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Fundamental Molecular Biology BL 424 Ch 4 Molecular Biology Student Learning Outcomes: 1. Explain essential principles of molecular biology: expression of genetic information: DNA → RNA → protein. 2. Explain basic tools of recombinant DNA: gene cloning, DNA sequencing, PCR. 3. Describe tools to detect specific nucleic acids and proteins: Southern, Northern, Western, hybridization 4. Describe how tools of recombinant DNA permit detailed analysis of gene function in prokaryotes and eukaryotes, including construction of transgenic organisms The structure of DNA DNA is genetic material: (Figs. 4.5, 4.6) • • • • • double-helical structure (antiparallel chains), complementary bases A-T, C-G semi-conservative replication 5’ → 3’ direction of synthesis; leading, lagging strands Fig 4.2 Chromosomes at meiosis and fertilization Eukaryotes: most cells of plants, animals are diploid: – 2 copies of each chromosome. Meiosis segregates chromosomes → haploid gametes; Fertilization restores diploid progeny. Haploid prokaryotes duplicate DNA; • divide by fission Fig. 4.2 Heredity, Genes, and DNA Classic Mendelian transmission genetics: Gene: determines polypeptide or structural RNA Alleles: alternate versions of genes, encode traits One copy (allele) specifying each trait is inherited from each parent. Genotype: genetic makeup of an individual. Phenotype resulting physical appearance. Fig 4.1 Inheritance of dominant and recessive genes Ex: Parental strains with identical alleles of gene specifying yellow (Y) or green (y) seeds, are crossed: YY x yy. Progeny (F1 generation) are hybrids: yellow seeds: yellow is termed dominant, green recessive. • Genotype of F1 generation is Yy. • Phenotype is yellow. Phenotype of F2 shows • Recessive and dominant: • 2 alleles per individual • 1 allele per gamete Fig. 4.1 Fig 4.3 Gene segregation and linkage Dihybrid crosses: • Genes on different chromosomes segregate independently • Genes on same chromosome mostly stay together - linked Fig. 4.3 Fig 4.8 Colinearity of genes and proteins Colinearity of genes and proteins: • revealed by positions of mutations • 5’-end of gene is NH2-end of protein • 3’ end of gene is COOH- end of protein Fig. 4.8 mutations of TrpA gene of E. coli Central dogma Central dogma of molecular biology: Genetic information DNA → RNA → Protein • RNA polymerase synthesizes RNA from DNA templates (transcription): • complementary base pairing T-A, A-U; C-G • Proteins are synthesized on ribosomes from mRNA templates (translation) • Ribosomal RNA (rRNA) sites of protein synthesis on ribosomes • Transfer RNAs (tRNAs) adaptor molecules that align charged amino acids on mRNA template Triplet code 3 nucleotides specify 1 amino acid; degenerate code Fig. 4.9 Fig. 4.10 Fig 4.13 Reverse transcription and retrovirus replication Retroviruses, group of RNA tumor viruses replicate via synthesis of a DNA intermediate, • Forms DNA provirus that integrates in host (Ex. HIV) • RT carried by virus; critical for forming DNA copy Reverse transcriptase (RT) can make DNA copies of any RNA molecule (cDNA from mRNA) • Clone copy of mRNAs of eukaryotic cells to study Fig. 4.13 Recombinant DNA Recombinant DNA technology (gene cloning) • Permits isolation, sequence, analysis and manipulation of individual genes from any cell. • Enables detailed molecular studies of structure and function of genes and genomes • Revolutionized understanding of cell biology • Series of tools: • • • • • Restriction enzymes, ligase Plasmids, other vectors Gel electrophoresis Transformation of bacteria, Introduction of DNA into other cell types Fig 4.14 EcoRI digestion and gel electrophoresis of λ DNA Restriction endonucleases (RE): • Enzymes cleave DNA at specific sequences – Ex. EcoRI cleaves 5’-GAATTC-3’ • About 100 different enzymes for specific recognition: • Fragments separated by gel electrophoresis • Smaller molecules move more rapidly • Stain DNA to visualize Fig. 4.14 Fig 4.16 Generation of a recombinant DNA molecule Recombinant DNA: gene cloning DNA fragment inserted into DNA molecule (a vector such as a plasmid) capable of independent replication in host cell. Recombinant plasmids introduced into E. coli (transformation); Select plasmid (antibiotic resistance) Plasmid replicates with bacteria: get millions of copies in culture Fig. 4.16 Fig 4.17 Joining of DNA molecules • RE often cleave staggered sites, leaving overhanging single-stranded regions (5’-PO4: 3’-OH) • DNA ligase seals ends (5’-PO4: 3’-OH) Fig. 4.17 Fig 4.18 cDNA cloning • Cloned inserts can be genomic DNA or cDNA • mRNA is copied using reverse transcriptase (RT) • Specific primer is often poly(dT) for eukaryotes (binds poly(A) on mRNA) • Add linker sequences for easier cloning. Fig. 4.18 Fig 4.19 Cloning in plasmid vectors * Review molecular cloning: Fig. 4.19 Fig 4.21 Expression of cloned genes in bacteria Bacterial expression vectors contain regulatable promoters Inserted genes are expressed at high levels Expression in eukaryotic cells may be needed if posttranslational modifications (phosphorylation, sugars) are required (also needs eukaryotic promoters). ** Consider cloning Fig. 4.21 DNA sequencing DNA sequencing gives order of bases understand genes, genomes, structure, function Dideoxy method uses premature termination of DNA synthesis. DNA synthesis is initiated with synthetic primer. Dideoxynucleotides included with normal nucleotides; each ddNTP labeled different fluorescent dye ddNTPs stop DNA synthesis because no 3 OH group for addition of next dNTP. Fig. 4.20 ddNTP Fig 4.20 DNA sequencing (Part 2) Dideoxynucleotides stop DNA synthesis because no 3 OH Get series of fragments, partial copies of target, terminated. Fragments separated by gel electrophoresis; laser beam excites fluorescent dyes, and records color at each position. Detection of Nucleic Acids and Proteins 3.Detection of specific nucleic acids, proteins • Polymerase chain reaction (PCR) amplifies DNA • Nucleic acid hybridization detects nucleic acids: • Southern – DNA on gel • Northern – RNA on gel • Microarrays - all the mRNAs • Antibodies detect proteins • Western – proteins on gel • Immunofluorescence • Immunoprecipitation Detection of Nucleic Acids and Proteins Polymerase chain reaction (PCR) amplifies DNA • • • • • Repeated replication of segment of DNA: specific primers Rounds of denature at 95oC, anneal to primer (55oC) synthesis of DNA (68oC) Heat-stable DNA polymerase from bacteria of hot springs (Thermus aquaticus (Taq) Fig. 4.23 PCR Fig 4.24 Detection of DNA by nucleic acid hybridization Nucleic acid hybridization uses complementary base pairing to detect specific nucleic acid sequences • DNA or RNA probes Fig. 4.24 Fig 4.25 Southern blotting Southern blotting detects specific genes (DNA). • • • • DNA digested with RE, Fragments separated by gel electrophoresis. DNA fragments transferred to membrane (blotted). Filter incubated with labeled nucleic acid probe Northern blotting detects RNA: separate RNA on gel, transfer, hybridize with specific probe • Sizes, amount mRNA • Different tissues Fig. 4.25 Fig 4.26 Screening a recombinant library by hybridization Recombinant DNA libraries: collections of clones containing all genomic or mRNA sequences of particular cell type. (vector can be plasmid, virus) Ex. Clone random fragments in vector, test for specific gene Fig. 4.26 Fig 4.27 DNA microarrays Hybridization to DNA microarrays allows 1000s of genes analyzed simultaneously. • DNA microarray on glass slide has oligonucleotides or fragments of cDNAs printed by robotic system in tiny spots • Compare expression in two cell types (cancer vs. normal) • Isolate mRNA • Use RT then PCR with different dyes Ex. Cancer red, Normal green If equal, yellow color Fig. 4.27 Fig 4.28 Fluorescence in situ hybridization In situ hybridization detects homologous DNA or RNA sequences in chromosomes or intact cells. Hybridization of fluorescent probes to specific cells or subcellular structures • seen by microscope Different probe for each human chromosome Fig. 4.28 Detection of Nucleic Acids and Proteins Antibodies detect specific proteins Antibodies - proteins from immune cells (B lymphocytes) react to foreign molecules (antigens). • Different antibodies recognize unique antigens Antibodies can detect proteins in intact cells. Cells stained with antibodies labeled with fluorescent dyes, or tags visible by electron microscopy. Fig. 4.31 Human Cells in culture: actin (blue), tubulin (yellow), nuclear stain (red) Fig 4.29 Western blotting Immunoblotting (Western blotting). Proteins separated by size on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS detergent binds, denatures proteins, gives – charge Small proteins faster Transfer to membrane Antibodies bind to specific proteins Fig. 4.29 Fig 4.30 Immunoprecipitation Immunoprecipitation Purifies specific proteins. Cells (radioactive proteins) incubated with antibodies Antigen-antibody complexes are isolated and electrophoresed. Co-immunoprecipitation asks which proteins are bound together in complexes; Antibody purifies one, ask which other proteins Fig. 4.30 Gene Function in Eukaryotes Analysis of gene function: • Revealed by altered phenotypes of mutant organisms. • Study function of cloned gene by reintroducing it into eukaryotic cells • Can use specific mutations in genes, deletions of genes, or add specific genes (can have conditional (ts) mutants) • Use embryonic stem cells in culture, then transfer to whole animals or plants Transgenic organisms have altered genomic DNA Genetically modified organisms (GMO) Fig 4.32 Cloning of yeast genes Model eukaryote yeast: Transform yeast with plasmids carrying selectable genes (prototrophic, LEU+) Yeast vectors are shuttle vectors that reproduce in E. coli Yeast: • grow as haploid or diploid • easily grown in culture, reproduce rapidly (90 min), • small genome. • Mutants available for every gene • ts mutants for essential genes Fig. 4.32 Fig 4.33 Introduction of DNA into animal cells Cloned DNA can be introduced into plant and animal cells (gene transfer, transfection). In most cells, DNA is transcribed for several days: transient expression. In 1% or less of cells, DNA integrates into genome and is stably transferred to progeny cells (can select) Fig. 4.33 Fig 4.34 Retroviral vectors Animal viruses, especially retroviruses, are vectors to introduce cloned DNAs into cells. Fig. 4.34 Fig 4.35 Production of transgenic mice Transgenic mice model system: Cloned genes in germ line of multicellular organisms Microinject cloned DNA into pronucleus of fertilized egg; Check offspring for gene (fur color, check by Southern blot). Easier to add a new gene – can be inserted anywhere Fig. 4.35 Fig 4.36 Introduction of genes into mice via embryonic stem cells Embryonic stem (ES) cells for transgenic mice: • Cloned DNA put into ES cells in culture – select drug-R • Stably transformed cells introduced into mouse embryos • Check gene is in germline, transfer to progeny Similar techniques to make other transgenic animals Fig. 4.36 Transgenic plants Transgenic plants (genetically modified crops, GMOs) have specific genes added or deleted. Add DNA to cells in culture with DNA gun, or use Ti plasmid with Agrobacterium (root nodule symbiont). Many plants can regenerate from callus tissue Fig. 4.37 Many GFP transgenic animals and plants now exist Widespread applications of GFP Fig 4.39 Gene inactivation by homologous recombination Specific mutagenesis - homologous recombination of synthetic DNA to make particular mutations: • Powerful tool in studying function of eukaryotic genes • Mutate one copy of gene to be cancer-causing oncogene • More difficult to delete both copies (knockout) • Easier to add a gene Fig. 4.39 specific mutagenesis Fig 4.40 Production of mutant mice by homologous recombination in ES cells Knockout mice Transgenic mice with both copies of a gene mutated: • Powerful tool • • May be lethal Techniques to have KO only in some tissues Fig. 4.40 Fig 4.41 Inhibition of gene expression by antisense RNA or DNA Antisense nucleic acids • Use RNA or single-stranded DNA complementary to mRNA of the gene of interest (antisense). • Hybridize with mRNA and block translation into protein RNA interference (RNAi) (discovered in C. elegans): • injection of double-stranded RNA inhibited expression of gene with complementary mRNA sequence • Involves RISC complex binding mRNA, cleaving (Fig. 4.36) Fig. 4.35 antisense Chapter 5 BL 424 Chapter 5 Genomes brief: Student learning outcomes: • Sequences of many genomes known • Explain structure of eukaryotic chromosomes includes telomeres, centromeres • Describe how eukaryotic DNA is linear, is compacted on nucleosomes (by histones) • Explain that eukaryotic genes have introns, exons – much of DNA is noncoding – Splicing occurs on the primary transcript – Alternative splicing provides additional proteins Fig 5.2 The structure of eukaryotic genes • Gene coding sequences (exons) are separated by noncoding sequences (introns). • Entire gene is transcribed to RNA; introns removed by splicing; only exons are included in mRNA. • Average human gene 8 introns (gene 27 kb, coding 2.5 kb) Fig. 5.2 Alternative splicing Alternative splicing • provides diversity of final proteins • different tissues, different times of development Fig. 5.3 DNA is organized in nucleosomes in eukaryotes • Eukaryotic DNA is linear, organized in nucleosomes • Histones (basic small proteins) bind DNA Fig. 5.11 Review Review questions: 4.7. Starting with 2 sperm, how many copies of a specific gene sequence will be obtained after 10 cycles of PCR? After 30 cycles? 4.12. Nucleic acids have net negative charge and are separated by electrophoresis on basis of size. Proteins have different charges, and so how are they separated by size in electrophoresis? 4.11. What is critical feature of cloning vector that permits isolation of stably transfected mammalian cells? 5.1. Many eukaryotic organisms have genomic sizes much larger than their complexity would seem to require; explain the paradox.