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

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 geneone 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
16
(No Transcript)
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 of
l 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 l and 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
4.27 DNA 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