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HHMI Research Studio Freshmen Biology Section Instructor: Michael Lehmann Mouse Lipin 1 controls adiposity Control Lipin 1 mutant Lipin 1 up Lipin 1 normal Peterfy et al., 2001: Nature Genetics 27, 121-124 Phan & Reue, 2005: Cell Metabolism 1, 73-83 Lipin’s domain structure is highly conserved SWR SWR SWR NLIP domain mLpin1 hLPIN1 mLpin2 hLPIN2 mLpin3 hLPIN3 D.m. C.e. S.c. S.p. A.t. P.f. CLIP domain mLpin1 hLPIN1 mLpin2 hLPIN2 mLpin3 hLPIN3 D.m. C.e. S.c. S.p. A.t. P.f. mLpin1 hLPIN1 mLpin2 hLPIN2 mLpin3 hLPIN3 D.m. C.e. S.c. S.p. A.t. P.f. mLpin1 hLPIN1 mLpin2 hLPIN2 mLpin3 hLPIN3 D.m. C.e. S.c. S.p. A.t. P.f. * dLipin Lipin1_human IFSSPNLVVRLNGKYYTWMAACPIVMTMITFQKPLTHDAIEQLMSQTVDGKCLPGDEKQE 656 IIDDPNLVVKIGSKYYNWTTAAPLLLAMQAFQKPLPKATVESIMRDKMP----------- 547 *:..*****::..***.* :*.*::::* :*****.: ::*.:* :.: dLipin Lipin1_human AVAQADNGGQTKRYWWSWRRSQDAAPNHLNNTHGMPLGKDEKDGDQAAVATQTSRPTSPD 716 --------KKGGRWWFSWR-GRNTTIKEESKPEQCLAGKAHSTGEQPPQLSLATR----- 593 : *:*:*** .:::: :. .:.. ** .. *:*.. : ::* Excision of “SWR peptide” from Lipin gene 1. Cut dLipin gene with restriction enzymes on either side of conserved sequence and remove excised sequence 2. Insert short DNA sequence to restore open reading frame (ORF) and ligate 3. Transform modified gene into bacteria 4. Select clones that harbor correctly modified gene by PCR (Polymerase Chain Reaction) Chemical composition of DNA • deoxyribose (sugar) • 4 organic bases (pyrimidines and purines) • phosphoric acid DNA = deoxyribonucleic acid RNA = ribonucleic acid DNA contains organic bases, pyrimidines and purines: 9 sugar Thymine 1 1 Nucleoside (Thymidine) in DNA: Thymine Nucleoside (Thymidine) in RNA!: Uracil Nucleoside (Uridine) Nucleosides1) + Cytosine + Adenine + Guanine + Ribose Ribose Ribose Ribose Uridine (U) Cytidine (C) Uracil 1) in DNA: prefix Deoxy…, e. g.: Thymine + Deoxyribose Deoxythymidine (dT) Adenosine (A) Guanosine (G) Nucleotides are the basic building blocks of DNA Phosphate group 1 Nucleotide (2’-Deoxythymidine monophosphate) DNA = deoxyribonucleic acid 2’-deoxy- 2’-deoxy- DNA is a polar molecule: 5’ 3’ Erwin Chargaff, 1949: Chargaff’s rules: A=T C=G ratio of pyrimidine to purine = 1:1 A+T ≠ C+G Watson and Crick, 1953: The double helix model of DNA structure based on: 1. Chargaff’s rules 2. X-ray diffraction analysis of DNA structure • Rosalind Franklin and Maurice Wilkens, 1953: • DNA has helical structure • DNA consists of two polynucleotide strands Watson and Crick, Nature, 1953: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” Denaturation of DNA = separation of DNA strands Renaturation of DNA = Hybridization: joining of two complementary DNA strands The two DNA strands are antiparallel: (10 base pairs) Sugar-phosphate backbone DNA strands of double helix serve as templates for synthesis of complementary strands Reverse transcription Francis Crick,1957: The “Central Dogma” of molecular biology Within each cell, genetic information flows from DNA to RNA to protein Ways to encode sequences of 20 different amino acids….. 4 nucleotides (A, T, C, and G) two-letter code: 42 = 16 three-letter code: 43 = 64 triplet = codon = 3-lettered code word Nirenberg & Leder, 1964: The triplet binding assay Nirenberg & Leder, 1964: The triplet binding assay The genetic code: Stop Stop Stop Met Stop codon = nonsense codon (UAG = amber; UAA = ocher; UGA = opal) Start codon = initiation codon = AUG (for N-formylmethionine in bacteria) The genetic code……. • is a triplet code; a codon consists of 3 nucleotides • is nonoverlapping: each nucleotide is part of only one codon • is commaless: no punctuation is used to separate the code words • includes start and stop codons that set the (open) reading frame (ORF) • is degenerate: an amino acid can be specified by more than one codon • is unambiguous: each codon specifies only one amino acid (one exception) • is universal: is used by all organisms and viruses (with minor exceptions) non-overlapping:5’-AGTTAGTTCCAGTAAGGTTAACC-3’ overlapping: 5’-AGTTAGTTCCAGTAAGGTTAACC-3’ The genetic code is nearly universal 1979: deviations in mitochondria Triplet normal code altered code UGA Stop trp mitochondria (human/yeast) Mycoplasma CUA leu thr mitochondria (yeast) AUA ile met mitochondria (human) AGA AGG arg stop mitochondria (human) UAA stop gln Paramecium Tetrahymena UAG stop gln Paramecium Where? Frameshift mutations THE FAT CAT ATE THE BIG RAT Insertion: Deletion: Insertion + Deletion: THE FAT CAA TAT ETH EBI GRA T T THE FAT CAT AET HEB IGR AT T THE FAT CAA TAE THE BIG RAT The genetic code is a triplet code THE FAT CAT ATE THE BIG RAT 3 Insertions: THE FAT TCA AGT ATE THE BIG RAT Cloning of DNA Restriction enzyme digest linearized vector Ligation recombinant DNA Transformation of E. coli Plasmid E. coli chromosome Ampicillin-resistant clones further selection Restriction and modification in bacteria progeny grows unrestricted on K12 axis of symmetry 5’ 5’ sticky ends • restriction sites are often palindromic Palindrome: MADAM I’ M ADAM OT TO Restriction fragments can have different ends Frequency of restriction sites 4n n = # of bp in site bp = base pairs 1000 bp = 1kb (or kbp) Restriction mapping cloned DNA fragment separate restriction fragments by gel electrophoresis _ (cathode) agarose gel + (anode) construct restriction map DNA ligase closes the remaining open phosphodiester bond nick Kary Mullis, 1986 PCR: Polymerase Chain Reaction • only minute amounts (as little as 1 DNA molecule) of starting material required! denaturation PCR cycle extension • Taq from thermophilic bacterium, Thermus aquaticus annealing PCR: Polymerase Chain Reaction 22 cycles > 4 million copies from a single DNA molecule Structure of a eukaryotic gene gene: nucleotide sequence that encodes a protein or a functional RNA; it includes regions that are required for the regulated expression of the gene upstream 5’ 3’ promoter -20 nontemplate (RNA-like) strand downstream +1 5’ 5’-UTR +20 ATG intron exon AUG template strand 3’-UTR: 3’ untranslated region (trailer) 3’-UTR 3’ 3’ 5’ hnRNA (pre-mRNA) transcription unit 5’-UTR: 5’ untranslated region (leader) UGA Each of the two strands of a DNA double helix can be the template strand Gene 1 5’ 3’ 5’ RNA 3’ 3’ RNA 5’ 3’ 5’ Gene 2 1) > 80 introns! < 1% of hnRNA (not drawn to scale!) 1) defect in genetic disorder called Duchenne muscular dystrophy Construction of a cDNA library Purify mRNA from specific cell type, e.g. cDNA = copy or complementary DNA Transform E. coli Insert cDNA in vector Life cycle of a retrovirus (provirus)