BIOL 3451 1st Edition Lecture 25 Outline of Last Lecture I. 19.2 Cancer Cells Contain Genetic Defects Affecting Genomic Stability, DNA Repair, and Chromatin ModificationsII. 19.3 Cancer Cells Contain Genetic Defects Affecting Cell-Cycle RegulationIII. 19.4 Proto-oncogenes and Tumor-Suppressor Genes Are Altered in Cancer CellsIV. 19.5 Cancer Cells Metastasize and Invade Other TissuesV. 19.6 Predisposition to Some Cancers Can Be InheritedVI. 19.7 Viruses Contribute to Cancer in Both Humans and AnimalsVII. 19.8 Environmental Agents Contribute to Human Cancers Outline of Current Lecture I. 19.8 Environmental Agents Contribute to Human CancersII. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning VectorsIII. 20.2 DNA Libraries Are Collections of Cloned SequencesCurrent LectureI. 19.8 Environmental Agents Contribute to Human Cancerso Both UVlight and ionizing radiation (X rays and gamma rays) induce DNA damage- Can cause skin cancer- Radon gas may be responsible for up to 50% of the ionizing radiation exposure to the US population and contribute to lung cancerII. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectorso Recombinant DNA: joining of DNA molecules, usually from different biological sources, that are not found together in natureo The basic procedure for producing recombinant DNA:- generating specific DNA fragments using restriction enzymes- joining these fragments with a vector- transferring the recombinant DNA molecule to a host cell to produce many copies that can be recovered from the host cell (called clones)1. Can be used to study the structure and orientation of the DNAo Recombinant DNA technology is used to isolate, replicate, and analyze geneso A restriction enzyme binds to DNA at a specific recognition sequence (restriction site) and cleaves the DNA to produce restriction fragments - Figure 20.1These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Most recognition sequences exhibit a form of symmetry descried as a palindrome, and restriction enzymes cut the DNA in a characteristic cleavage pattern1. Fig 20.12. Mostly 4-6 nucleotides long (some have 8 or more)o DNA ligase joins restriction fragments covalently to produce intact DNA molecules- Fig 20.2o Vector: carrier DNA molecules that can replicate cloned DNA fragments in a host cell- must be able to replicate independently - Needs several restriction enzyme sites to allow insertion of a DNA fragment- should carry a selectable gene marker to distinguish host cells that have taken them up from those that have noto Plasmid: extrachromosomal double-stranded DNA molecule that replicates independently from the chromosomes within bacterial cells (Fig 20.3a)- Used for DNA cloning usually have been engineered to contain:1. Number of convenient restriction sites2. A marker gene to select for its presence in the host cell 3. Fig. 20.3bo Transformation: how plasmids are introduced and it is done by:- Using calcium ions and brief heat shock to pulse DNA into cells- Electroporation: uses brief but high-intensity pulse of electricity to move DNA into bacterial cellso Plasmid DNA and DNA to be cloned are cut with the same restriction enzyme- DNA restriction fragments from the DNA to be cloned are added to the linearized vector in the presence of DNA ligase- A recombinant DNA is produced, which is then introduced into bacterial hostcells by transformation - Figure 20.4o Recombinant DNA can be readily identified by using selectable marker genes- Genes that provide resistance to antibiotics such as ampicillin and genes such as the lacZ genes are very effective selectable markers- Blue-white selection is used to identify cells containing recombinant and nonrecombinant DNA - Figure 20.5o Phage vectors were among the earliest vectors used in addition to plasmids- The central third of lambda (λ) phage vectors can be replaced with foreign DNA without affecting the ability to infect cells and replicate1. Lambda vectors can carry up to 45 kb of cloned DNAo Expression vectors are engineered to express a gene of interest to produce large quantities of the encoded protein in a host cell- Available for both prokaryotic and eukaryotic host cellso Plant and animal cells can serve as hosts for recombinant DNA, in addition to bacteria and yeast- Rhizobium radiobacter (formerly Agrobacterium tumefaciens) can be used totransform plant cells with T-DNA containing foreign DNA- Rhizobium contains the Ti plasmid (tumor inducing) with genes that induce tumors- Tumor-inducing genes can be removed from the vector so the recombinant vector does not produce tumorso Plants- The vector when mixed with plant cells enters inside the cell, and the foreignDNA gets inserted into the plant genome - The plant cells can be grown in tissue culture and eventually regenerate a mature plant carrying a foreign gene- These cells are then grown by tissue culture to give a mature plant carrying aforeign geneo Bacteria- E. coli used as a prokaryotic host cell for plasmids- Yeast is used as a host for DNA cloning and expression of eukaryotic genes because: 1. grown easily and manipulated2. genetics have been studied intensively3. sequenced genome4. posttranslationally modify eukaryotic proteins5. considered safeo Humans- A variety of different human cell types can be grown in culture and used to express genes and proteins1. Used for gene or protein functional analysis (i.e. drug testing)a. Especially with cancerIII. 20.2 DNA Libraries Are Collections of Cloned Sequenceso A genomic library contains at least one copy of all the sequences in the genome of interest- DNA libraries represent a collection of cloned DNA samples derived from a single source that could be a particular tissue type, cell type, or single individual- constructed by cutting genomic DNA with a restriction enzyme and ligating the fragments into vectors, which are chosen depending on the size of the genomeo YACs were commonly used to accommodate large sizes of DNA necessary to span the 3 billion ppb of DNA in the human genomeo Whole-genome shotgun cloning approaches and next-generation sequencing are replacing traditional genomic librarieso All DNA fragments in a genomic sample are sequenced without the need to insert DNA fragments into vectors