1. 目 录目 录 Chapter two Structures and Functions of Nucleic Acids

2. 目 录目 录 Main contents 1. The components of nucleic acids 2. The structures and functions of DNA 3. The structures, and functions of RNA 4. The physicochemical properties of nucleic acids 5. Nucleases and Ribozymes 6. Genomics and Human Genome Project Disease casesQuestionsDemands Introduction

3. 目 录目 录 Nucleic acid Polynucleotide or polymers of nucleoties which can carry, transmit and express the genetic information

4. 目 录目 录 1868 Fridrich Miescher the isolation of nuclein from pus leukocytes 1944 Avery the confirmation of DNA as genetic molecules 1953 Watson and Crick the discovery of DNA double helix structure 1968 Nirenberg the discovery of genetic codons 1975 Temin and Baltimore reverse transcriptase 1981 Gilbert and Sanger the method for DNA sequencing (dideoxy chain terminator sequencing) 1985 Mullis the creation of PCR technology 1990 human genome project (HGP) start-up in America 1994 Chinese human genome project start-up 2001 HGP were nearly finished 1.The discovery of nucleic acids and the research advance

5. 目 录目 录 2. The sorts and distribution of nucleic acids more than 90% in nucleus, and other found outside of nucleus such as mitochondria, chloroplast, plasmid Distributed mainly in cytoplasm, minority in nucleus Deoxyribonucleic acid, DNA Ribonucleic acid, RNA Carry genetic information, determine the genotype of individual or cell Transmit the genetic information and gene expression, sometime work as the carrier of genetic information such as RNA virus

6. 目 录目 录 The Monomeric Units of Nucleic Acids Section One Nucleotides

7. 目 录目 录 Chemical composition of nucleic acids 1. Element component C 、 H 、 O 、 N 、 P （ 9~10% ） 2. Molecular component —— base ： purines ， pyrimidines —— ribose ： ribose ， deoxyribose —— phosphate 3. Building units (monomeric units): nucleotides

8. 目 录目 录 Chemical composition of nucleic acid nucleic acid ribonucleotide ribonucleoside phosphate base Pentose Elementary unit of nucleic acid is nucleotide DNA: dAMP, dGMP, dCMP, dTMP RNA: AMP, GMP, CMP, UMP The composition of nucleotide base Pentose phosphate

9. 目 录目 录 Purine adenine, A guanine, G Bases

10. 目 录目 录 Pyrimidine cytosine, C uracil, U thymine, T

11. 目 录目 录 Pentose （ RNA ） 1´1´ 2´2´3´3´ 4´4´ 5´5´ ribose （ DNA ） deoxyribose

12. 目 录目 录 deoxyribonucleoside ： dAR, dGR, dTR, dCR 1. The structures of nucleotides (1) The formation of ribonucleoside It can be formed by glycoside bond joined base to ribose (ribonucleoside) or deoxyribose (deoxyribonucleoside) 1´1´ 1 ribonucleoside ： AR, GR, UR, CR

13. 目 录目 录

14. 目 录目 录 (2) The naming of nucleoside or nucleotides

15. 目 录目 录

16. 目 录目 录

17. Biological active substances containing nucleotide ： NAD + 、 NADP + 、 CoA-SH 、 FAD etc, containing AMP nucleotides ： NMP ， NDP ， NTP cyclic nucleotide: cAMP ， cGMP AMP ADP ATP cAMP NADP + NAD + Some important free nucleotide and their ramifications in organisms

18. 目 录目 录 5´end 3´end (3)The linkage of nucleotides The nucleotides are linked together by phosphodiester bonds to form polynucleotides, namely nucleic acids C G A

19. 目 录目 录 5′end 3′end C G A 2. Primary Structure of nucleic acids Definition The linear sequence of (deoxy)nucleotides ( or base sequences ) in DNA (RNA) is termed primary structure of DNA (RNA). Linkage bond phosphodiester linkage

20. AG P 5 P T P G P C P T P OH 3 writing 5 pApCpTpGpCpT- OH 3 5 A C T G C T 3 目 录目 录

21. 目 录目 录 Tautomerism between the lactam and lactim 1.2 Properties of nucleosides and nucleotides Absorbance of ultraviolet light at 260 nm

22. 目 录目 录 Section Two Dimensional Structure and Function of DNA

23. 目 录目 录

24. 目 录目 录 The secondary structure of DNA----double helix structure –The research background and historic significance of DNA helix structure –The key points of DNA double helix structure The superhelix structure of DNA and the its composition in chromatin –The DNA superhelix structure –The spatial structure of DNA in prokaryote –DNA constitution in the nucleus of eukaryote cells DNA functions

25. 目 录目 录 5′end 3′end C G A 1. Primary Structure of nucleic acids Definition The linear sequence of deoxynucleotides ( or base sequences ) in DNA is termed primary structure of DNA. Linkage bond phosphodiester linkage Backbone: ---Pi-dR-Pi-dR---

26. 目 录目 录 2. Secondary Structure of DNA---- Double Helix Model

27. 2.1 Background 目 录目 录 The analysis of base composition of DNA Chargaff rules The analysis of chemical and physical data on DNA building model Primarily x-ray diffraction data collected by Rosalind franklin and Maurice Wilkins

28. 目 录目 录 Chargaff rules a. A = T, G = C; b. The base composition is different in different organism species. c. The base composition of different organs in same individual is same.

29. 目 录目 录 2.2 The key points of DNA Double Helix Model (1) The DNA double helix is oriented to right-handed running. (2) The two DNA strands are organized in an antiparallel arrangement (i. e. The two strands run in opposite directions, one strand is oriented 5’  3’ and the other is oriented 3’  5’ ). 3’ 5’ 3’ 5’ minor groove major groove

30. 目 录目 录

31. 目 录目 录 (3) The bases of the two strands form hydrogen bonds to each other; A pairs with T and G pairs with C. For each round of the helix, there are 10 pairs of bases 3’ 5’ 3’ 5’ minor groove major groove 2.2 The key points of DNA Double Helix Model

32. 目 录目 录 Base pairing rules TA GC A=TG≡C Hydrogen bond

33. (4) The stable forces are hydrogen bonds between base pairs and base stack force. (5) There are major grooves and minor grooves in DNA double helix. 目 录目 录 3’ 5’ 3’ 5’ minor groove major groove 2.2 The key points of DNA Double Helix Model

34. 目 录目 录 The summary on DNA double helix （ Watson, Crick, 1953 ） (1) right-handed oriented double helix, antiparallel (2) Backbone outside, bases inside (3) base-pairing, A=T, G≡C (4) A running of the helix containing 10 pairs of bases (5) Structure stable depends on the vice-bonds (6) There are minor grooves and major grooves hydrogen bonds, base stack force

35. 目 录目 录 2.4 Polymorphism ofsecondary structure of DNA

36. 目 录目 录 Forms of DNA Form Pitch(nm) Residues inclination of base per turn pair from horizontal A 2.8 11 20 ° B 3.4 10 0° Z 4.5 12 7 ° RNA-DNA 2.8 11 20° hybrid Source: From Davidson, The biochemistry of the Nucleic Acids, 8thed., revised by Adams, et al. Copyright ©Chapman & Hall, London.

37. 目 录目 录 （ 1 ） Superhelix structure of DNA in prokaryote ------Circular double stranded superhelical DNA 2.5 The Superhelix Structure of DNA

38. 目 录目 录 The nucleosome in eukarytic cells DNA double helix  superhelix  nucleosome  chromatin  chromosome （ 2 ） Superhelix structure of DNA in eukaryote ------Nucleosomes The nucleosomes in eukarytic cells consist of DNA and proteins

39. 目 录目 录 The elementary unit of chromosome is nucleosome which consists of DNA and histone. Histone includes H1, H2A, H2B, H3, H4. Two molecules of each H2A, H2B, H3, H4 constitute the core particle of nucleosome.

40. 目 录目 录 Core particles Linkage DNA H1, H2A, H2B, H3, H4 histones

41. 目 录目 录

42. 目 录目 录 10 nm fiber Transv erse section lengthwisesection 30 nm fiber chromosome 300nm solenoid chromatin The folding of nucleosomes, the chromatin condensation, the formation of chromosome

43. 目 录目 录 Genes consist of regulatory region and large protein-coding segments. Genome is a whole sequence of DNA in an organism. Genetic code: The letters A,G,T and C correspond to the nucleotides found in DNA. They are organized into three-letter code words called codons, and the collection of these makes up the genetic codes. 2.6 Functions of DNA

44. 目 录目 录 DNA functions: 1. Template of replication 2. Template of transcription 3. To accept some mutations

45. 目 录目 录 Structures and Functions of RNA Section Three

46. 目 录目 录 Structure and Function of RNA

47. 目 录目 录 The primary structures of RNA belong to single stranded linear polynucleotide, but contain part self-complementary pairing, namely hairpin structures 1. Structure of RNA Elementary unit of nucleic acid is nucleotide RNA: AMP, GMP, CMP, UMP

48. 目 录目 录 Structure Characters of mRNA: (1) 5’-cap sequence: m’GpppNm The cap functions are to boost the binding of ribosome with mRNA, and to increase the stability of mRNA. 2. Types and Functions of RNAs 2.1 Messenger RNA

49. 目 录目 录 Cap structure of mRNA 5’-end Guanine

50. 目 录目 录 Structure Characters of mRNA: (2) 3’-end sequence: poly A The 3’-end functions are related to increase the stability of mRNA and the lifetime worked as template for translation. 5’ cap structure 3’ poly A 5’ uncoding region3’ uncoding region coding region

51. hnRNA ( intron ) mRNA (3) Mature process of mRNA * ( exon ) 目 录目 录

52. 目 录目 录 (4) mRNA functions: As a template of protein synthesis, it contains triplet codes. So, mRNA carries the information for the primary structure of proteins, serves as template of protein synthesis during translation.

53. 目 录目 录 DNA mRNA 蛋白 转录 翻译 Prokaryotic cells Transcript translation protein 细胞质 细胞核 DNA 内含子 外显子 转录 转录后剪接 转运 mRNA hnRNA 翻译 蛋白 真核细胞 Transcript Prost-transcript modification translation protein Eukaryotic cells extron intron

54. 目 录目 录 (1) Composition of transfer RNA, tRNA It’s the smallest RNA among RNAs, only consists of 70~90 nucleotides. 2.2 Transfer RNA (tRNA) tRNAs contain some unusual (modified) bases, such as 7-methylguanine, pseudouridine, dehydrouridine,

55. 目 录目 录 N,Ndimethylguanine Dihydrouridine unusual bases N 6 -isopreneadenine 4-thiouracil

56. 目 录目 录 The tRNA molecules serve as adapters for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. (2) Functions of transfer RNA, tRNA Two roles: Activating amino acids Recognizing codons in mRNA

57. 目 录目 录 ◆ Contain rare bases, such as DHU, pseudouridine (  ), m G, m C ◆ Stem-loop structure ( local double strands) Secondary structure: cloverleaf pattern ◆ Anticodon in the anticodon loop ◆ The base sequence of an anticodon can reversely complement with codon on mRNA. (3) The structure characters of tRNA

58. 目 录目 录 The cloverleaf pattern of tRNA ( secondary structure) Amino acid arm DHU loop TΨC loop Anticodon loop variable loop

59. 目 录目 录 5’ 3’ AUG Tyr The anticodon at the end of a base- paired stem recognizes the triplet nucleotide or codon of the template mRNA. 5’ 3’ UAC mRNA GCACCAUCGAUG Met CGU Ala 5’ GGU 5’ Pro

60. 目 录目 录 The cloverleaf pattern of tRNA ( secondary structure)

61. 目 录目 录 The tertiary structure of tRNA

62. 目 录目 录 (1) rRNA structure (2) rRNA functions a. A component of ribosomes b. Ribosomes work as the apparatus of protein synthesis 2.3 Ribosomal RNA (rRNA)

63. 目 录目 录 Eukaryotes 5S rRNA 28S rRNA 5.8S rRNA 18S rRNA Prokaryotes 5S rRNA 23S rRNA 16S rRNA (3) rRNA sorts ( dependent on the sedimentation coefficient, S) Large subunit small subunit Large subunit small subunit

64. 目 录目 录 The components of ribosome Prokaryote ( E coli.) Eukaryote （ mouse liver ） Small subunit 30S40S rRNA16S1542 nucleotides18S1874 nucleotides proteins21Occupy 40% of total weight 33Occupy 50% of total weight Large subunit 50S60S rRNA23S 5S 2940 nucleotides 120 nucleotides 28S 5.85S 5S 4718nucleotides 160nucleotides 120nucleotides proteins31Occupy 30% of total weight 49Occupy 35% of total weight

65. 目 录目 录 A large number of discrete, highly conserved, and small stable RNA species are found in eukaryotic cells. The majority of these molecules exist as ribonucleoproteins and are distributed in the nucleus, in the cytoplasm, or in both. 2.4 Other Small Stable RNA

66. 目 录目 录 The major sorts of RNA and their functions In the nucleus or in the cytoplasm mitochondrion functions Ribosomal RNA rRNA mt rRNA the components of ribosomes Messenger RNA mRNA mt mRNA the template for translation Transfer RNA tRNA mt tRNA activating AA and recognizing codons on mRNA heterogeneous nuclear RNA hnRNA the precursor of mRNA Small nuclear RNA snRNA related to the splicing and transfering of hnRNA Small nucleolus RNA SnoRNA related to the processing and modifying of rRNA Small cytoplast RNA scRNA/7SL-RNA the components of signal discriminator for proteins located in endoplasmic reticulum

67. 目 录目 录 Small nuclear RNAs are significantly involved in mRNA processing and gene regulation. Of the several snRNAs, U1, U2, U44, U5, and U6 are involved in intron removal and the processing of hnRNA into mRNA. hnRNA ( heterogeneous nuclear RNA ) snRNA ( small nuclear RNA)

68. The Properties of Nucleic Acid Section Four 目 录目 录

69. 目 录目 录 1. The general properties of nucleic acids 1.1 Acidic molecules 1.2 Macromolecules 1.3 High viscosity (DNA) 1.4 Ultraviolet absorption ( 260 nm)

70. 目 录目 录 Extinction coefficient Ultraviolet absorbent spectrum of various bases (pH 7.0) Cytosine Adenine Guanine Thymine Uracil Wavelength

71. 目 录目 录 2. DNA denaturation Definition Double helix of DNA Single strand The denaturation factors heating, chemical reagents, ultraviolet light

72. 目 录目 录 The essence of DNA denaturation is the breaking of hydrogen bonds between the double strands of DNA Natural DNA Denatural DNA heating Cooling slowly

73. 目 录目 录 The changes of DNA properties after denaturation * Hydrogen linkage broken * A 260  ( hyperchromic effect ) * Double helix  single strand * Tm ( melting temperature ) * Tm and G + C Tm = 69.3 + 0.41 ( % G+ C ) < 20 bp, Tm = 4 ( G + C ) + 2 ( A + T )

74. (1) The determination of the amount of DNA or RNA OD 260 =1.0 is corresponded to 50μg/ml double stranded DNA 40μg/ml single stranded DNA （ or RNA ） 20μg/ml oligonucleotides (2) To estimate the purification of nucleic acid samples Purified DNA : OD 260 /OD 280 = 1.8 Purified RNA: OD 260 /OD 280 = 2.0 The application of OD 260 目 录目 录

75. For example: the change of ultraviolet absorption spectrum of DNA induced by denaturation The ultraviolet absorption spectrum of DNA  Hyperchromic effect ： the phenomenon of OD 260 increase companied by DNA denaturation in solution 目 录目 录 Natural state Denatured state Absorbent value Wave length

76. Heated denaturation of DNA Melting curve: a graph in which absorbance versus temerature is plotted 目 录目 录 Temperature-optical density profile for DNA

77.  Tm: Tm is the temperature at which 50% of DNA in solution are denatured by heated, or called melting temperature, Tm. The value of Tm is related to the contents of G and C. 目 录目 录

78. 目 录目 录 3. The renaturation of DNA and nucleic acid hybridization (1) Renaturation Single strand of DNA  double helix ( annealing ) The best annealing temperature: 25 ℃ < Tm

79. 目 录目 录 Hybridization double stranded molecules of DNA-DNA denaturationrenaturation Various source DNA Renaturation process

80. 目 录目 录 (2) Nucleic Acid Hybridization Definition The process of forming a double stranded structure from two polynucleotide strands ( either DNA or RNA) from different source is termed hybridization Hybridization Principle ( To see the above slice or next slice)

81. 目 录目 录 Double stranded DNA 1 Double stranded DNA 2 Double stranded DNA heating single stranded DNA Heterogeneous double stranded DNA cooling slowly Cooling slowly Adding marked single stranded DNA probes Forming the marked heterogeneous stranded DNA The denaturation and renaturation of DNA, nucleic acid hybridization

82. 目 录目 录 (3) The application of Hybridization technology ★ Determining whether a certain sequence occurs more than once in the DNA of a particular organism ★ Demonstrating a genetic or evolutionary relationship between different organisms ★ Determining the number of genes transcribed in a particular mRNA ★ Determining the location of any given DNA sequence by annealing with a complementary polynucleotide probe

83. 目 录目 录 Nucleases catalytic RNA and DNA Section 5

84. 目 录目 录 (1) Definition Nucleases are the enzymes capable of degrading nucleic acids. (2) Catalogue DNase Those which exhibit specificity for deoxyribonucleic acid are referred to as deoxyribonucleases RNase Those which specifically hydrolyze ribonucleic acids are ribonucleaes. Endonuclease Exonuclease 1. Nucleases

85. 目 录目 录  Classification according to the substrates deoxyribonuclease, DNase Specially degrade DNA ribonuclease, RNase Specially degrade RNA 。 –Classification according to the cut sites Endonuclease restriction endonuclease (RE) and nonRE Exonuclease 5’-3’ exonuclease or 3’-5’ exonuclease

86. 目 录目 录 2. Ribozymes  There are five classes of ribozymes  Ribozymes are RNA molecules with catalytic activity ★ Three classes carry out self-processing reactions. ★ Two classes ( RNase P and rRNA )are true catalysts that act on separate substrates (containing proteins)

87. 目 录目 录 3. Deoxyribozymes  These special DNA sequences with catalytic capability are called deoxyribozymes.  Some single-stranded DNA molecules are capable of adopting intricate tertiary structures and performing efficient catalysis.  At present, deoxyribozymes are only found in laboratories.

88. 目 录目 录 Genomics and the Human Genome Project Section 6

89. 目 录目 录 1. Characteristics of Eukaryotic Genome DNA  Genomes in eukaryotes are much larger than in prokaryotes  Only a few percent ( 2-4%) of DNA in a mammalian cell may suffice for all genes.  Eukaryotes genes do not overlap but are spaced, on the average, 40 000 bp apart.  There are split genes that consist of exons and introns in eukaryote genomes.

90. 目 录目 录 2. The human Genome Project  1990, the project started  An international program  Make a sequence map for the total 3 billion bp of human genome  Resource for the investigation of hereditary diseases as well as normal gene structure and expression

91. 目 录目 录 1. Hemoglobin diseases ( 血红蛋白病 ) Disease Cases 2. Horsebean disease ( 蚕豆病 ) 3. X-Fragile Syndrome (X- 脆性综合征 )

92. 目 录目 录 ♣ Types of nucleic acids and their functions Demands ♣ Basic units for nucleic acid composition ♣ The differences between DNA and RNA ♣ Secondary structure characters of DNA ♣ Structure characters of mRNA, tRNA, rRNA ♣ Physicochemical properties of DNA, RNA

93. 目 录目 录 Questions ： 1. Describe the sorts, functions of nucleic acids in nature. What are the characteristics of their chemical components or structures respectively? 2. What are the double helix of DNA? How to prove the structural characters of DNA double helix? 3. Why is it said that DNA are the carriers of genetic substances ? How to demonstrate it ?

94. 目 录目 录 4. Which important physical and chemical properties of nucleic acids are there that can be utilized ? 5. Illustrate those important free mononucleotides existing in organisms 6. Do you know how to compare DNA with RNA? 7. What are the differences between nucleases and ribozymes?

95. 目 录目 录 Friedrich Miescher (1844-1895) Friedrich Miescher worked at the Physiological Laboratory of the University of Basel and in Tübingen and is most well known for his discovery of the nucleic acids.

96. 目 录目 录 Friedrich Miescher （ 1844 － 1895 ） From an early age Friedrich was recognized as being highly intelligent, but he was shy and introspective - perhaps in part as the result of a serious hearing impairment he had suffered from since boyhood. Despite this handicap he took great interest in music. Miescher had originally intented to study lymphocytes, he collected laudable pus from used bandages in the nearby hospital. He obtained a precipitate from the cell nuclei solution. Obviously this material must have come from the nucleus, and he therefore named it nuclein. Using elementary analysis, one of the few methods available to characterize an unknown compound. He extracted high-molecular-weight DNA from damaged cells eventually.

97. 目 录目 录 Oswald T. Avery (1877-1955). Courtesy of The Rockefeller University Archives. Reichard, P. J. Biol. Chem.2002;277:13355-13362

98. 目 录目 录 O.T. Oswald Theodore Avery (1877 ～ 1955) Oswald Theodore Avery (1877-1955) was a distinguished bacteriologist and research physician and one of the founders of immunochemistry. He is best known, however, as a discoverer that deoxyribonucleic acid (DNA) serves as genetic material. The Oswald T. Avery Collection is a part of the Joshua Lederberg Papers, which are at the National Library of Medicine and available digitally. The collection was assembled by Nobel laureate Dr. Lederberg because of the strong connection between Dr. Avery's work and his own. The work of Avery and his lab, observes Dr. Lederberg, was "the historical platform of modern DNA research" and "betokened the molecular revolution in genetics and biomedical science generally.

99. 目 录目 录

100. 目 录目 录 Rosalind Franklin （ 1920-1958 ） Born in July of 1920, Rosalind Franklin graduated from Cambridge University and in 1951 went to work as a research associate for John Randall at King's College. A chemist by training, Franklin had made original and essential contributions to the understanding of the structure of graphite and other carbon compounds. It was Franklin alone whom Randall had given the task of elucidating DNA's structure. The technique with which Rosalind Franklin set out to do this is called X- ray crystallography. With this technique, the locations of atoms in any crystal can be precisely mapped by looking at the image of the crystal under an X-ray beam. By the early 1950s, scientists were just learning how to use this technique to study biological molecules. Rosalind Franklin applied her chemist's expertise to the unwieldy DNA molecule. After complicated analysis, she discovered (and was the first to state) that the sugar-phosphate backbone of DNA lies on the outside of the molecule. She also elucidated the basic helical structure of the molecule.

101. 目 录目 录 More advanced reading: Nelson, D. L., and Cox, M. M. (2000) Lehninger Principles of Biochemistry, third edition, Worth Publishers. Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002) Biochemistry, Fifth edition, W. H. Freeman and Company, New York. Mathews, C. K., van Holde, K. E., and Ahern, K. G. (2000) Biochemistry, third edition, Benjamin/Cummings (www.aw-bc.com/mathews/).