Ch. 3. MOD Fundamentals of Molecular Biology.pptx

Princples of molecular biology for a cell bio college course

1. Ch. 3 – Fundamentals of Molecular Biology

2. Heredity, Genes and DNA One gene copy ( allele ) specifying each trait is inherited from each parent. Example: Plants with identical alleles specifying yellow ( YY ) or green ( yy ) seeds, are crossed. The progeny (F 1 generation) are hybrids ( Yy ) and have yellow seeds: yellow is dominant , green is recessive . Homozygous Heterozygous

3. FIGURE 3.1 Inheritance of dominant and recessive genes (1)

4. Heredity, Genes and DNA Genotype is the genetic makeup of an individual. Phenotype is the resulting physical appearance. The genotype of the F 1 generation is Yy . The phenotype is yellow. Diploid – 2n Haploid – 1n

5. FIGURE 3.2 Chromosomes at meiosis and fertilization (1)

6. Heredity, Genes and DNA Avery, Macloid and McCarty established that Griffith transforming molecule was in fact DNA after they discovered that chloroform could permit its purification from proteins

7. FIGURE 3.4 Transfer of genetic information by DNA

8. Heredity, Genes and DNA Watson and Crick compiled the discoveries of Erwin Chargaff and Rosalind franklin to determine the structure of DNA. Now with its structure determined, theory's into how the molecule works (especially how it’s replicated) could be evaluated.

9. FIGURE 3.5 The structure of DNA

10. FIGURE 3.7 Experimental demonstration of semiconservative replication

11. Expression of Genetic Information The pathway for the flow of genetic information: DNA → RNA → Protein is now known as the central dogma of molecular biology. RNA is synthesized from DNA templates ( transcription ); proteins are synthesized from RNA templates ( translation ).

12. Expression of Genetic Information RNA polymerase catalyzes synthesis of messenger RNA ( mRNA ) from a DNA template. Ribosomal RNA ( rRNA ) is a component of ribosomes, sites of protein synthesis. Transfer RNAs ( tRNAs ) serve as adaptor molecules that align amino acids along the mRNA template.

13. FIGURE 3.8 Synthesis of RNA from DNA

14. Expression of Genetic Information Charging of a tRNA molecule is accomplished by a large family of enzymes: amino-acyl tRNA synthetases Each aa has its own enzyme

15. FIGURE 3.9 Function of transfer RNAs

16. Expression of Genetic Information How can four nucleotide bases specify the sequence of 20 amino acids? The nucleotides are used as triplets (codons) to encode the different amino acids: the genetic code . Thus, addition or deletion of 1 or 2 nucleotides causes frameshift mutations

17. FIGURE 3.10 Genetic evidence for a triplet code

18. Expression of Genetic Information Experiments working with RNA polymers of just one nucleotide would result in peptides made of only one amino acid. This is how the language of codon usage began to be translated .

19. FIGURE 3.11 UUU encodes phenylalanine

20. Expression of Genetic Information All 64 possible triplets (called codons ) were assigned in this way. 61 specify an amino acid; three are stop codons that signal the termination of protein synthesis. The code is degenerate: many amino acids are specified by more than one codon.

21. TABLE 3.1 The Genetic Code First Position Second position (U) Second position (C) Second position (A) Second position (G) Third position U Phe Ser Tyr Cys U Phe Ser Tyr Cys C Leu Ser stop stop A Leu Ser stop Trp G C Leu Pro His Arg U Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G A Ile Thr Asn Ser U Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G G Val Ala Asp Gly U Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G

22. Expression of Genetic Information Genes – Regions of transcribed DNA Open Reading Frames (ORF) – regions of translated RNA UTR – Untranslated regions can be either 5’ or 3’ as they flank the ORF

23. FIGURE 3.12 Basic model for protein-encoding gene expression

24. Expression of Genetic Information RNA tumor virus requires DNA synthesis in infected cells. These viruses (now called retroviruses ) replicate via a DNA intermediate, called a DNA provirus. Reverse transcriptase is used experimentally to generate DNA copies ( cDNA ) of any RNA molecule. This has allowed study and manipulation of eukaryotic mRNAs.

25. FIGURE 3.13 Reverse transcription and retrovirus replication

26. rDNA Technology Recombinant DNA technology allows scientists to isolate, sequence, and manipulate individual genes from any type of cell. It has enabled detailed molecular studies of the structure and function of eukaryotic genes and genomes, and revolutionized our understanding of cell biology.

27. rDNA Technology Restriction endonucleases : enzymes that cleave DNA at specific palindromic sequences. First identified in bacteria, where they provide defense against the entry of foreign DNA. Bacteria have a variety of restriction endonucleases that cleave DNA at more than 100 distinct recognition sites.

28. TABLE 3.2 Recognition Sites of Representative Restriction Endonucleases Enzyme a Source Recognition site Cleavage products Bam HI Bacillus amyloliquefaciens H 5′-GGATCC-3′ 3′-CCTAGG-5′ 5′-G 3′-CCTAG GATCC-3′ G-5′ Eco RI Escherichia coli RY13 5′-GAATTC-3′ 3′-CTTAAG-5′ 5′-G 3′-CTTAA AATTC-3′ G-5′ Hae III Haemophilus aegyptius 5′-GGCC-3′ 3′-CCGG-5′ 5′-GG 3′-CC CC-3′ GG-5′ Hpa II Haemophilus parainfluenzae 5′-CCGG-3′ 3′-GGCC-5′ 5′-C 3′-GGC CGG-3′ C-5′ Not I Nocardia otitidis-caviarum 5′-GCGGCCGC-3′ 3′-CGCCGGCG-5′ 5′-GC 3′-CGCCGG GGCCGC-3′ CG-5′ a Enzymes are named according to their species of isolation, followed by a number to distinguish different enzymes isolated from the same organism.

29. rDNA Technology Restriction mapping : Using restriction enzymes to elucidate a DNA molecule: Generation of a scaffold for sequencing Cut and then shuttle DNA from one system to another (cloning) Eco RI recognizes the sequence GAATTC. This sequence is present at five sites in DNA of bacteriophage λ , so the DNA is digested into 6 fragments ranging from 3.6 to 21.2 kb long. 1 kilobase (kb) = 1000 base pairs

30. FIGURE 3.14 Eco RI digestion and gel electrophoresis of λ DNA and human genomic DNA The fragments can be separated by gel electrophoresis : A gel of agarose or polyacrylamide is placed between two electrodes and the sample is added to the gel. Nucleic acids are negatively charged so they migrate toward the positive electrode. Smaller molecules move more rapidly, allowing the fragments to be separated by size.

31. rDNA Technology Using DNA ligase , fragments of human DNA can be cloned in plasmid vectors (small circular DNA molecules that can replicate independently in bacteria) Recombinant plasmids with human DNA inserts can be introduced into E . coli , where they replicate along with the bacteria to yield millions of copies of plasmid DNA.

32. FIGURE 3.16 Single-stranded overhangs facilitate molecular cloning (1)

33. FIGURE 3.15 Plasmid vectors

34. rDNA Technology cDNA libraries can be generated of entire transcriptomes easily thanks to reverse Transcriptase, restriction enzymes plasmid vectors and DNA ligase

35. FIGURE 3.17 Cloning in plasmid vectors

36. FIGURE 3.18 cDNA cloning (1)

37. rDNA Technology Dideoxy sequencing, also called chain terminating sequencing, is possible because of the fact that dideoxy nucleotides will bind to a growing strand of DNA, but then terminate: Use PCR to generate all the possible polymerization sizes of a DNA strand, each new strand terminating with a ddNTP that fluoresces Separate the sizes in an agarose capillary Measure the fluorescence of each fragment as it comes out of capillary and assemble the sequence.

38. FIGURE 3.19 DNA sequencing

39. rDNA Technology In Class: Cloning and gel electrophoresis exercise