Overview of Molecular Biology Expression of Genetic Information Recombinant DNA Detection of Nucleic Acids Gene Function

1. Overview of Molecular BiologyExpression of Genetic InformationRecombinant DNADetection of Nucleic AcidsGene Function in Eukaryotes

2. Introduction • What is molecular biology? • A means of attempting to determine the mechanism of transmission and expression of genetic information which ultimately dictates cell structure and function. • All cells share a number of basic properties, which allows scientists to choose simple organisms as model systems. • Numerous experiments have established that similar molecular mechanisms are operative in organisms as diverse as E. coli and humans.

3. Heredity, Genes, and DNA • All organisms inherit the genetic information specifying their structure and function from their parents. • Each trait is determined by a pair of inherited factors called genes (Gregor Mendel). • An allele specifies each trait and is one gene copy that is inherited from each parent. • Chromosomes are the carriers of genes and consist of long DNA molecules and associated proteins.

4. 4.1 Inheritance of dominant and recessive genes • A dominant allele determines the phenotype of an organism when more than one allele is present. • A recessive allele is masked by a dominant allele. • Genotype is the genetic composition of an organism. • Phenotype is the physical appearance of an organism.

5. Genes and Chromosomes • Chromosomes are the carriers of genes and consist of long DNA molecules and associated proteins. • A diploid organism or cell carries two copies, while a haploid organism carries one copy of each chromosome • Meiosis is the division of diploid cells to haploid progeny, consisting of two sequential rounds of nuclear and cellular division. Fig. 4.2. Chromosomes at meiosis and fertilization

6. 4.3 Gene segregation and linkage • The fundamentals of mutation, genetic linkage, and the relationships between genes and chromosomes were largely established by experiments performed with the fruit fly, Drosophila melanogaster. • A mutation is a genetic alteration.

7. Identification of DNA as the Genetic Material • The one gene–one enzyme hypothesis states that each gene specifies the structure of a single enzyme. • The first evidence leading to the identification of DNA as the genetic material came from studies in bacteria. • Transformation is the transfer of DNA between genetically distinct bacteria. Fig.4.4. Transfer of genetic information by DNA. The original Expt. was performed in Pneumococcus.

8. The Structure of DNA • DNA is a helical (turns every 3.4 nm) molecule composed of four nucleic acid bases linked to phosphorylated sugars: • two purines (A, G) • and two pyrimidines (C, T) linked to phosphorylated sugars. • The base pairing between the two strands of a DNA is complementary. Fig. 4.5. The structure of DNA. DNA is a double helix with the bases on the inside and the sugar-phosphate backbones on the outside of the molecule.

9. Replication of DNA • How does DNA direct its own replication? • Semiconservative replication occurs when one strand of parental DNA is conserved in each progeny DNA molecule. • Replication is catalyzed by DNA polymerase. Fig. 4.6. Semiconservative Replication of DNA.

10. 4.7 Experimental demonstration of semiconservative replication

11. Expression of Genetic Information • Genes act by determining the structure of proteins. • Proteins are polymers of 20 amino acids, the sequence of which determines their structure and function.

12. Colinearity of Genes and Proteins • The relationship between genes and enzymes was that the order of nucleotides in DNA specified the order of amino acids in a protein. • The first direct link between a genetic mutation and an alteration in the amino acid sequence of a protein was made in 1957 -- Sickle-cell anemia patients Figure 4.8. The sequence of DNA dictates the amino acid sequence of a protein.

13. The Role of Messenger RNA • What directs protein synthesis? • RNA is a likely candidate for such an intermediate because the similarity of its structure to that of DNA suggested that RNA could be synthesized from a DNA template. Figure 4.9. Syntheis of RNA from DNA.

14. The Role of Messenger RNA • The central dogma of molecular biology states that RNA molecules are synthesized from DNA templates, and proteins are synthesized from RNA templates. • Transcription is the synthesis of an RNA molecule from a DNA template. • Translation is the synthesis of a polypeptide chain from an mRNA template. • There are 3 types of RNA: • MessengerRNA • Transfer RNA • Ribosomal RNA

15. The Genetic Code • The genetic code is the correspondence that takes place between nucleotide triplets and amino acids in proteins. • Codons are the basic units of the genetic code. Fig. 4.8. Genetic evidence for a triplet code was found after a series of mutations consisting of additions of one, two, or three nucleotides were studied in the rII gene of bacteriophage T4

17. Recombinant DNA • Restriction Endonucleases • Generation of Recombinant Molecules • DNA Sequencing • Expression of Cloned Genes

18. Restriction Endonucleases • Restriction endonucleases are enzymes that cleave DNA at specific sequences.

19. 4.14 EcoRI digestion and gel electrophoresis ofl DNA Restriction Enzymes are used to cut a piece of DNA into fragments. Gel electrophoresis is a common method in which molecules are separated based on the rates of their migration in an electric field.

20. 4.15 Restriction maps of land adenovirus DNAs • Restriction maps of DNA molecules show the locations of cleavage sites for multiple different restriction endonucleases.

21. Generation of Recombinant DNA Molecules • Molecular cloning is a process wherein a DNA fragment of interest is inserted into a vector. • A vector is a DNA molecule that is capable of independent replication in a host cell. • A plasmid is one type of vector – it is a small circular DNA molecules that can replicate independently in bacteria. • A recombinant molecule, or molecular clone, is composed of the DNA insert linked to vector DNA sequences.

22. 4.16 Generation of a recombinant DNA molecule

23. Generation of Recombinant DNA Molecules • Creating recombinant molecules: • Digestion of DNA with restriction endonucleases. • Use of Gel Electrophoresis to isolate DNA fragments • Ligation of digested DNA fragment to digested vector DNA – DNA Ligase. Fig. 4.17. Joining of DNA molecules.

24. Generation of Recombinant DNA Molecules • mRNA sequences can be closed too. • Complementary DNA (cDNA) is the DNA product of reverse transcription. Fig. 4.18. Cloning of cDNA.

25. Vectors for Recombinant DNA • Many different types of vectors can be used for cloning DNA. This may be variable depending on on the size of the insert DNA and the purpose of the experiment. • Plasmids – most common tool for cloning • Expression vectors – for gene expression • Viral vectors – to produces virus particles • Bacteriophage vectors are also used for the isolation of either genomic or cDNA clones from eukaryotic cells. • Cosmid vectors accommodate large inserts (45 kb).

26. 4.19 Cloning in plasmid vectors • An origin of replication is the DNA sequence that signals the host cell DNA polymerase to replicate the DNA molecule.

27. DNA Sequencing • Determination of the nucleotide sequences. • Dideoxynucleoties are nucleotides that lack the normal 3¢ hydroxyl group of deoxyribose.

28. Expression of Cloned Genes • To study protein function, it is often necessary to express a gene in eukaryotic cells. • An expression vector is a plasmid or a phage vector that contains sequences that drive transcription and translation of the inserted gene in bacterial cells. Fig. 4.21. Expression of cloned genes in bacteria.

29. Detection of Nucleic Acids and Proteins • Understanding the role of genes within cells requires analysis of the intracellular organization and expression of individual genes and their encoded proteins. • Polymerase Chain Reaction (PCR) • Nucleic Acid Hybridization • Antibodies as Probes for Proteins • Western blotting • Immunoprecipitation

30. Amplification of DNA by Polymerase Chain Reaction • PCR is a process that allows individual DNA fragments to be propagated in bacteria and isolated in large amounts. • PCR amplification provides an extremely powerful method of detecting small amounts of specific DNA or RNA molecules in a complex mixture of other molecules. • The DNA polymerases used in PCR reactions are heat-stable enzymes from bacteria such as Thermus aquaticus.

31. Nucleic Acid Hybridization • Nucleic acid hybridization is the formation of double-stranded DNA and/or RNA molecules by complementary base pairing – a means of identifying a specific sequence of DNA/RNA • Southern blotting-- is a technique that is widely used for detection of specific genes in cellular DNA. • Northern Blotting – a technique to detect RNA • Screening a Recombinant DNA library to identify a gene of interest. • DNA microarrays allow tens of thousands of genes to be analyzed simultaneously.

32. 4.25 Southern blotting

33. Nucleic Acid Hybridization • Recombinant DNA libraries are collections of clones that contain all the genomic or mRNA sequences of a particular cell type. Fig. 4.26. Screening a recombinant library by hybrization.

34. 4.27DNA Microarrays

35. Antibodies as Probes for Proteins • Antibodies are proteins produced by cells of the immune system that react against molecules (antigens) that the host organism recognizes as foreign substances. • Immunoblotting (also called Western blotting) is another variation of Southern blotting. • SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a method in which proteins are separated.

36. Antibodies as Probes for Proteins Fig. 4.29. Western blot.

37. Antibodies as Probes for Proteins • In immunoprecipitation, antibodies are used to isolate the proteins against which they are directed. Fig. 4.30. Immunoprecipitation.

38. Gene Function in Eukaryotes • Understanding the function of a gene requires analysis of the gene within cells or intact organisms. • Transgenic mice carry foreign genes that have been incorporated into the germ line. • Gene transfer or transfection is the introduction of foreign DNA into animal cells. • Liposomes are lipid vesicles that can incorporate DNA and fuse with the plasma membrane. • Electroporation is the exposure of cells to a brief electric pulse that transiently opens pores in the plamsa membrane.

39. 4.33 Introduction of DNA into animal cells

40. 4.34 Retroviral vectors

41. Mutagenesis of Cloned DNAs • In classical genetic studies, mutants are the key to identifying genes and understanding their function. • Reverse genetics involves the introduction of any desired alteration into a cloned gene in order to determine the effect of the mutation on gene function. • In homologous recombination, the cloned gene replaces the normal allele, so mutations introduced into the cloned gene in vitro become incorporated into the chromosomal copy of the gene.

42. Interfering with Cellular Gene Expression • Antisense nucleic acids are RNA or single-stranded DNA complementary to the mRNA of the gene of interest. • RNA interference (RNAi) is the degradation of mRNAs by short complementary double-stranded RNA molecules.

43. 4.43 Direct inhibition of protein function