Imagine a book that is falling from a shelf. At a particular moment during its fall, the book has a kinetic energy of 24 J and a potential energy with respect to the floor of 47 J. (a)How does the book’s kinetic energy and its potential energy change as it continues to fall?
(b)What is its total kinetic energy at the instant just before it strikes the floor? (c) If a heavier book fell from the same shelf, would it have the same kinetic energy when it strikes the floor? [Section 5.1]
• Generalized Transduction results from a rare error during phage assembly • A fragment of bacterial DNA can mistakenly enter a protein coat. • This creates a transducing particle which carries the bacterial DNA instead of the phage nucleic acid, and so it is not a phage • A transducing particle will attach to another bacterial cell and inject the DNA it contains which is a bacterial DNA • The bacterial DNA may then integrate into the host chromosome by homologous recombination • 8.7. Transduction Conjugation: DNA transfer between bacterial cells • Requires contact between donor, recipient cells • Plasmid are most frequently transferred to other cells by conjugation • Conjugative plasmids direct their own transfer from donor to recipient cells • They are replicon • Independent of cell chromosomal replication • F plasmid (fertility) of E. coli most studied 8.8. Conjugation Conjugation (continued…) F plasmid of E. coli + – • F cells have, F do not Encodes proteins required for conjugation including F pilus • Sex pilus • Brings cells into contact • Enzyme cuts plasmid • Single strand transferred • Complementary strands synthesized • Both cells are now F + 8.9. The Mobile Gene Pool Genomics reveals surprising variation in gene pool (the sum of all genes) of even a single species Perhaps 75% of E. coli genes found in all strains • Termed core genome of species Remaining make up mobile gene pool or mobilome which can be transferred to other locations within the cell or other cells. • Plasmids, transposons, genomic islands, phage DNA 8.9. The Mobile Gene Pool Plasmids found in most Bacteria, Archaea Some Eucarya Usually dsDNA with origin of replication Generally nonessential; cells can survive their loss (curing) Carry few to thousands of genes Low-copy-number plasmids: • One or a few per cell High-copy-number plasmid: • Many, perhaps 500 Most have narrow host range • Single species Some broad host range • Includes Gram– and Gram+ 8.9. The Mobile Gene Pool Resistance plasmids (R plasmids) Encode resistance to antimicrobial medications, heavy metals (mercury, arsenic) which all are found in hospital environments Often two parts • R genes: encode the resistance traits • RTF (resistance transfer factor) – Codes for conjugation • Often broad host range • Normal microbiota can harbor R plasmids, and then transfer them to pathogens 8.9. The Mobile Gene Pool Transposons provide mechanism for moving DNA and transferring various genes • Can move into other replicons in same cell • The simplest type of transposon is insertion sequence (IS) • Encodes only transposase enzyme for transposition, Flanking the genes are inverted repeats • Composite transposons include one or more genes flanked by ISs • Integrate into their new location via non-homologous recombination • Simply inserts into a stretch of DNA, doesn’t need replace the existing similar sequences • 8.9. The Mobile Gene Pool Transposons yielded vancomycin resistant Staphylococcus aureus strain Patient infected with S. aureus • Susceptible to vancomycin • Also had vancomycin resistant strain of Enterococcus faecalis • Transferred transposon- containing plasmid to S. aureus • Transposon jumped to plasmid in S. aureus 8.9. The Mobile Gene Pool Genomic islands: large DNA segments in genome • Originated in other species • Nucleobase composition very different from genome G-C base pair ratio characteristic for each species • May provide different characteristics Utilization of energy sources Acid tolerance Development of symbiosis Ability to cause disease – Genomic islands that encode ability to cause disease are called Pathogenicity islands A Glimpse of History “Dirty War” in Argentina (1976–1983) • Military junta killed thousands of citizens • Collapsed in 1983 • Democratically elected government unsealed records • Confirmed ~200 children survived killings, had been kidnapped and placed with pro-junta families • Mary-Claire King (then at UC Berkeley) analyzed DNA sequences to reunite children with relatives • But parents usually dead or missing • Chromosomal DNA difficult to use • Used mitochondrial DNA (mtDNA), which is inherited only from the mother • Biotechnology Biotechnology • The use of microbiological and biochemical techniques to solve practical problems and produce useful product • Recombinant DNA techniques • Methods used to manipulate DNA to intentionally genetically alter organisms through genetic engineering – Often to give them more useful traits – Possible to genetically alter organisms – Can isolate genes from one, manipulate them, and transfer to another • Genetic engineering: the process of deliberately altering an organism’s genetic information using in vitro techniques • Numerous uses: agriculture, medicine, law enforcement Biotechnology 9.1. Fundamental Tools Used in Biotechnology Basic components of molecular biologist’s “tool kit”: • Restriction enzymes • Gel electrophoresis Restriction Enzymes 9.1. Fundamental Tools Used in Biotechnology • Restriction enzymes (continued…) • Name represents bacterium from which isolated 9.1. Fundamental Tools Used in Biotechnology Basic components of molecular biologist’s “tool kit” (continued…) Gel electrophoresis • Used to separate DNA fragments according to size, allows identification • DNA is put into wells in gel • Gel subjected to electrical current • DNA moves through the gel • DNA migrates toward (+) electrode – Fragments are separated according to size » Large fragments remain high in the gel » Small fragments migrate lower • DNA must be stained to be visible • Stained with ethidium bromide solution • Gel Electrophoresis: can also separate RNA, and proteins • 9.2. Applications of Genetic Engineering Numerous uses for genetically engineered bacteria • Protein production • DNA production • Research tools 9.2. Applications of Genetic Engineering DNA cloning • Isolate DNA • Cut with restriction enzymes • Join DNA (insert) with plasmid (vector) to generate independently replicating recombinant molecule • Introduce into host (e.g., E. coli) 9.2. Applications of Genetic Engineering DNA cloning (cont…) Cloning into a High – Copy Number Vector • Can place gene (insert) into high-copy-number vector • Host bacteria will make large amounts of the protein • Each gene copy can be transcribed and translated 9.2. Applications of Genetic Engineering Protein production • Produce commercially important proteins • Pharmaceutical proteins – Human insulin • Vaccines – Hepatitis B vaccine • Commercially valuable proteins – Chymosin used in the production of cheese 9.2. Applications of Genetic Engineering Protein production by genetically engineered organisms : many pharmaceutical proteins are now produced by microorganism: • Gene for human insulin cloned into bacteria: safer, more economical • Previously, insulin extracted from pancreatic glands of cattle and pigs sometimes caused allergic reactions • Vaccine production : clone gene for specific proteins • E.g., vaccines for hepatitis B and cervical cancer • Foot-and-mouth disease of domestic animals • Cheese production: organisms produce chymosin (rennin), an enzyme used in cheese production • Traditionally enzyme was obtained from stomachs of calves • Restriction enzymes, bovine somatoropin, a GH that increase milk in cows are also produced by genetically engineered microbes. • Synthetic bacteria may someday produce wide range of commercially valuable substances • E.g., synthetic chromosome in Mycoplasma mycoides 9.2. Applications of Genetic Engineering DNA production • Aids in research • Clone DNA segment into bacterium (e.g., E. coli) • Allows easy production of DNA for study • Random samples of DNA from environment can be cloned and then sequenced • Termed “shotgun cloning” • First step of metagenomics, the study of total genomes in the sample • Aids in study of ~99% of bacteria that have not been grown in culture • 9.2. Applications of Genetic Engineering Researching Gene Function and Regulation • Gene fusion of gene being studied and a reporter gene • Encodes observable product – E.g., green fluorescent protein (GFP) 9.2. Applications of Genetic Engineering Genetically engineered eukaryotes • Yeasts serve as important eukaryotic model for gene function and regulation • Plants or animals that receive engineered gene termed transgenic organism • Examples of genetically altered plants include Pest-resistant plants – Corn, cotton and potatoes Herbicide-resistant plants – Soybeans, cotton and corn Plants with improved nutrient value – Rice Plants as edible vaccines – Rice and potatoes 9.4. Concerns Regarding Genetic Engineering and Other DNA Technologies Any new technology needs to be scrutinized to ensure its safety and effectiveness • Recombinant DNA Advisory Committee (RAC) formed by NIH nearly three decades ago • Numerous advances have been made Technologies could be used for malicious purposes • Concerns over sequencing human DNA • Genetically modified (GM) organisms hold promise Concerns over possible allergens Unintended effects on environment – Pollen from pest - resistant plants may or may not harm butterflies – Herbicide-resistant genes may transfer to weeds 9.5. DNA Sequencing DNA sequencing: determining the sequence of DNA Human Genome Project produced great advances • Rapid growth in field of genomics • Sequencing of numerous organisms • Spawned new field of bioinformatics to analyze data • Allows determination of amino acid sequences Comparisons of different proteins, various organisms • Evolutionary relatedness of organisms • Technology so efficient that new Human Microbiome Project aims to determine biological diversity of normal microbiota and also comparing the composition of the microbiota in health and disease. • 9.5. DNA Sequencing Dideoxy chain termination most common method • Automated and fast • In vitro DNA synthesis • Template DNA • DNA polymerase • Primer • Deoxynucleotides dNTPs • Dideoxynucleotides ddNTPs • 9.5. DNA Sequencing Dideoxy chain termination most common method (continued…) • Dideoxynucleotides (ddNTPs) Serve as chain terminators – Lack 3′OH – Synthesis stops – Yields mixture of DNA of different lengths – Denature DNA – Separate ssDNA via electrophoresis – Read fluorescent label on ddNTPs to obtain sequence 9.5. DNA Sequencing Gel electrophoresis • Laser reads color • Determines sequence 9.6. Polymerase Chain Reaction (PCR) PCR is used to rapidly increase the amount of a specific DNA segment in a sample Creates millions of copies of given region of DNA in matter of hours • Technique exploits specificity of primers • Allows for selective replication of chosen regions – Termed target DNA • Large amounts of DNA can be produced from very small sample • 9.6. Polymerase Chain Reaction (PCR) Allows amplification to millions of copies of DNA in a matter of hours • Products can be visualized via gel electrophoresis • Allows detection of specific sequences • Can detect organisms without culturing – E.g., pathogens • Requires Double stranded DNA (contains target DNA, and serves template DNA), polymerase, primers (to choose where synthesis starts) , and deoxynucleotides (dATP, dGTP, dCTP, dTTP) 9.7. Probe Technologies DNA probes locate specific nucleotide sequences in a nucleic acid sample attached to a solid surface • Probe is single-stranded piece of DNA • Will hybridize to complementary sequence • Labeled with marker • Numerous different probe technologies 9.7. Probe Technologies Colony Blotting Uses probes to detect specific DNA sequences in colonies grown in agar plates • Colonies are transferred in place (“blotted”) on nylon membrane to add the probe Colony blots are used to determine which cells contain gene of interest (the sequence being studied) 9.7. Probe Technologies Fluorescence in situ Hybridization (FISH) Uses fluorescently labeled probe to detect certain nucleotide sequences • Detects sequences inside intact cell fixed to microscope slide Specimen is viewed using fluorescence microscope Probe often binds to rRNA sequences since rRNA is numerous in multiplying cells • Rapid identification in specimen without culturing • FISH is used to detect either related organisms or specific species • FISH can be used to identify specific properties of bacteria • Mycobacterium tuberculosis in sputum sample • 9.7. Probe Technologies DNA Microarrays • Studies of gene expression • Also used to detect DNA sequences • A DNA microarray is a glass slide contains a large number of short DNA fragments that function in a manner analogous to a probe • Enable researchers to screen sample for numerous sequences simultaneously • mRNA is isolated, converted to labeled cDNA, hybridized to array • Array has numerous short probes specific to each gene of interest • Allows comparisons of conditions