How Cell Read the Genome

1. How Cell Read the Genome 潘台龍博士 長庚大學中醫系 pan@mail.cgu.edu.tw

3. Goal of Human Genome Project

4. Introduction transcription translation DNA RNA Protein DNA and RNA polymerase genetic code (codon) messenger RNA (mRNA) transfer RNA (tRNA) ribosome RNA (rRNA) amino acids-peptide bond-protein

5. Deoxyribose nucleic acid (DNA) and ribonucleic acid (RNA) • Pentose Ribose-RNA Deoxyribose-DNA • Base Purines Pyrimidines Adenine (A) Uracil (U) Guanine (G) Thymine (T) Cytosine (C) • Phosphate Nucleotides

6. DNA is the Genetic Substance Alfred Hershey & Martha Chase 35S & 32P

7. DNA Double helix of two antiparallel chains with complementary nucleotide sequences (1953 James D Watson and Francis H.C)

8. Structure of DNA • Base-pair complementarity between a larger purine (A & G) and a smaller pyrimidine (C or T) • Natural DNA: A with T by two hydrogen bonds, G with C by three hydrogen bonds

9. Double helical DNA Stability of natural DNA  right-handed  sugar-phosphate backbone-outside  bases-inside  0.34 nm between bases  B form of DNA in most of the time in cells = a complete 3600 every 3.4 nm (10 bases)  A form of DNA in non-aquwous solution = a coplete 3600 in 2.3 nm (11 bases) Z (zigzag) DNA  left-handed Bent DNA  Flexible about long axis  Bent by/without DNA binding protein

10. Denaturation of DNA Melting tempreature (Tm) =separation of DNA double strands  Increased Tm =G-C rich DNA (3 hydrogen bonds)  As DNA denatures, UV absorption increases. Other factors to destabilize the double helix  low ion concentration  alkaline solution formamide Renaturation of DNA  By lowering the temperature or increasing the ion concentration  Other single strand DNA not related in sequence never affect the renaturation of two complimentary strands of DNA.

11. Many DNA molecules in all bacteria and many virus are circular  Nick (one of the strands is cut) is necessary to unwind and separate two strands (one for circular, one for linear).  Natural nicking upon DNA replication  Experimental cleave with deoxyribonuclease

12. 4 rules of the synthesis of nucleic acids transcription DNA RNA replication RNA polymerase 1. Copy of temprate: DNA synthesis from DNA temprate retroviruss: DNA synthesis from RNA temprate 2. Nucleic acid strand growth is in one direction : 5’-3’ 3. Special enzymes called polymerase make RNA or DNA RNA synthesis by copying DNA by RNA polymerase DNA polymerase requires a primerto make DNA 4. Duplex DNA synthesis requires a special growing fork. Leading strand-5’ to 3’-in the direction of the fork lagging strand (Okazaki)-discontinuous-DNA ligase

13. DNA replication Template strand 5‘ 3‘ 3‘ 5‘ primer

14. Replication bubble of DNA under EM

16. DNA Sequencing • Like PCR, it utilizes DNA polymerase and thermal cycling • Only reads the sequence of 1 strand of DNA using 1 primer • Utilizes the Sanger dideoxy termination method • Reaction generates a population of dye-terminated DNA fragments

18. Cycle Sequencing

19. DTCS DeoxyNucloetide DideoxyNucloetide

20. DTCS Extension and Termination

21. Extension and Termination • Simultaneous reactions terminate at different lengths • Reactions generate multiple fragments of all sizes from 1 to 1000+ bases

22. Separation and Detection • Fragments are separated by capillary gel electrophoresis • Laser-induced fluorescence of dye terminators is sequentially read by the PMT sensor

23. Sequencing Results www.ncbi.nlm.nih.gov

24. Overview of Telomere and Telomerase • Background: Several studies have reported telomere and telomerase in a variety of human malignant tumor. • Telomere, located at the end of eukaryotic chromosomes, is considered important in protecting and stabilizing the chromosomal ends. • Telomerase is a ribonucleoprotein polymerase that can compensate for telomere losses. • Telomerase activity is present in almost all carcinomas and can be detected in some pre-neoplasias and early stage cancers.

25. Telomere dynamics and chromosomal instability in human epithelial cancers

26. Action of Telomere and Telomerase

27. One Gene, One Outcome??? One Gene, One Protein???

29. What is Single Nucleotide Polymorphism ? • Many of differences among people have a genetic basis - alterations in the DNA that change the way important proteins are made. • Sometimes the alterations involve a single base pair (the smallest building block of DNA) and are shared by many people. Such single base pair differences are called "single nucleotide polymorphisms", or SNPs. However, the majority of the SNPs do not produce physical changes in people with affected DNA. • Estimate ~ 15M SNPs in total throughout human genome (one SNP every 200 bases).

30. Pharmacogenomics Genetics of Drug Efficacy and Toxicity

31. Genetics of Drug Response

32. Future Potential of Pharmacogenomics

34. RNA Similar to DNA in chemical makeup However, additional hydroxyl group at the 2’ position and thymine (T) in DNA is replaced by uracil (U). More chemically labile than DNA (cleaved into mononucleotides even by alkaline solution)

35. Structure and function of RNA 2 dimensional structure  stem-loop and hairpin 3 dimensional structure (pseudoknot)  small RNA: transfer RNA (tRNA)  large RNA: ribosome RNA (rRNA) Function  carry out genetic expression and convert to proteins Flexible about long axis  folded domains-catalytic ability (cut RNA an chain) e.g. phosphotransferase (cleave and unite-”splicing”)  Various function in RNA bound to protein: the largest is ribosomes ( small nuclear ribonucleoproteins (“snurps”)

36. Transcription of RNA

37. Eukaryotic primary RNA transcripts are processed to form functional mRNAs DNA RNA Protein transcription translation • RNA processing • Modification of primary RNA is necessary for mRNA to be • functional and capable of being translated into protein. • 5’ cap and 3’ poly-A polymerase • Exon and intron • exon: amino-acid sequence coding segment • intron: protein non-coding segment • Removal of intron (splicing) is required to make • functional mRNA • 5’ and 3’ untranslated regions also exist

38. mRNA carries information from DNA in a three-letter genetic code (codon) transcription translation DNA RNA Protein A.C.G.U A.C.G.T • Triple code (codon) : 4x4x4=64 • 61 codons encode 20 amino acids • synonymous and degenerate : • e.g. leucine, serine, and arginine. Each have six. • Initiator(AUG) • Terminator ( UAA, UGA,UAG) • Reading frame (from initiator to terminator) • Frame shift yields different polypeptides.

39. Modification of RNA Transcription

40. 5‘ methylated cap RNA processing 7-methylguanylate

41. Evidence of Splicing

43. Regulation of RNA Transcription

44. High Through-put Screening

45. cDNA Microarray Platform

46. Gene Analysis by Bioformatic

47. Protein synthesis: the three roles of RNA in translation transcription translation DNA RNA Protein • Messenger RNA (tRNA) • encodes the genetic information copied from DNA • Transfer RNA (tRNA) • The amino acids specified by the sequence of an mRNA • are each attached to specific tRNAs, then carried to and • deposited at the growing end of a polypeptide chain • Ribosome RNA (rRNA) • 1. attracts mRNA, catalyzes peptide-bond formation and • binds a set of proteins to form ribosomes. • 2. Ribosomes bound tRNAs can move along an mRNA to • translate its encoded genetic information into protein.

48. Overview in Protein Synthesis

49. Structure of tRNA

50. How to work in tRNA ?