Chap 6

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6 Principles of Genetic Manipulation of Organisms: Recombinant. DNA (rDNA) ... rDNA technology is often referred to as genetic engineering. 3. The product of ...

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Chap. 6 Principles of Genetic Manipulation of Organisms: Recombinant DNA (rDNA) Technology

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Purpose and expected outcomes Recombinant DNA (rDNA) technology allows scientists to transfer genes from one organism to any other, circumventing the sexual process. rDNA technology is often referred to as genetic engineering. The product of recombinant DNA manipulation is called a “transgenic organism” In this chap., you will learn: (1) The basic steps in genetic engineering or rDNA technology. (2) The enabling technologies of genetic engineering. (3) The importance of microorganisms in genetic engineering. (4) The fundamental difference between conventional breeding and genetic engineering. General steps in rDNA procedure (Fig. 6-1) The DNA of interest that is to be transferred (also called “foreign DNA”, “insert DNA”, “cloned DNA”, or “transgene ”) is obtained by extracting the DNA from the organism and cutting out the specific DNA sequence using restriction enzymes. The transgene is inserted into a special DNA molecule call a “cloning vector” to produce a new recombinant DNA molecule. The cloning vector-inset DNA construct is transferred into, and maintained in, a host cell by the process of transformation. The vector replicates, producing identical copies (clones) of the insert DNA. The host cells that have incorporated the foreign DNA are identified and isolated from untransformed cells. The cloned DNA can be manipulated such that the protein product it encodes can be expressed by the host cell.

Restriction endonucleases: cutting DNA 1. Restriction endonucleases are found in bacteria where they play a defensive role against invading bacteriophage (a virus that attacks bateria) by digesting the foreign DNA. 2. Restriction endonucleases cut DNA at specific sites called “recognition sites) (Fig. 6-2). a. The nucleotide sequence at these sites is palindromic (read the same from either end). b. Usually consists of 4, 5, 6, or 8 nucleotide pairs. c. Blunt or sticky ends. d. The sticky ends can be re-annealed through complementary binding with the enzyme T4 DNA ligase (from bacteriophage T4) producing the ligation. 3. Restriction endonucleases are named according to a certain protocol following the binomial nomenclature (genus and species).

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a. The genus is represented by the first letter (uppercase), followed by the first two letters of the species (lowercase), then the order of characterization (roman numerals). b. Ex: HpaI and HpaII represent the first and second type II restriction enzymes isolated from Haemophilus parainfluenzae. 4. Three categories of restriction endonucleases: a. Type I: cleavage site is located more than 1,000 bp away from the recognition site and thus cleavage does not occur at a specific sequence. b. Type II: the recognition sites or sequences are usually short (4-5 bp) and often palindromic. Require no ATP for restriction. The workhorses of rDNA technology. c. Type III: have 2 subunits, one for recognition and methylation and the other for restriction. Cleavage occurs 24-26 bp downstream from the recofnition site. 5. Unlike the three type of restriction enzymes (proteins), there is a unique class of non-protein enzyme s called “ribozymes”, which are RNA enzymes with the capacity for cleaving specific phosphodiester bonds. Gene isolation 1. A number of strategies may be used to isolate or clone a gene. a. Activation tagging: The gene to be isolated is first inactivated by transposon insertion, resulting in the formation of a mutant. The DNA sequence of the transposon is used to identify the clones that contain the gene of interest. b. cDNA screening: A cDNA library is first created. A probe is then designed and used to screen the library to hybridize to the sequence of interest. c. Map-based gene cloning: The DNA markers are used to screen a genomic library to isolate clones that contain the target gene. d. Transformation-associated recombination: Based on the natural ability of yeast cells to find and combine similar DNAs, regardless of their origin. Yeast cells are transformed with pieces of DNA along with a small fragment of the target DNA/ As the yeast cells reproduce, only DNA that complements the small piece of DNA introduced into the cell are maintained (cloned). Cloning vectors 1. Vectors are entities for carrying the target DNA into a host cell. 2. All vectors consist of certain essential features (Table 6-1). 3. Commonly used vectors include plasmids, bacteriophages, BACs, YACs, fosmids, P1, and

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PACs. 4. Plasmid cloning vector: (a) Plasmids are double-stranded, circular DNA molecules that occur in bacteria. (b) They are extrachromosomal structures and can replicate autonomously. (c) The size range from less than 1 to more tha 500 kb. (d) There is a sequence that functions as an origin of replication (ori) in plasmid. (e) Usually has at least one selectable genetic maker (may be a gene for antibiotic resistance). (f) pUC18 (Fig. 6-3): has a polylinker sit e (or multiple cloning site) (a cluster of many restriction endonuclease recognition sites). (g) E. coli lacZ gene: i. with insertion of target DNA → inactivates the lacZ gene → white colonies ii. without insertion → lacZ gene function normally → blue colonies 5. Viral vectors: (a) Viruses that infect bacteria (bacteriophage, phage) have been engineered as vectors for cloning longer piece of DNA, useful in the creation of DNA libraries, which can be screened by means of DNA probes or immunological assays. (b) The E. coli virus lambda (λ) phage is a widely used cloning vehicle. (c) It has been determined that the middle 1/3 of the λ DNA consists of 20-kb sequence (out of 50-kb) that is required for the integration-excision events (Fig. 6-4). (d) The M13 bacteriophage vector is a single-stranded phage. When it infects a bacterium, the single strand (+ strand) replicates to produce a double-stranded molecule called “replicative form (RF)”, which is essentially similar to plasmids. 6. Vectors for cloning very large DNA fragments: (a) One of the most commonly used vectors for cloning large fragments of DNA is the bacterial artificial chromosome (BAC). (b) Cosmids: i. a hybrid between a plasmid and a phage. ii. Consists of the cos sequence of phage λ (required for packing the phage DNA into the phage protein coat), the plasmid sequence for replication, and an antibiotic resistance gene. iii. Can handle about 40 kb of cloned DNA. iv. Ex: pJB8-5 (Fig. 6-5). (c) Bacterial artificial chromosome: i. independently replicating plasmids that are involved in the transfer of genetic information during bacterial conjugation (temporary fusion of cells for transfer of genetic information). ii. BAC vectors carry the F factor genes for replication and copy number (carrying up to 1 Mb fragment), as well as antibiotic resistance marker and

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restriction enzyme sites (Fig. 6-6). (d) Yeast artificial chromosome: i. YAC contains yeast telomeres for the distribution of replicated YACs to daughter cells at cell division (Fig. 6-7). ii. YACs also contain a selectable marker on each arm (TRP1 and URA3) and a cluster of unique restriction sites. iii. YACs are capable of receiving DNA inserts longer than 1 Mb. (e) Shuttle vectors: i. The hybrid vectors, constructed with origins of replication from different sources, can replicate in more than one host cell. ii. Contain genetic markers that are selectable in different host systems. iii. Useful in studying gene expression.

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Bacterial transformation Transformation is a process of introducing free DNA into a host cell. Genetic transformation of prokaryotes can be accomplished by one of several methods: (a) CaCl2 -heat transformation. (b) Electroporation (Fig. 6-8). (c) Conjugation Transgene delivery

1. Direct gene transfer: (a) Protoplast transformation (b) Tissue/cell electroporation (c) Silicon carbide fiber vortexing (d) Microprojectile bombardment (biolistics) (shotgun transformation): gene gun 2. Mediated (indirect) gene transfer (a) Biological vectors: i. Viral vectors: cauliflower mosaic virus (CaMV) ii. Agrobacterium-mediated transformation: most common technique for transforming plants (b) Requirements for successful transformation by Agrobacterium mediation i. Efficient plant regeneration system ii. Determination that the cells are susceptible to Agrobacterium transformation iii. An efficient and sensitive selection method iv. Stable transformation (c) Methodology of Agrobacterium transformation i. Incubate bacteria with plant cells (a few hours to about two days) ii. Wash cells and treat with antibiotic (to remove bacteria)

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iii. Culture in the presence of selectable agents iv. Regenerate transformed cells (d) Chemically mediated transformation i. Polyethylene glycol (PEG) and polyvinyl alcohol (PVA) are commonly used chemicals ii. Together with Ca2+ and high pH, these chemicals permeabilize the cell membrane to allow the uptake of foreign DNA. iii. Low transformation frequency (e) Electroporation-mediated DNA uptake i. Linearized plasmid DNA gives a higher transformation frequency than supercoiled DNA ii. Lower temperature is more favorable iii. Higher DNA concentration increases transformation frequency (f) Gene delivery into mammalian cells i. Animal and plant cells can uptake DNA by “transfection” ii. Common methods for DNA uptake in mammalian cells include electroporation, coprecipitation with calcium phosphate, endocytosis, direct microinjection, and direct encapsulation of DNA into artificial membranes (liposomes) followed by fusion with cell membranes. iii. Maybe also be transferred by using YACs and vector-based on retrovirus (RNA cirus). Tissue culture and selection 1. Tissue culture: (a) An integral part of most transformation systems in plants. (b) The transgene is usually delivered into undifferentiated cells of primary explants before callus initiation or into proliferating embryogenic tissue (Fig. 6-10). (c) The embryogenic cells then divide to produce one or several somatic embryos and subsequently develop into full plants (Fig. 6-11). 2. Selection systems: (a) Cloning vectors have selectable markers (Fig. 6-12). (b) Selectable markers may be grouped as follows: i. Antibiotic selection: (1) vectors with markers that confer resistance to antibiotics such as aminoglycosides, kanamycin, neomycin, and paromomycin (Fig. 6-13). (2) On of the most common antibiotic genes is nptII, which encodes the enzyme neomycin phosphotransferase. ii. Herbicide selection: (1) genes encode modified target proteins that are insensitive to the herbicide,

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or an enzyme that degrades or detoxifies the herbicide in the plant. (2) Ex: bacterial phosphinothricin acetyltransferase gene (bar); bacterial and plant EPSP synthase genes; acetolactate synthase genes. iii. Scorable gene- mediated selection: (1) Scorable marker genes (reporter genes) are typically used as markers for rapid visual confirmation for transient expression following DNA delivery. (2) They are expressed in cells without integration into the genome. (3) Often encode enzymes that have distinct substrate specificities. (4) Ex: CAT (chloramphenicol acetyltransferase); GUS (β-glucuronidase); LUC (luciferase) (Fig. 6-14); GFP (green fluorescent protein) iv. Positive selection: (1) benign markers (2) based on metabolic pathways (3) Ex: the mannose selection system, phosphomannose isomerase gene (pmi) is used as a selectable marker, with mannose as the selecting agent. (c) Transgene integration: i. The transgene first enters the nucleus and then may become integrated into the genome randomly, but predominantly in transcriptionally active regions. ii. Once inside the nucleus, the foreign DNA may be transcribed or become integrated into the chromosome. Transgene expression 1. Three major classes pf promoters used: (a) Constitutive promoters: i. Have high affinity for RNA polymerase and consequently promote frequent transcription of the adjacent region. ii. Mainly used to drive the expression of the selectable marker gene. (b) Tissue-specific and developmentally regulated promoters: (c) Inducible promoters: Stability of transgene expression 1. Progressive transgenic silencing or failure may occur. 2. Usually, one strategy to reduce the cha nce of transgee instability is to ensure that the introduced foreign DNA sequence matches the isochore of the host genome. That is, the coding regions should have the right codon usage and GC content. 3. Use genomic clones instead of cDNA since there is evidence that introns, 5’ unsaturated regions, and specific sequences downstream of the polyadenylation site all impact the expression levels and stability of mRNA.

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Marker-independent transgenic production 1. Two approaches: (a) development of new markers (benign markers) (b) post-transformational removal of marker genes in transgenic products: may be accomplished artificially by the techniques of transposition and site-specific recombination. Key concepts Websites tutorial See p. 93-94. References 1. Crystal, R.G. 1995. Transfer of genes to humans: Early lessons and obstacles to success. Science, 270:404-410. 2. Hamilton, A.J. and D.C. Baulcombe. 1999. A species of small antisense RNA in post-transcriptional gene silencing in plants. Science, 286:950-952. 3. Hansen, G. and M.S. Wright. 1999. Recent advances in the transformation of plants. Trends in Plant Science, 4:226-231.