue plays a role in formation of a covalent bond that is

characteristic for a lariat structure as described in the underlined part (b). Describe characteristic nature of this covalent bond within two lines.

(End of Problem 2)

14


15

Problem 3 A. Read the following questions of (1)-(10), and choose the correct term(s),

number(s), or sentence(s) from A - E. Write the answers such as (1)-A, and (2)-B, C. The number of correct term(s), number(s), or sentence(s) is not always one.

(1) Choose the correct description(s) of human genome.

A. Human somatic cells carry diploid genome. B. The human haploid genome consists of approximately 30 trillion base pairs. C. Gene products functioning in mitochondria are coded on the mitochondrial

genome. D. Mitochondria and nucleus share common genetic codes. E. The expression profile of human genes is determined only by the nucleotide

sequence information of the genome.

(2) Choose the initiation codon(s) on mRNA in the universal genetic code.

A. 5'-UAA-3' B. 5'-AGU-3' C. 5'-AUG-3' D. 5'-CAG-3' E. 5'-UGA-3'

(3) Choose the stop codon(s) on mRNA in the universal genetic code.

A. 5'-UAA-3' B. 5'-AGU-3' C. 5'-AUG-3' D. 5'-CAG-3' E. 5'-UGA-3'

(4) Choose basic rule(s) or law(s) in genetics.

A. Law of dominance B. Murphy’s law C. Gauss’ law D. Hardy-Weinberg law E. Dalton’s law

(5) Choose the correct description(s) about SNP (single nucleotide polymorphism).

A. SNPs are found in healthy human population B. The number of human SNPs is about 24,000. C. Allele frequencies of SNP are almost the same between Japanese and

Caucasians. D. SNPs are used for the identification of individuals. E. SNPs are not associated with risks of diseases.

16

(6) What is the estimated frequency of heterozygote of a SNP with minor allele frequency of 5%?

A. 0.25% B. 4.75% C. 9.5% D. 90.25% E. 95%

(7) Blood type is determined by the type of protein modifications on the surface of red

blood cells. The modifications are regulated by variations in the ABO gene. What are the estimated frequencies of blood types O, A, B, and AB, when type A and type B alleles of the ABO gene are 40% and 20%, respectively? Choose the appropriate combination of the frequencies.

A. O type 16%, A type 40%, B type 20%, AB type 24% B. O type 16%, A type 48%, B type 20%, AB type 16% C. O type 32%, A type 40%, B type 20%, AB type 8% D. O type 48%, A type 20%, B type 10%, AB type 22% E. O type 72%, A type 16%, B type 4%, AB type 8%

(8) The risk of autosomal recessive diseases is increased in cousin marriage. Please

choose the relative risk of an autosomal recessive disease in cousin marriage compared with non-consanguineous marriage when the frequency of the disease is one in 10,000 births. Answer the closest relative risk when all carriers and patients are born normally and all homozygotes develop the disease.

A. 6.25-fold B. 12.5-fold C. 25-fold D. 50-fold E. 100-fold

(9) My aunt (my father’s sister) developed breast cancer, and a subsequent genetic test

identified a pathogenic germline mutation in BRCA1, a responsible gene for hereditary breast and ovary cancer, an autosomal dominant disease. Please calculate the probability that my daughter carries the mutation. Answer the closest probability, when other clinical or genetic information is not available and de novo mutation did not occur in the pedigree.

A. 0% B. 6.25% C. 12.5% D. 25% E. 50%

17

(10) The onset of an autosomal dominantly-inherited disease X starts from 30 years old, and linearly increases up to 100% at 80 years old. My grandmother of my mother’s side was diagnosed of this disease at 75 years old. My mother is healthy and does not show any symptoms or phenotypes of disease X at 50 years old. Calculate my risk of this disease at 80 years old, and choose the closest risk, when de novo mutation did not occur in the pedigree.

A. 0% B. 12.5% C. 14% D. 19% E. 25%


18

B. Read the following text related with gene therapy, and answer the questions 1 - 5.

Today’s hottest genome-engineering method, CRISPR/Cas9, has recently been

used to treat mouse models of the most common form of muscular dystrophy, Duchenne’s muscular dystrophy. Duchenne’s muscular dystrophy is a fatal X-Linked recessive muscle disease. The disease is caused by mutations in the gene encoding Dystrophin, a large intracellular protein. In the absence of Dystrophin, muscles degenerate, causing weakness and myopathy. Many therapeutic approaches for Duchenne’s muscular dystrophy have failed, at least in part because of the size of the Dystrophin protein and the necessity for lifelong restoration of Dystrophin expression in the myriad skeletal muscles of the body as well as the heart.

CRISPR/Cas9 is currently used for genome editing in various organisms. In the

presence of single guide RNAs (gRNAs), CRISPR-associated(Cas) nuclease (Cas9) is directed to specific sites in the genome, causing a double-strand break (DSB). Then, variable insertion-deletion mutations are created at the target site via non-homologous end joining (NHEJ).

In the current study, the authors delivered the required components of CRISPR/Cas9 to muscle using gene-therapy vectors based on the adeno-associated virus (AAV), an efficient gene-delivery vehicle for mouse muscle. The AAV vectors were delivered in the mdx mouse model of Duchenne’s muscular dystrophy, which

19

carries a nonsense mutation in exon 23 of Dystrophin. In the gene-correction strategy, the gRNAs guided the Cas9 protein to cut the two noncoding introns that flank exon 23 (Figure) (2). The cut ends were rejoined by the cell’s endogenous nonhomologous end-joining functions, permitting the reading frame of Dystrophin mRNA to remain intact. The initial efficiency of gene correction was estimated to be about 2%. But Dystrophin levels increased over time, perhaps because of continued correction, since the AAV constructs remain present in the muscle fibers for many months.

Obstacles to the CRISPR/Cas9 approach (3) include a lower efficiency compared with other approaches that have been proposed. Dystrophin expression was “rescued” in only a fraction of the treated cells. Although the accuracy of the CRISPR/Cas9 system continues to improve, there is a risk of off-target cutting at undesired genomic locations. Thus, additional work is required before the CRISPR system could be deemed safe enough for clinical application. Furthermore, immune responses against the AAV capsid, the Cas9 protein, and to Dystrophin itself represent potential hurdles (4). There are conceivable solutions for these problems, as well as alternative approaches that might avert them. The increasing number of approaches that could cure fatal disorders like Dystrophin would lead to eventual success.

(modified from New England Journal of Medicine 374;17 1684-1686) Question 1. Read the following text and choose the most appropriate word for ( 1 ) - ( 5 ) form A-P and answer as 1-A and 2-B. A Nonsense mutation in exon 23 of Dystrophin causes ( 1 ) in the open reading frame and results in the translation of abnormal protein. In addition, ( 2 ) causes mRNA degradation and subsequently reduces protein quantity. Because Dystrophin gene is located on chromosome X, Dystrophin mutation causes disease among ( 3 ), but Dystrophin mutation does not usually cause Duchenne’s muscular dystrophy among ( 4 ). When Duchenne’s muscular dystrophy is affecting 1/4000 of ( 3 ), the frequency of mutation carriers among ( 4 ) is one among ( 5 ), if disease penetrance is 100 %. A. frameshift, B. stop codon, C. amino acid substitution, D. alternative splicing, E. nonsense mediated decay, F. epigenetic silencing, G. micro RNA, H. reprograming, I. DNA methylation, J. histone code, K. 1000, L. 2000, M. 4000, N. 8000, O. male, P. female Question 2. Explain how CRIPSR/Cas9 system can improve the phenotype of muscular dystrophy in mdx mouse. Question 3. Describe the possible obstacles of the CRISPR/Cas9 approach in the treatment of human Duchenne’s muscular dystrophy other than immune response. Question 4. Explain how Dystrophin itself will induce immune responses after the treatment with CRISPR/Cas9 system.

20

Question 5. Give an example of potential application of CRISPR/Cas9 system other than gene therapy.

(End of Problem 3) (Blank page for draft)

21

Problem 4 A Select one appropriate term or sentence from the list below for the following

questions (1)-(10). For each question, write your answer as “(1)-A” in the answer sheet.

(1) Type I interferons (IFNs) play a critical role in the innate immune response against viral infections. Which one of the following statements concerning the mechanisms of action of type I IFNs is TRUE?

A. Type I IFNs activate a ribonuclease that degrades viral mRNA. B. Type I IFNs inhibit viral protease activity. C. Type I IFNs inhibit viral polymerase activity. D. Type I IFNs act against viruses with an RNA genome but not against viruses

that have DNA as their genome. (2) A large amount of a certain immunoglobulin isotype is found associated with the

mucosal surface of the respiratory tract. What is this immunoglobulin isotype? A. IgA B. IgD C. IgE D. IgG E. IgM

(3) Phagocytosis of microorganisms such as bacteria can be enhanced by binding of

antibodies or complement factors (mainly C3b). What is this process called? A. Complement activation B. Opsonization C. Neutralization D. Agglutination E. Precipitin reaction

(4) Which of the following statements concerning the characteristics of viruses is

FALSE? A. Viruses can grow only in living cells. B. The proteins on the surface of the virus mediate the entry of the virus into host

cells. C. Antibiotics typically are effective against viruses. D. The genomes of viruses can be composed of either DNA or RNA.

(5) Which of the following viruses is transmitted by airborne droplet nuclei? A. Epstein-Barr (EB) virus B. Hepatitis C virus C. Measles virus D. Poliovirus E. Dengue virus

22

(6) Which of the following diseases is transmitted by ticks?

A. Japanese encephalitis B. Severe fever with thrombocytopenia syndrome (SFTS) C. Hemorrhagic fever with renal syndrome (HFRS) D. Zika virus disease E. West Nile fever

(7) Which of the following statements concerning hepatitis B virus is TRUE?

A. The hepatitis B virus vaccine contains live attenuated virus as the immunogen. B. Hepatitis B immune globulin (Human) injection is used to prevent hepatitis B in

exposed person. C. Hepatitis B virus is an enveloped RNA virus. D. Hepatitis B virus is transmitted primarily by the fecal-oral route.

(8) What activates the classical complement activation pathway by interaction with C1

complement component?

A. Antigens B. Haptens C. Factor B D. Antigen-IgG complexes E. Bacterial lipopolysaccharides

(9) Which of the following statements concerning influenza A virus is FALSE?

A. Influenza A virus is transmitted by airborne respiratory droplets. B. The natural reservoirs of influenza A viruses are wild aquatic birds such as

ducks. C. Influenza A virus is an enveloped RNA virus. D. Antigenic drift of influenza A viruses may result in a worldwide epidemic.

(10) Which of the following statements concerning virus vector is FALSE?

A. Adenoviral vector can deliver genetic material into both dividing and non-

dividing cells, although its DNA does not integrate into the genome. B. Retroviral and Lentiviral vectors have ability to integrate gene material into

both dividing and non-dividing cells. C. Productive infection by Adeno-Associated virus occurs only in the presence of

a helper virus. D. Herpes Simplex virus (HSV) vector has a large packaging capacity for multiple

or large transgenes, and its features make HSV an attractive vector for cancer therapy.


23

B. Answer the questions of (1) – (2). (1) Read the following text and answer the questions (1-1)–(1-3) related to the underlined words, respectively. Once a virus enters a host cell, cell-mediated responses are required to clear virus-infected cells. The death of infected cells generates cellular debris containing both host and viral material. The cellular debris is ingested and degraded by antigen-presenting cells(1-1), and fragments of antigen are presented on the cell surface via major histocompatibility complex (MHC) class II molecules for interaction with CD4+ T cells. Activated CD4+ T cells, through release of cytokines(1-2), are able to provide help for activation and differentiation of CD8+ T cells into cytotoxic T cells（often called cytotoxic T lymphocytes, or CTLs）. Cytotoxic T cells search out virus-infected cells that have the same MHC class I/peptide complex that stimulated their own activation. Once such a cell is found, cytotoxic T cells can use multiple mechanisms to destroy those targeted cells(1-3). （1-1）List three cells which can act as antigen-presenting cells. （1-2）Which one of the following cytokines is NOT released by CD4+ T cells?

A. Interleukin-2 B. Interleukin-4 C. Interleukin-5 D. Interferon β E. Interferon γ

（1-3）CD8+ cytotoxic T cells (CTLs) have two different ways to destroy cells infected

with viruses. Explain these two ways within 80 words, respectively. （2） Natural killer (NK) cells can detect aberrant host cells and target them for destruction. Answer the following questions (2-1) – (2-2) regarding NK cells. （2-1）Which one of the following statements concerning NK cells is FALSE?

A. CD16 (FcγRIII) is involved in one of the activation pathways of NK cells. B. NK cells express T cell antigen receptor (TCR). C. NK cells do not express B cell antigen receptor (BCR).

（2-2）Describe how NK cells are able to distinguish aberrant cells (such as virus-infected cells and tumor cells) from normal host cells within 100 words.


24

C. Read the following text and answer the questions (1) – (3). One day, a mouse died with severe viral diarrhea. I isolated viruses from feces of the mouse, and infected it to human and mouse cultured cells. The human cells were normal, while the mouse cells showed apparently cell death. Further, when the RNA fraction was extracted from the infected mouse cells, was transfected into the human and mouse cultured cells, both of the cells died. (1) Explain what kind of genome the virus has, and describe briefly the reason.

(2) Answer the name of the virus, which you explained in (1) and causes the diarrhea.

(3) Explain the reason why the virus isolated from feces could not infect to human

cultured cells.

(End of Problem 4)

25

Problem 5 A. Read the following questions (1)-(10) and choose the single most appropriate

answer from A-D.

(1) Which is not established as a method for reprogramming of somatic cells to an undifferentiated state?

A. Stimulation of cells with the acid solution B. Generation of induced pluripotent stem cells (iPS) by gene transfer C. Cell fusion with ES cells D. Nuclear transplantation

(2) Which cell is in the most undifferentiated state that is close to a fertilized egg?

A. ES cell B. Jurkat cell C. iPS cell D. HeLa cell

(3) Choose one correct sentence about DNA methylation in mammalian cells.

A. The methylation of DNA functions at the stage of translation of genes. B. The methylation of DNA is a reversible reaction. C. DNA of sperm and ova are in hypomethylation states than in that of a fertilized

egg. D. All the CpG sites in mammalian DNA are methylated.

(4) Choose one correct sentence.

A. The acethylation of histones is important for transcriptional activities of genes. B. Euchromatin is highly condensed and in transcriptionally inactive state. C. The methylation of lysine residues in histones results in suppression of gene

translation. D. The methylation of lysine residues in histones is a reversible reaction.


A. Parthenogenesis is a natural phenomenon seen in all biological species. B. The ontogeny is possible from a single type of gamete, e.g. two sperm cells or

two ovum cells, even in the mammals. C. Initialization of gene modification is necessary for genomic imprinting. D. A genomic imprinting is unique machinery to the mammals with a placenta.

26


A. The autophagy is one of the immune systems in which cells eliminate pathogenic microorganisms.

B. Cells cannot degrade a big structure such as mitochondria by the autophagy. C. The autophagy is required for adaptation for starvation and early embryonic

development. D. The ubiquitin-proteasome system is a non-selective proteolysis system.

(7) Choose one which is NOT differentiated from hematopoietic stem cells.

A. Adipocyte B. Mast cell C. Osteoclast D. Erythrocyte

(8) Choose one which is NOT involved in sexual reproduction.

A. Meiotic division B. Germ cell C. Somatic cell division D. Zygote

(9) Choose one incorrect sentence.

A. ES cells exist in an ovary. B. Stem cells, which differentiate into epidermal cells, exist around basement

membrane between dermis and epidermis. C. Stem cells, which differentiate into small intestinal epithelial cells, exist in

crypts. D. Stem cells, which differentiate into erythrocytes and leukocytes, exist in bone

marrow. (10) Choose one which is NOT differentiated from neural stem cells.

A. Neuron B. Astrocyte C. Microglia D. Oligodendrocyte


27

B. Read the text below and answer the following questions (1)-(7), which are associated with the underlines (1)-(7).

To investigate functional roles of a particular gene, the techniques of RNA interference (RNAi)(1) and gene-targeting in mice are widely used. The use of ES cells manipulated by homologous recombination(2)(3)(4) is one of the methods for generation of gene-targeted mice. By use of this technique, it is possible to generate the reporter mice in which the target gene is replaced by the gene encoding the fluorescent protein(5). The mammalian genomes contain approximately 23,000 genes, and the project targeting all of the individual genes(6)(7) is in progress worldwide. (1) Briefly explain about the phenomenon of RNA interference (RNAi). (within 50

words)

(2) Briefly explain about the phenomenon of homologous recombination. (within 50 words)

(3) Choose all biological processes, in which homologous recombination occurs in

mammals.

A. Meiotic division B. Somatic cell division C. Repair of DNA which were damaged by chemicals and/or radiation D. Virus infection

(4) Choose one which is used in the process of generation of gene-knockout mice.

A. Mosaic mouse B. Chimera mouse C. Clone mouse D. Fancy mouse

(5) GFP is widely used in the generation of reporter mice. Choose the organism from

which the GFP gene was originally discovered.

A. Escherichia coli B. Nematode C. Firefly D. Aequorea Victoria (jellyfish)

(6) The gene-deficient mouse for a certain gene was not obtained by mating between

heterozygous male and female mice. Briefly explain the possible reasons for it. (within 30 words)

(7) In the case of (6), some conditional targeting techniques can be applied for

generation of gene-deficient mice. Briefly explain one technique for the conditional targeting.

(End of Problem 5)

28


29

Problem 6 A. Select the single best answer from the list for the following questions (1)-(10). (1) Which of the following phenomena is most suitable to account for the increasing

number of cancer patients?

A. Reduction of air pollution B. Improvement of the average life expectancy C. Promotion of bans on smoking and the separation of smoking areas D. Development of transportation network

(2) Which of the following phenomena is not suitable to account for the difficulties of

cancer treatment?

A. Metastasis to distant organs B. Individual variation of cancer C. Aerial infection of oncogene D. Accumulation of genetic mutations

(3) Which of the following biological reactions is associated with tumor growth in

vivo?

A. Immunological tolerance B. Apoptosis C. Cellular senescence D. Cell shrinkage

(4) The oncogene Src is often described as “v-Src” or “c-Src” in research papers. Choose the correct description of the abbreviation of the “v-” and “c-”.

A. The “v-” stands for “viral” and the “c-” stands for “cancerous”. B. The “v-” stands for “viral” and the “c-” stands for “cellular”. C. The “v-” stands for “venous” and the “c-” stands for “cancerous”. D. The “v-” stands for “venous” and the “c-” stands for “cellular”.

(5) Which of the following functions is not appropriate to account for the function of

tumor suppressor genes?

A. Growth inhibition of transformed cells B. Apoptosis inhibition of transformed cells C. DNA mismatch repair of transformed cells D. Cell cycle arrest of transformed cells

30

(6) The following are descriptions of epithelial–mesenchymal transition (EMT) in cancer. Which is the wrong statement?

A. EMT promotes metastasis and invasion of cancer cells. B. EMT correlates with the expression pattern of cadherins. C. EMT makes cancer cells more sensitive to chemotherapy. D. EMT makes cancer cells to be more primitive state.

(7) The following are descriptions about the relationship of microRNA (miRNA) and cancer. Which is the wrong statement?

A. miRNA suppresses gene expression, and the expression level of miRNA is low in cancer cells.

B. miRNA acts on specific complementary messenger RNA (mRNA), and is expected as a promising nucleic acid drug for targeted cancer therapy.

C. Tumor cell derived miRNA can be detected in peripheral blood samples, and is expected to be applied for cancer diagnosis.

D. miRNA is 20 to 25 bps in length, and is a degenerative product of mRNA. (8) Which of the following substance is not suitable as an anticancer drug targeting epi-

genome abnormalities?

A. DNA methyltransferase inhibitor B. Histone deacetylase inhibitor C. Histone acetylase inhibitor D. DNA polymerase inhibitor

(9) Which of the following characters is not suitable as the cancer stem cell character?

A. Loss of metastatic potential B. Capability of self renewal C. Capability of asymmetric cell division D. Resistance to chemotherapy and radiation therapy

(10) It is well known that tumors are consisted of various kinds of host normal cells,

and that tumor surrounding host normal cells regulate tumor microenvironment. Which of the following descriptions is not suitable as the role of tumor microenvironment during tumor development?

A. Tumor microenvironment is associated with tumor heterogeneity B. Tumor surrounding host normal cells create uniform tumor population in vivo. C. Regulatory T cells (Treg) in tumor microenvironment is associated with

immunity against tumors. D. Cancer associated fibroblasts (CAFs) in tumor microenvironment is associated

with tumor malignancy.


31

B. Read the text below and answer the following Questions (1)-(4). It is known that molecularly targeted drugs for cancer have a great impact on the decrease in the cancer mortality rate. These molecularly targeted drugs are developed to interfere the functions of mutated genes that are specifically expressed or overexpressed in cancer(1). The molecularly targeted drugs exhibit anti-tumor activity by suppressing the functions of proteins derived from oncogenes or the mutated genes(2). Unfortunately, the anti-tumor activity of the molecularly targeted drugs will not continue forever. Within several years, cancers develop drug-resistant clones, thereby resulting in recurrence. From the numerous efforts to uncover the drug resistant mechanisms, several genetic and epigenetic mechanisms(3) and the overcoming drugs(4) have been identified. (1) “Personalized medicine” is becoming more and more necessary along with the

increasing number of molecularly targeted drugs. Explain about “personalized medicine”, especially from the view point of molecularly targeted drugs. (Maximum 100 words)

(2) Imatinib has already been used in clinic to suppress Bcr-Abl oncogene product. Explain briefly the reason why inhibition of Bcr-Abl function by Imatinib led to the suppression of tumor growth. (Maximum 100 words)

(3) Genetic mutation(s) have often found in drug-resistant recurrent cancer cells.

Explain why the genetic mutation(s) of the target gene leads to the resistance to molecularly targeted drugs. (Maximum 100 words)

(4) To find out drug resistance-overcoming agents, several researchers take a drug

repositioning approach (also known as drug repurposing approach). The drug repositioning is one of the drug screening approaches to find out candidate drugs from library of compounds that had been passed the safety tests but could not show efficacy in human clinical trials. Explain the advantage of drug repositioning approach to find out new drugs from the other drug screening approaches. (Maximum 100 words)

(End of Problem 6)

32


Problem 7 �

(1) Given an integer x , we calculate a polynomial, 0

ii

i

n

f a x=

=∑ (n , (0 )ia i n≤ ≤ are natural

numbers). A single addition or multiplication of two values takes a unit time regardless of the number of the digits in the representation of the values. (A) Let i

i if a x= . Find the time complexity (with regard to n ) of an algorithm that

calculates 0 1, , , nf f f… individually and then calculates 0

n

ii

f f=

=∑ .

(B) Let1

(when ) (when )

ni

i i

ga i n

g x a i n+

=

+ <

!= "#

. Notice that 0f g= holds. Find the time complexity

(with regard to n ) of an algorithm that calculates 0g hence f by calculating

1 0, , ,n ng g g− … in this order.

(2) Given two 12n+ -bit integers a and b , let ,hi hia b be the highest 2n bits of a and b ,

and ,lo loa b the lowest 2n bits of a and b , respectively. We calculate the product of a and b �when n is fairly large. Assume that computation time for addition or shift is negligible compared to that for multiplication. (A) The multiplication of 12n+ -bit integers can be constructed from the multiplication of 2n

-bit integers as follows: 22 1 02 2n nab r r r= + + , where 2 hi hir a b= , 1 hi lo lo hir a b a b= + ,

0 lo lor a b= . We can recursively apply this reduction until every multiplication only

involves integers small enough to fit in the machine word of the computer. Let T (2n+1) be the number of machine-word multiplications required in the multiplication of two

12n+ -bit integers. Express 1(2 )nT + in terms of (2 )nT .

(B) Solve the recursive equation you answered in (A), and express (2 )nT in Landau’s Onotation.

(C) We reduce the number of multiplications in the calculation in (A) by calculating 1r as

1 2 0 ( )( )hi lo hi lor r r a a b b= + − − − . Express (2 )nT in Landau’s O notation, and prove it.

Problem 8

Answer the following questions about linear algebra. �

(1) Denote by ! the zero vector. Let ! denote a two-dimensional vector that is not !. !! ! is the orthogonal projection of a point ! on !. Prove the following propositions. (1.1) !! !! ! = !! ! for any two-dimensional point !. (1.2) !! !! ! = ! for any non-zero two-dimensional vector ! orthogonal to !.

�

�

�

�

�

�

�

�

�

�

�

(2) Assume that a real symmetric matrix ! satisfies !! = ! . Prove that the eigenvalues of�!�are either 0 or 1. (3) Denote by !!,!! the column vectors corresponding to the bases of a two dimensional subspace of the three dimensional space. Describe the projection matrix to the subspace using ! = [!!,!!].

�

�

�

!�

!�

!!(!)�

Problem 9 Suppose that ! is an array with !!(≥ 1) distinct integers. The quicksort algorithm for sorting

! in the ascending order has the following three steps.

A) Select and remove an element ! from !. Call ! a pivot.

B) Divide ! into arrays !! and !! such that ! ≤ ! for ! ∈ !! and ! ≤ ! for ! ∈ !!.

C) Sort !! and !! in the ascending order using quicksort, and return the concatenation of

!!, !, and!!!.

Answer the following questions.

(1) How would you implement Step B in quicksort?

(2) In Step A, if we always set the first element in ! to pivot !, show an input array that the

algorithm sorts in !(!!) worst-case time, and prove this property.

(3) In Step A, if we select a position in ! at random and set the element at the position to

pivot !, prove that the expected time complexity is !(! log !) for an arbitrary input

array.

(4) Design an algorithm that calculates the !-th smallest element in ! in !(!) expected

time, and prove this property.

Problem 10 �We define the shortest distance from a vertex i to a vertex j on a graph as the number of edges in a path from i to j that contains the smallest number of edges, except that the shortest distance is +∞ �when no such path exists and that it is 0 when i and j �are identical. �(1) Let us consider the directed graph shown below.

(A) Show the adjacency matrix.

(B) Show a matrix S , whose element ,i js �is the shortest distance from a vertex i to a

vertex j . �(2) Suppose we are given a simple directed graph ( , )G V E= , where the vertex set

{1,2, , }V n= … �and E is the edge set. E is represented by a matrix ( )(0) (0),i jD d= , where

(0),

0 (if )1 (if an edge

(otherwiexists

e)

s )i j

i jd i j

=!"

=

∞

→%"+&

��

(A) Let ( )

, {1,2, , } { , }ki jV k i j= … U ��Let ( )

,ki jE be the set of edges in E that start from and

end at a vertex in ( ),k

i jV . Let ( ),ki jd be the shortest distance from a vertex i to a vertex

j �on a directed graph ( ) ( ) ( ), , ,( , )k k ki j i j i jG V E= , and let ( )( ) ( )

,k k

i jD d= .�Express (1)D in

terms of� (0)D .

(B) ( 1)kD + �can be computed from ( )kD �as follows. Fill in the two blanks.

( )( 1) ( ), ,min , + k ki j i jdd + = �

(C) Given G , show an algorithm to compute the all-pair shortest distances, and find its

time complexity with regard to n .

� �

Problem 11 Let ! = !!,!!,!!,⋯ ! be a sequence of mutually independent random variables such that each !! takes the value of 1 with probability !, and 0 with probability 1− ! ! 0 < ! < 1 . We define a sequence !! ! = 0,1,2,⋯ as follows:

!! = 1 !! = !!!!! + !! !, ! ≥ 1

Here, ! is a positive real number. Answer the following questions. (1) Find the expected value Ε !! of !!. (2) Find the variance Var !! = Ε !!! − Ε !!

!of !!. (3) Express !! as a function of ! and the elements of ! ! ≥ 1 . (4) Find Ε !! ! ≥ 1 . (5) Find !! = lim!→! Ε !! as a function of ! and !.

Problem 12

Consider an algorithm that calculates a longest palindrome that is a substring of a given string

mxxx …1= .

Here, we define a string nyyy …1= to be a palindrome if for any j ( nj ≤≤1 ) jnj yy −+= 1 .

Any string of one character is a palindrome and a string of two characters 21yy is a palindrome if

and only if 21 yy = .

Answer the following questions. .

(1) When ji xx… is a palindrome, show the condition that is necessary and sufficient for

11 +− ji xx … to be a palindrome.

(2) Variable ),( jiR mjimi ≤≤≤≤ ,1 is 1 if ji xx… is a palindrome, otherwise 0. Show the

formula for each of the blanks, A , B and C , in the following iterative equation.

),( baδ is 1 if ba = , otherwise 0.

1( −iR , A () δ= B (), 1 Rxj+ C ), j

(3) Show all the initial values of ),( jiR and the procedure of the iterations for calculating

),( jiR mjimi ≤≤≤≤ ,1 by dynamic programming using the above iterative equation.

(4) Explain how to get a longest palindrome that is a substring of mxxx …1= using ),( jiRmjimi ≤≤≤≤ ,1 .

graduate school of frontier sciences, the university of

Documents