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林光輝: 生化科教授 醫學大樓 Room 0861 Tel: 5177, 5975 e-mail:khlin@mail.cgu.tw PowerPoint Presentation
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林光輝: 生化科教授 醫學大樓 Room 0861 Tel: 5177, 5975 e-mail:khlin@mail.cgu.tw

林光輝: 生化科教授 醫學大樓 Room 0861 Tel: 5177, 5975 e-mail:khlin@mail.cgu.tw

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林光輝: 生化科教授 醫學大樓 Room 0861 Tel: 5177, 5975 e-mail:khlin@mail.cgu.tw

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  1. 林光輝: 生化科教授 醫學大樓 Room 0861 Tel: 5177, 5975 e-mail:khlin@mail.cgu.edu.tw

  2. 上課投影片網址 長庚大學—系所介紹—基礎醫學研究所-生物化學暨細胞分子生物學組-師資介紹-林光輝-講義上網-Biochem

  3. C Chapter 8Nucleotides and Nucleic Acids Basic information: Griffith--pneumococcus bacteria; Avery,MacLeod, MacCarty (1944) * Hershey, Chase - bacteriophages (1952) * Chargaff - ratios of purines to pyrimidines = 1, A/T=1, G/C=1 (late 1940s) Wilkins, Franklin - X-ray diffraction provided clues to the shape of the DNA molecule (1950) * Watson, Crick-double helix (1953)

  4. X-ray diffraction :Franklin and Wilkins

  5. Nucleic acids: monophosphates: Base + sugar + phosphate H-bond properties UV-light absorbing properties Sugars: 5-Carbon pentose: D-ribose: RNA 2'-deoxy-D-ribose: DNA Nucleoside:N-glycosidic linkage of base to carbon of sugar

  6. Nomenclature Pyrimidines:+ idine: Cytidine, uridine, thymidine Purines: + osine: Adenosine, guanosine Nucleotides: Typically 5' nucleoside phosphate 5‘-ribonucleoside monophosphates AMP, GMP, CMP, UMP: cAMP, cGMP: 3',5'-Cyclic ribonucleoside monophosphates: Regulation of cellular metabolism

  7. 5'-Ribonucleoside diphosphates ADP, GDP, CDP, UDP: 5’-Ribonucleoside triphosphates ATP (energy currency), GTP (protein synthesis and signal transduction), CTP (lipid synthesis), UTP (carbohydrate and polysaccharide synthesis) 5’-Deoxyribonucleoside- triphosphates dATP, dGTP, dCTP, dTTP: For DNA synthesis

  8. Structure of nucleotides N-glycosidic bond

  9. Nomenclature Base Nucleoside Abbreviation adenine adenosine A guanine guanosine G cytosine cytidine C uracil uridine U thymine thymidine T Important to know: U in RNA only T in DNA only

  10. More Nomenclature Nucleotide = base + sugar + phosphate Nucleoside = base + sugar

  11. 5-methycytosine is a mutation hot spot

  12. GATC CTAG 5’ - GATC - 3’ 3’ - CTAG - 5’ = Double stranded DNA is written with the 5’ to 3’ on top If only one strand of DNA is given, it is assumed to be in the 5’ to 3’ orientation GATC CTAG GATC =

  13. DNA replication

  14. Double stranded DNA: Strands antiparallel: Base pairs AT and GC *B-form DNA: Right-handed helix Parameters:0.34 nm/base pair, 10 bp/turn H-bonded base pairs AT and GC inside Base pairs perpendicular and stacked Negative charged phosphate and sugar outside Major and minor groove

  15. *A-form DNA: Right-handed helix Parameter: 0.224 nm/base pair, 11 bp/turn Base pairs tilted dsRNA and DNA/RNA hybrids

  16. *Z-form DNA: Left-handed helix Dinucleotide repeat: Pyrimidine-purine Conformation: Purine -syn, pyrimidine -anti (p284) Zigzag pattern of sugar-phosphate backbone Gene regulation or in genetic recombination

  17. Adenosine: A mediator of the sleeping • inducing effect of prolonged wakefulness • Function: • Blood vessel dilation. • Slow the heart rate (adenocardTM) • 具有強力的抗心律不整及冠狀動脈擴張作用。臨床上則是利用其抗心律不整之作用。 • 3. Sleepiness

  18. Peptide nucleotide (PNA) • Sugar phoaphate backbond—NA • Peptide backbond—PNA • Function: • Resistant to nuclease or protease • Diagnostic probes • Antisense drugs

  19. Nucleic acids: Nucleoside monophosphates in phosphodiester linkage 5' to 3'  DNA: Genetic material: double stranded dsDNA: Strands antiparallel Interchain H bonds form base pairs Chargaff's rules:A=T, G=C, Purines = Pyrimidines X-ray diffraction of Franklin and Wilkins and model building of Watson and Crick

  20. 1/1

  21. What is RNA? • Ribonucleic acid • Ribonucleotides (Ribose, base, & phosphate) • Types • Coding: messenger RNA (mRNA) • Non-coding: • Ribosomal RNA (rRNA) • Transfer RNA (tRNA) • Small nuclear RNA (snRNA) • Small nucleolar RNA (snoRNA) • Interference RNA (RNAi) • Short interfering RNA (siRNA) • Micro RNA (miRNA)

  22. RNA can form secondary structures by internal base-pairing MCB 1.6

  23. RNA can also be used: • - to store genetic information (viruses) • for catalytic activity (self-splicing, RNaseP) • Regulation

  24. RNA:Typically single stranded: Produced during transcription mRNA: Carries information encoded in genes to direct protein synthesis on ribosomes *Derive from heterogeneous nuclear RNA (hnRNA) mature mRNA processed by splicing (removal of introns and joining of exons), capping (5' end), and polyA tail addition (3' end)

  25. Basic differences between Eucaryotes and Procaryotes (Bacteria) MCB 3.15

  26. DNA Pol vs RNA Pol • DNA replication Gene transcription • Ori promoter • dNTP NTP • Proofreading NO • Replicate dsDNA Only template • Primer require NO

  27. Different at transcriptional level ofProkaryotic vs Eukaryotic cell • One RNA Pol Three • Continuous gene Discontinuous gene • NO intron/Extron Yes • mRNA unstable more stable • No processing Yes(capping,polyA • addition,splicing) • Trancription/Translation NO coupled Polycistron Moncistron

  28. rRNA Components of ribosome: Protein synthesis Small subunit: Single rRNA: 16S (1500 nt) or 18S (1800 nt) Large subunit: Two or three 23S (2900 nt) or 28S (5000 nt), 5S, 5S rRNA: 120 nt, 5.8S rRNA: 160 nt (eukaryotic cytoplasmic ribosomes). Blue color: Prokaryotic; Pink, eukaryotic cell.

  29. tRNA:carriers of activated amino acids used by ribosome for protein synthesis. *Small (73 to 94 nucleotides) RNA charged with amino acids * Cloverleaf secondary structure *Acceptor stem,CCA 3’: end at which amino acid is attached *Anticodon stem and loop: three-base anticodon: decodes mRNA

  30. tRNA

  31. DNA vs RNA Composition: * T in DNA not U to distinguish from T formed by deamination of C * 2' OH in RNA accounts for instability of RNA phosphodiester bond Hydrolysis *RNA:Sensitive to base hydrolysis but resistant to acid hydrolysis *DNA:Resistant to base hydrolysis: Depurinates by acid hydrolysis (apurinic base)

  32. DNA denaturation: Strand separation and loss of base stacking Temperature, pH, ionic strength: Disrupt H bonds Hyperchromic shift of UV absorbance upon denaturation Tm: Midpoint of transition.

  33. Renaturation * Second order kinetics, * Nucleic acid hybridization Measure evolutionary relationships Identify specific gene