StemCell Therapy

MOLECULAR GENETICS You Are Here* molecular basis of inheritance Genes ---> Enzymes ---> Metabolism (phenotype) Central Dogma of Molecular Biology* DNA -transcription-->RNA-translation--> Protein Concept Activity -17.1 Overview of Protein Synthesis - INFORMATION FLOW

What is a GENE = ? DNA is the genetic material... [ but what about, retroviruses, as HIV & TMV, contain RNA ] - a discrete piece of deoxyribonucleic acid - linear polymer of repeating nucleotide monomers nucleotides* --> A adenine,C cytosine T thymidine,G guanine --> polynucleotide*

Technology with a Twist - Understanding Genetics

INFORMATION PROCESSING & the CENTRAL DOGMA - the letters of the genetic alphabet... are the nucleotides A, T, G, & C of DNA - the unit of information is CODON = genetic 'word' a triplet sequence of nucleotides 'CAT' in a polynucleotide 3 nucleotides = 1 codon (word) = 1 amino acid in a polypeptide - the definition of (codon) word = amino acid - Size of Human Genome: 3,000,000,000 base pairs or 1.5b in single strand of DNA genes 500,000,000 possible codons (words or amino acids) - average page your textbook = approx 850 words thus, human genome is equal to 588,000 pages or 470 copies of bio text book reading at 3 bases/sec it would take you about 47.6 years @ 8h/d - 7d/w WOW... extreme nanotechnology Mice & humans (indeed, most or all mammals including dogs, cats, rabbits, monkeys, & apes) have roughly the same number of nucleotides in their genomes -- about 3 billion bp. It is estimated that 99.9% of the 3billion n's of human genome is same person to person.

Experimental Proof of DNA as Genetic Material...

1. Transformation Experiments of Fred Griffith... (1920's) Streptococcus pneumoniae -pathogenic S strain & benign R transforming 'principle'* (converting R to S cells) is the genetic element 2. Oswald Avery, Colin MacLeod, & Maclyn McCarty... (1940's) suggest the transforming substance* is DNAmolecules, but... 3. Alfred Hershey & Martha Chase's* 1952 bacteriophage experiments*... VIRAL REPLICATION*[ phage infection & & lytic/lysogenic* ] a genetically controlled biological activity (viral reproduction) they did novel experiment... 1st real use radioisotopes in biology* CONCLUSION - DNA is genetic material because (32P) nucleic acid not (35S) protein guides* viral replication Sumanas, Inc. animation - Lifecycle of HIV virus

Structure of DNA ..... Discovery of Double Helix... Watson's book Nobel prize* -JD Watson, Francis Crick,Maurice Wilkins, but [ Erwin Chargaff & RosyFranklin]... Race for the Double Helix "Life Story" - a BBC dramatization of the discovery of DNA. used two approaches to decipher structure: 1. model building - figure* (are the bases in/out; are the sugar-P's in/out?) 2. x-ray diffraction*pattern* favor a DNA helix of constant diameter* we know now: DNA is a double stranded, helical, polynucleotide chains, made of... 4 nucleotides - A, T, G, C (purine & pyrimidines) in 2 polynucleotide strands (polymer chains) head-tail polarity [5'-----3'] - strands run antiparallel held together via weak H-Bonds & complimentary pairing - Chargaff's rule*..... A:T G:C A + G / T + C = 1.0 Fig's:sugar-P backbone*,base*pairing, dimensions*, models of DNA structure* john kyrk's animation of DNA & Quicktime movie of DNA structure literature references & myDNAi timeline*

Replication of DNA... (Arthur Kornberg - 1959 Nobel - died 10/26/07) copying of DNA into DNA is structurally obvious??? [figure*] Patterns of Replication* = conservative, semi-conservative, & dispersive Matt Meselson & Frank Stahl1958 - experimental design* can we separate 15N-DNA from 14N-DNA - (OLD DNA from NEW DNA)? sedimentation of DNA's (sucrose gradients --> CsCl gradients* & picture*) we can predict results... figure* & overview& all possible results Sumanas, Inc. animation - Meselson-Stahl DNA Replications*

Model of Replication is bacterial with DNA polymerase III... several enzymes* form a Replication Complex (Replisome) & include: helicase - untwists DNA topoisomerase [DNA gyrase] - removes supercoils, single strand binding proteins - stabilize replication fork, Primase - makes RNA primer POL III - synthesizes new DNA strands DNA polymerase I - removes RNA primer 1 base at a time, adds DNA bases DNA ligase repairs Okazaki fragments (seals lagging strand 3' open holes) Concept Activity - DNA Replication Review Structure of DNA polymerase III* copies both strands simultaneously, as DNA is Threaded Through a Replisome* a "replication machine", which may be stationary by anchoring in nuclear matrix Continuous & Discontinuous replication occur simultaneously in both strands

EVENTS: 1. DNA pol III binds at the origin of replication site in the template strand 2. DNA is unwound by replisome complex using helicase & topoisomerase 3. all polymerases require a preexisting DNA strand (PRIMER) to start replication, thus Primase adds a single short primer to the LEADING strand and adds many primers to the LAGGING strand 4. DNA pol III is a dimer adding new nucleotides to both strands primers direction of reading is 3' ---> 5' on template direction of synthesis of new strand is 5" ---> 3' rate of synthesis is substantial 400 nucleotide/sec 5. DNA pol I removes primer at 5' end replacing with DNA bases, leaves 3' hole 6. DNA ligase seals 3' holes of Okazaki fragments on lagging strand the sequence of events in detail* and DNA Repair* Rates of DNA synthesis: myDNAi movie of replication* native polymerase: 400 bases/sec with 1 error per 109 bases artificial: phophoramidite method (Marvin Caruthers, U.Colorado); ssDNA synthesis on polystyrene bead @ 1 base/300 sec with error rate of 1/100b

GENE Expression the Central Dogma of Molecular Biology depicts flow of genetic information Transcription - copying of DNA sequence into RNA Translation- copying of RNA sequence into protein DNA sequence -------> RNA sequence -----> amino acid sequence TAC AUG MET triplet sequence in DNA --> codon in mRNA ---->amino acid in protein Information : triplet sequence in DNA is the genetic word [codon] Compare Events: Procaryotes* vs. Eucaryotes* = Separation of labor Differences DNA vs. RNA (bases & sugars) and its single stranded Flow of Gene Information (FIG*) - One Gene - One enzyme (Beadle & Tatum) 18.3-Overview: Control of Gene Expression

Transcription - RNA polymerase Concept Activity 17.2 - Transcription RNA*polymerase - in bacteria Sigma factor* binds promoter & initiates* copying* [pnpase] transcription factors* are needed to recognize specific DNA sequence [motif*], binds to promoter DNA region [ activators & transcription factors*]* makes a complimentary copy* of one of the two DNA strands[sense strand] Quicktime movie of transcription*myDNAi Roger Kornberg's movie of transcription (2006 Nobel)* Kinds of RNA [table*] tRNA - small, 80n, anticodon sequence, single strand with 2ndary structure* function = picks up aa & transports it to ribosome rRNA - 3 individual pieces of RNA - make up the organelle = RIBOSOME primary transcript is processed into the 3 pieces of rRNA pieces(picture*) & recall structure of ribosome

Other classes of RNA: small nuclear RNA (snRNP's)- plays a structural and catalytic role in spliceosome* there are 5 snRNP's making a spliceosome [U1, U2, U4, U5, & U6]; they and participate in several RNA-RNA and RNA-protein interactions

SRP (signal recognition particle) - srpRNA is a component of the protein-RNA complex that recognizes the signal sequence of polypeptides targeted to the ER - figure*

small nucleolar RNA (snoRNA) - aids in processing of pre-rRNA transcripts for ribosome subunit formation in the nucleolus

micro RNA's (micro-RNA)- also called antisense RNA & interfereing RNA; c7-fig 19.9 short (20-24 nucleotide) RNAs that bind to mRNA inhibiting it. figure* present in MODEL eukaryotic organisms as:roundworms, fruit flies, mice, humans, & plants (arabidopsis); seems to help regulate gene expression by controlling the timing of developmental events via mRNA action also inhibits translation of target mRNAs. ex: siRNA --> [BARR Body*]

TRANSLATION - Making a Protein process of making a protein in a specific amino acid sequence from a unique mRNA sequence...[ E.M. picture* ] polypeptides are built on the ribosome (pic) on a polysome [ animation*] Sequence of 4 Steps in Translation... [glossary] 1. add an amino acid to tRNA -- > aa-tRNA - ACTIVATION* 2. assemble players [ribosome*, mRNA, aa-tRNA] - INITIATION* 3. adding new aa's via peptidyl transferase - ELONGATION* 4. stopping the process - TERMINATION* Concept CD Activity - 17.4 Events in Translation Review the processes - initiation, elongation, & termination myDNAi real-time movie of translation*& Quicktime movie of translation Review figures & parts: Summary fig* [ components, locations, AA-site, & advanced animation ] [ Nobel Committee static animations of Central Dogma ]

GENETIC CODE... ...is the sequence of nucleotides in DNA, but routinely shown as a mRNA code* ...specifies sequence of amino acids to be linked into the protein coding ratio* - # of n's... how many nucleotides specify 1 aa 1n = 4 singlets, 2n= 16 doublets, 3n = 64 triplets Student CD Activity - 11.2 - Triplet Coding S. Ochoa (1959 Nobel) - polynucleotide phosphorylase can make SYNTHETIC mRNA Np-Np-Np-Np <----> Np-Np-Np + Np Marshall Nirenberg (1968 Nobel)- synthetic mRNA's used in an in vitro system 5'-UUU-3' = pheU + C --> UUU, UUC, UCC, CCC UCU, CUC, CCU, CUU the Genetic CODE* - 64 triplet codons [61 = aa & 3 stop codons] universal (but some anomalies), 1 initiator codon (AUG), redundant but non-ambiguous, and exhibits "wobble*".

GENETIC CHANGE - a change in DNA nucleotide sequence (= change in mRNA) - 2 significant waysmutation & recombination [glossary] 1. MUTATION - a permanent change in an organism's DNA*that results in a different codon = different amino acid sequence Point mutation* - a single to few nucleotides change... - deletions, insertions, frame-shift mutations* [CAT] - single nucleotide base substitutions* : non-sense = change to no amino acid (a STOP codon) UCA --> UAA ser to non mis-sense = different amino acid UCA --> UUA ser to leu Sickle Cell Anemia* - a mis-sense mutation... (SCA-pleiotropy) another point mutation blood disease - thalassemia - Effects = no effect, detrimental (lethal), +/- functionality, beneficial

2. Recombination (Recombinant DNA)newly combined DNA's that [glossary]* can change genotype via insertion of NEW (foreign) DNA molecules into recipient cell 1. fertilization*- sperm inserted into recipient egg cell* --> zygote [n + n = 2n] 2. exchange of homologous chromatids via crossing over* = new gene combo's 3. transformation* - absorption of 'foreign' DNA by recipient cells changes cell 4. BACTERIAL CONJUGATION* - involves DNA plasmidsg* (F+ & R = resistance) conjugation may be a primitive sex-like reproduction in bacteria[Hfr*] 5. VIRAL TRANSDUCTION - insertion via a viral vector(lysogeny* &TRANSDUCTION*) general transduction - pieces of bacterial DNA are packaged w viral DNA during viral replication restricted transduction - a temperate phage goes lytic carrying adjacent bacterial DNA into virus particle 6. DESIGNER GENES - man-made recombinant DNA molecules

Designer Genes - Genetic Engineering - Biotechnology

RECOMBINANT DNA TECHNOLOGY... a collection of experimental techniques, which allow for isolation, copying, & insertion of new DNA sequences into host-recipientcells by A NUMBER OF laboratory protocols & methodologies

Restriction Endonucleases-[glossary]*... diplotomic cuts (unequal) at uniqueDNA sequences Eco-R1-figure* @ mostly palindromes... [never odd or even] 5' GAATTC 3' 5' G. . . . . + AATTC 3' 3' CTTAAG 5' 3' CTTAA .. .. G 5' campbell 7/e movie* DNA's cut this way have STICKY (complimentary) ENDS & can be reannealed or spliced* w other DNA molecules to produce new genes combosand sealed via DNA ligase. myDNAi movie of restriction enzyme action*

Procedures of Biotechnology? [Genome Biology Research] A. Technology involved in Cloning a Gene...[animation* & the tools of genetic analysis] making copies of gene DNA 1. via a plasmid*[ A.E. fig& human shotgun plasmid cloning & My DNAi movie*] 2. Librariesg... [ library figure* & BAC's* &Sumanas animation - DNA fingerprint library] 3. Probesg... [ cDNAg & reverse transcriptaseg & DNA Probe Hybridizationg... cDNA figure*& cDNA library* & a probe for a gene of interest* finding a gene with a probe among a library*] 4. Polymerase Chain Reactiong & figure 20.7* & animation*+Sumanas, Inc. animation* the PCR song PCR reaction protocol & Xeroxing DNA & Taq polymerase

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Molecular Genetics - DNA, RNA, & Protein

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