1. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Molecular Genetics DNADNA ChromosomesChromosomes Genes and genomeGenes and genome Protein synthesisProtein synthesis DNA replicationDNA replication Changes in DNA sequence: phages and transposonsChanges in DNA sequence: phages and transposons Genetic engineeringGenetic engineering

2. Larry M. Frolich, Ph.D. Biology Department, Yavapai College DNA and GENETICS Cells divide and pass on instructions coded in DNA of chromosomes Each chromosome is a huge DNA molecule with coded information DNA has dual role: –DNA replicates to pass on information –DNA is transcribed to make proteins that run cell metabolism

3. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Prokaryote DNA/chromosome Single (usually) chromosome is one long DNA molecule Small secondary DNA molecules are called plasmids Localized in region of cell called "nucleoid" but not bound by membrane as is nucleus of eukaryotes DNA molecule is packaged in loose loops within cell DNA of prokaryotes is invisible in light microscope

4. Larry M. Frolich, Ph.D. Biology Department, Yavapai College DNA and chromosomes Long DNA molecules (millions of base pairs long) in nucleus are called chromosomes Each chromosome is organized and packaged or wrapped up with proteins giving it a certain shape In humans, 23 pairs of chromosomes –1 of each pair from mother –1 of each pair from father Total view of all 23 pairs is called karyotype

5. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Each chromosome is a single DNA molecule wrapped up within a special group of proteins giving it a particular shape Chromosomes

6. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Genes and Genome Genome: –entire DNA of cell (all DNA molecules) –also includes DNA of mitochondria, chloroplasts in eukaryotes –Thought question: Are viruses, phages, transposons part of genome? Gene: –Region along DNA molecule that codes for 1 protein –usually 1000's of base pairs long –E. coli lac operon is first gene whose regulation and function was understood at molecular level in coding of DNA (see more below)

7. Larry M. Frolich, Ph.D. Biology Department, Yavapai College DNA is transcribed to make proteins that run cell metabolism DNA is transcribed to mRNA mRNA is translated to amino acid sequence Amino acid sequence folds up into protein Proteins catalyze reactions of cell metabolism This process is called “gene expression”—the information in one region of the DNA—a “gene”—is being expressed so that the cell’s metabolism can function

8. Larry M. Frolich, Ph.D. Biology Department, Yavapai College 2 steps of gene expression 1.Transcription – DNA is read to make a mRNA in the nucleus of our cells 2.Translation – Reading the mRNA to make a protein in the cytoplasm

9. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Transcription Happens in nucleus DNA double helix “opens up” mRNA transcript is made from DNA template

10. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Translation Happens outside nucleus Ribosomes (special RNA particles or organelles) do the translation They glom onto mRNA and line up amino acids according to mRNA sequence (see next slide for “code”

11. Larry M. Frolich, Ph.D. Biology Department, Yavapai College RNA-protein translation code Every three RNA bases codes for one amino acid This code is very evolutionary conservative—works almost the same in all forms of life

12. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Overview of transcription and translation Details in web link video animations REMEMBER: A particular region of DNA that has the code to make a particular protein is called a “gene.”

13. Larry M. Frolich, Ph.D. Biology Department, Yavapai College How does cell decide when to activate which genes to produce what proteins? DNA must be unpackaged and uncoiled in order to be transcribed to mRNA Lampbrush chromosome shows loops of DNA that are being transcribed What determines which regions or genes are going to be transcribed and translated? This is called regulation of gene expression

14. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Regulation of gene expression Gene expression is regulated—not all genes are constantly active and having their protein produced The regulation or feedback on gene expression is how the cell’s metabolism is controlled. This regulation can happen in different ways: 1. Transcriptional control (in nucleus): e.g. chromatin density and transcription factors 2. Posttranscriptional control (nucleus) e.g. mRNA processing 3. Translational control (cytoplasm) e.g. Differential ability of mRNA to bind ribosomes 4. Posttranslational control (cytoplasm) e.g. changes to the protein to make it functional When regulation of gene expression goes wrong— cancer!

15. Larry M. Frolich, Ph.D. Biology Department, Yavapai College The lac operon: model for gene expression Region along DNA molecule Includes three protein synthesis coding region-- sometimes called "genes" as well as region of chromosome that controls transcription of genes Genes for proteins involved in the catabolism or breakdown of lactose Thus, when lactose is absent, no transcription of gene since no need for these proteins (a in figure below) When lactose is present, transcription of genes takes place so proteins are available to catalyze breakdown of lactose (b in figure below) See animation on course website

16. Larry M. Frolich, Ph.D. Biology Department, Yavapai College DNA is structured to replicate DNA is “double helix”— two complementary strands wound in a spiral Strands separate and DNA replicates by filling in other half of each separated strand Famous Watson-Crick model (Nobel prize)

17. Larry M. Frolich, Ph.D. Biology Department, Yavapai College DNA replicates to pass on information (to daughter cells in mitosis)

18. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Changes in DNA sequence Mutation—change in DNA sequence. Example: Changes in DNA allow bacteria to resist effects of antibiotics Mutation changes DNA sequence, which in turn changes protein sequence that codes for a specific protein in one of the many cell metabolic pathways

19. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Mutations can spread through a population Mutation allows cell to survive in a new environment--in this case, in the presence of penicillin. This type of beneficial mutations can result in more individual cells that are resistant to penicillin and can thereby grow colonies The offspring or daughter cells then continue to spread throughout the population, especially where penicillin is present. This is natural selection leading to evolutionary change!! We have seen it happen...in the laboratory...in the "wild." Fifty years, ago, almost no bacteria were resistant to penicillin. Today, some penicillin resistant cells are found in virtually all bacterial populations.

20. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Horizontal gene transfer Antibiotic resistant genes can also spread by "horizontal gene transfer," even between different species of bacteria.horizontal gene transfer Three ways that genes can transfer between adult cells: transformation--cell takes up DNA from environment, presumably released by another dead cell...or provided, in a laboratory, by a researcher. Transduction--phage delivers DNA to cell Conjugation--"bacterial sex." Cells form connection to transfer genes.

21. Larry M. Frolich, Ph.D. Biology Department, Yavapai College

22. Larry M. Frolich, Ph.D. Biology Department, Yavapai College

23. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Transposons "Jumping genes" can move from one location to another within the cell's DNA. For example, in bacteria, can move from main chromosome to plasmid. Always have palindrome to remove and insert into DNa Complex transposons carry genes for particular traits Example: "R factor" is transposon that carries gene that confers resistance to antibiotics (in the case of penicillin, presumably by changing the cell wall synthesis process and avoiding the killing effect of penicillin) Transposons can be carried by phage (transduction) or passed cell to cell (conjugation), even among different species of bacteria. Here is replication and transfer by conjugation of a plasmid carrying "F factor" which gives "fertility" or the ability to conjugate to a cell. This plasmid also contains a transposon or jumping gene that allows it to reintegrate into the cell's main chromosome or DNA molecule:

24. Larry M. Frolich, Ph.D. Biology Department, Yavapai College

25. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Recombinant DNA tools: Restriction Enzymes Cut DNA at particular, usually palindromic sequence: Cells make restriction enzymes normally to allow for plasmids, transposons, even viruses to insert into genome Molecular biologists use restriction enzymes to cut DNA at the same "restriction site" in order to create specific fragments of DNA These fragments can then be run out on a gel, multiplied using PCR, and even "recombined" or inserted into a new organisms, usually E. coli. If the restriction fragment contains a protein coding sequence, then that E. coli cell will produce that protein:

26. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Use of restriction enzyme to insert HGH into bacteria

27. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Common used restriction enzymes EnzymeOrganism Recognition Sequence Blunt or Sticky End EcoRIEscherichia ColiGAATTCSticky BamHIBacillus amyloliquefaciensGFATCCSticky BglIIBacillus globigiiAGATCTSticky PvuIProteus vulgarisCGATCGSticky PvuIIProteus vulgarisCAGCTGBlunt HindIIIHaemophilus influenzae RdAAGCTTSticky HinflHaemophilus influenzae RfGANTCSticky Sau3AStaphylococcus aureusGATCSticky AluIArthrobacter luteusAGCTBlunt TaqIThermus aquaticusTCGASticky HaeIIIHaemophilus aegyptiusGGCCBlunt NotINocardia otitidis-caviarumGCGGCCGCSticky

28. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Recombinant DNA tools: Reverse transcriptase Reverse transcriptase Used by RNA viruses to transcribe RNA into DNA (note reverse of central dogma) DNA can then insert into cell's genome Cell then makes RNA transcripts of that DNA and new RNA viruses are manufactured and released from cell Molecular biologists use reverse transcript to make cDNA--DNA made from RNA transcripts. This cDNA is the DNA that codes for proteins (not the entire genome codes for proteins It is useful to have only the protein- coding regions in order to figure out what a particular gene does or to be able to force a bacterial cell to make that protein.

29. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Recombinant DNA tools: Gel electrophoresis

30. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Recombinant DNA tools: Gel electrophoresis

31. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Recombinant DNA tools: PCR—polymerase chain reaction Basically is the process of replicating DNA, but done in a test tube for a particular DNA sequence (usually a restriction fragment) Allows for making many copies of a particular gene In PCR, a heat-stable DNA polymerase is used, most commonly Taq Polymerase from the thermophilic microbe Thermus aquaticus.

32. Larry M. Frolich, Ph.D. Biology Department, Yavapai College Recombinant DNA tools: Genetic maps, Genetic sequences, and Gene libraries Using combination of above techniques to fully characterize the genome of a particular cell A genetic map puts the restriction fragments and the genes they might contain in order along the chromosome A gene library is a bank of cells, each of which contains a cloned gene, often on a plasmid that can be easily manipulated, perhaps because it has an antibiotic resistance gene attached as well A genetic sequence is the complete DNA code of a particular region of a chromosome or the entire genome. It is obtained by knocking the last nucleotide off of a restriction fragment, one at a time, and then identifying the tailing nucleotide using specific molecular markers.