Biology of Genetic Engineering:

Transcription

1 Biology of Genetic Engineering: Once recombinant DNA molecules have been constructed in vitro the desired sequence can be isolated In some experiments, hundreds of thousands of different DNA fragments may be produced and isolation of particular sequence would seem to be an almost impossible task It is bit like looking for the proverbial needle in a haystack with added complication that the needle is made of the some material as the haystack Fortunately the methods available provide a relatively simple way to isolate specific gene sequences Three things have to be done to isolate a gene from a collection of recombinant DNA sequences: The individual recombinant molecules have to be physically separated from each other The recombinant sequences have to be amplified to provide enough material for further analysis and The specific fragment of interest has to be selected by some sort of sequence dependant method

2 In this chapter, we will consider the first two requirements which in essence represent the systems and techniques involved in gene cloning This is essential part of most genetic manipulation programs Even if the desired result is transgenic organism, the gene to be used must first be isolated and characterized and therefore cloning systems are required The biology of gene cloning is concerned with the selection and use of: a suitable carrier molecule or vector a living system or host in which vector can be propagated and methods for getting DNA into cells Vectors: Plasmid Vectors; Plasmids are widely used as cloning vehicles Plasmids are replicons which are stably inherited in any extra chromosomal state Most plasmids exist as double-stranded circular DNA molecules

3 Therefore, desirable properties of plasmid cloning vehicles are: o o o Low molecular weight Ability to confer readily selected phenotypic traits on host cells Single sites for a large number of restriction endonucleases preferably in genes with a readily scorable phenotype For genetic engineering, naturally occurring plasmids have been extensively modified to produce vectors that have the desired characteristics pbr322: an important plasmid in the history of gene manipulation It has all features of a good vector such as: o o o o Low molecule weight i.e. 4363bp Antibiotic resistance genes i.e. amplicine and tetracycline An origin of replication and Several single-cut restriction endonucleases recognition sites Fig 5.1 Nicholl 3 rd Ed

4 Mexican creators: Bolivar and Rodriguez

5 puc Family: More exotic, derived from pbr 322 Fig 5.2 Nicholl 3 rd Ed Although plasmid vectors have many useful properties and are essential for gene manipulation, they do have a number of disadvantages One of the major drawbacks is the size of DNA fragment that can be inserted into plasmids is 5kb before cloning efficiency or insert stability are effected Genomic library Bacteriophage Vectors: They are more specialized than plasmid In the 1940s Max Delbruck and the phage group laid the foundation of Modern Molecular Biology by studying bacteriophages These are literally eaters of bacteria and are viruses that are dependent on bacteria for their propagation so called bacteriophages or phages Structurally phages fall into three main groups: o o o Tailless Head with tail an Filamentous

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7 Fig 5.3 Nicholl 3 rd Ed The genetic material may be single or double stranded DNA or RNA. Double-stranded DNA (ds DNA) being found most often In typical ds DNA phages, the genome makes up about 50% of the mass of the phage particle. Thus phages represent relatively simple systems when compared to bacteria and for this reason they have been extensively used as models for the study of gene expression Phages may be classified as either virulent or temparate depending on their life cycles Fig 4.11 Primrose 6 th Ed The genome of phage is 48.5 kb in length and encodes some 46 genes The entire genome has been sequenced (this was the first major sequencing project to be completed and represents one of the milestones of molecular genetics) and all the regulatory sites are known At the ends of the linear genome there are short (126p) singlestranded regions that is complementary These act as cohesive or sticky ends which enable circularization of the genome following infection The region of the genome that is generated by the association of the cohesive ends is know as the cos site

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10 Fig 5.4 Nicholl 3 rd Ed The genome circularizes and the phage initiates either the lytic or lysogenic cycle depending on a number of factors that include the nutritional and metabolic state of the host cell and the multiplicity of infection (m.o.i) the ratio of phage to bacteria during absorption To determine the number of bacteriophage present in a suspension, serial dilutions of the phage stock are mixed with an excess of indictor bacteria (m.o.i. is very low) and placed onto agar On incubation, the bacteria will grow to form what is termed a bacterial lawn Phage that grown in this lawn will cause lysis of the cells that the phage infects and as this growth spreads a cleared area or plaque will develop Fig 5.6 Nicholl 3 rd Ed Plaques can then be counted to determine the number of plaque forming units (p.f.u.) in the stock suspension Two main classes of vectors obtained by manipulating nonessential parts of genome: Insertion vectors and Replacement vectors

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13 Fig 5.7 Nicholl 3 rd Ed Further innovations have been made by adding different characteristics on basis of selection and/or screening can be done Fig 5.8 Nicholl 3 rd Ed Fig 5.9 Nicholl 3 rd Ed The filamentous phage M13 differs from λ both structurally and in its life cycle Fig 5.3 Nicholl 3 rd Ed The M13 genome is a single stranded circular DNA molecule 6407bp in length The phage will infect only E.coli that has F-pili (threadlike protein appendages found on conjugation-proficient cells When DNA enters the cell, it is converted into double stranded molecule known as the replicative form or RF, which replicates until there are about 100 copies in the cell At this point DNA replication becomes asymmetric and single stranded copies of the genome are produced and extruded from the cell as M13 particles The bacterium is not lysed and remain viable during this process, though growth and division are slower than in non-infected cells

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15 ci λ repressor- plaque formation and morphology

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18 Two aspects of M13 infection are of value to the genetic engineer i. The RF is essentially similar to a plasmid and can be isolated and manipulated using the same technique ii. A single stranded DNA produced during infection is useful in techniques such as DNAsequencing by dideoxy method Unlike phage λ, M13 does not have any non-essential genes Only part available for manipulation is a 507bp intergenic region which has been used to construct vectors Fig 5.10 Nicholl 3 rd Ed Cosmids, Phasmids and Other Advanced Vectors: Today the Molecular Biologist has available an enormous range of vectors and these are notable for three reasons: Many of them combine elements from both plasmid and phages and are known as phasmids or if they contain an M13 ori region then called phagemids Many different features that facilitates cloning and expression can be found combined in a single vector Purified vector DNA plus associated reagents can be purchased from molecular biology suppliers There are two general uses of cloning vectors: cloning large pieces of DNAand manipulating genes

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20 Vectors for Cloning Large Fragments of DNA: Cosmid Vectors / Hybrid Vectors: Fig Klug BACs-Bacterial Artificial Chromosome: - A multi purpose vector that has been developed for mapping and analysis of complex eukaryotic genomes is based on the F factor of bacteria and is called a bacterial artificial chromosome - What are F factors? - Independently replicating plasmids that are involved in the transfer of genetic information during bacterial conjugation - Because F factors can carry fragments of bacterial chromosome up to one Mb in length, they have been engineered to act as vectors for eukaryotic DNA Yeast Vectors: Fig Klug - Although yeast is a eukaryotic organism, it can be manipulated and grown in much the same way as bacterial cells

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23 - Genetics has been studied intensively - Genetic map of yeast chromosomes - Large number of mutants available - A naturally occurring plasmid is available - 2 µm plasmid - A number of vectors has been developed, with the choice of vectors depending on the particular application Viral Vectors: Fig 5.11 Nicholl 3 rd Ed Table 5.1 Primrose 6 th Ed Two classes of animal viruses i. DNAcontaining viruses ii. RNA containing viruses Table 5.5 Nicholl 3 rd Ed Fig Primrose 5 th Ed

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27 tsa- temperature sensitive antigen

28 Plant Vectors: Plant transformation with the Ti plasmid of Agrobacterium tumefaciens OR Agrobacterium-mediated transformation Currently the most widely used plant cell vectors are based on Ti plasmid What is Ti plasmid? The Gram negative soil bacterium - Agrobacterium tumefaciens is a phytopathogen that as a normal part of its life cycle, genetically transforms plant cells The genetic transformation leads to the formation of crown gall tumors, which interfere with the normal growth of an infected plant This agronomically important disease affects only dicotyledonous plants (dicots) including grapes, stone fruit trees (peaches) and roses Fig 17.1 Glick 3 rd Ed Crown gall formation is the consequence of the i. transfer ii. integration and

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30 iii. expression of genes of a specific segment of bacterial plasmid DNA called the T-DNA (transferred DNA) into the plant genome The T-DNA is actually part of the tumor-inducing (Ti) plasmid that is carried by most strains of A. tumefaciens Depending on the bacterial strain that is host to the T. plasmid, the length of T-DNA region can vary from approximately Kb Strains of A. tumefaciens that do not posses a Ti plasmid cannot induce crown gall tumors Fig 17.2 Glick 3 rd Ed Fig 17.3 Glick 3 rd Ed Ti Plasmid Derived Vector Systems Fig 17.7 Glick 3 rd Ed / Fig 18.7 Glick 4 rd Ed A Simple Experimental Procedure for Agrobacterium-Mediated Transformation: Fig Primrose 6 th Ed

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33 The recipient A. tumefaciens strain carries a modified (defective or disarmed) Ti plasmid that contains a complete set of vir genes but lacks portion or all T DNA region so that T DNA can not be transferred

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35 Chloroplast Transformation: So far, we have exclusively considered DNA transfer to the plants nuclear genome However, the chloroplast is also a useful target for genetic manipulation Because thousands of chloroplasts may be present in photosynthetic cells and this can result in levels of transgene expression up to 50 times higher than possible using nuclear transformation Further more, transgenes integrated into chloroplast DNA do not appear to undergo silencing or suffers from position effects that can influence the expression levels of transgenes in the nuclear DNA Chloroplast transformation also provides a natural containment method for transgenic plants. Since the transgenes cannot be transmitted through pollen Direct DNA transfer methods-use of vector containing chloroplast homology regions, allowing targeted integration into the chloroplast genomes, and the use of selectable marker gene and amino glycoside adenyltransferase (AAD) which confer resistance to streptomycin and spectinomycin Table 17.4 Glick 3 rd Ed

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37 Plant Viruses as Vectors: As an alternative to stable transformation using agrobacterium or direct DNA transfer, plant viruses can be employed as gene transfer and expression vectors There are several advantages to the use of viruses. For example: o o o o o o o Viruses are able to adsorb to and introduce their nucleic acid into intact plant cells Infected cells yield large amounts of virus so recombinant viral vectors have the potential for high level transgene expression Viral infections are often systemic. The virus spreads throughout the plant, allowing transgenic expression in all cells Viral infections are rapid, so large amounts of recombinant protein can be produced in a few weeks All known plant viruses replicate episomally; there the transgene they carry are not subject to the position effects that often influence the expression of integrated transgene Since plant viruses neither integrate into nor pass through the germ line, plants can not be stably transformed by viral infection and transgenic lines can not be generated However, this limitation can also be advantageous in terms of containment

38 DNA Viruses as Expression Vectors: The vast majority of plant viruses have DNA genomes However, the two groups of DNA viruses that are known to infect plants The caulimo viruses and gemini viruses were the first to be developed as vector, because of the case with which their small DNA genomes could be manipulated in plasmid vectors Fig Primrose 6 th Ed RNA Viruses as Expression Vectors: Most plant RNA viruses have a filamentous morphology: i. Tobacco mosaic virus (TMV) ii. iii. Bromo mosaic virus (BMV) Potato mosaic virus (PMV) Method for Getting DNA into Cells: Fig 1 Chapter 6 Walker 3 rd Ed Fig 2 Chapter 6 Walker 3 rd Ed Fig 14.4 Primrose 5 th Ed

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41 Various methods to let DNA go into cells: - Chemical - Physical - Viral

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43 Fig 3 Chapter 6 Walker 3 rd Ed Fig 5.13 Nicholl 2 nd Ed Fig 16.5 Primrose 5 th Ed / Fig 11.1 Primrose 2 nd Ed Fig 13.9 Nicholl 3 rd Ed Fig 16.4 Brum Table 17.1 Glick 3 rd Ed Fig 17.9 Glick 3 rd Ed Table 17.2 Glick 3 rd Ed Table 17.3 Glick 3 rd Ed Host Cells: The type of host cell used for a particular application will depend mainly on the purpose of the cloning experiment If the aim is: To isolate a gene for structural analysis, the requirements may call for a simple system that is easy to use

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47 Large in size copied of GH gene/mouse metalothionine promoter were injected -Subsequently fuse to form the diploid zygote nucleus of fertilized egg - Embryo culture in vitro until they have undergone a number of division / Surrogate mother This was the ground work for development of genetically engineered livestock carrying genes that improve their food value. For example Pig - leaner meat as increased amount of GH stimulates the conversion of nutrients into proteins rather than fat Fig 13.9 Nicholl 3 rd Ed

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53 To express the genetic information in a higher eukaryotic such as a plant, a more specific system will be required These two aims are not necessarily mutually exclusive Often a simple primary host is used to isolate a sequence that is then introduced into a more complex system for expression Table 5.1 Nicholl 3 rd Ed Concept Map 5 Nicholl 3 rd Ed x x x x

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