I SATELLITI DI SCIENZE NATURALI I CLIL BIOTECHNOLOGY CLIL RICERCA E DIDATTICA DELLE SCIENZE PER LA SCUOLA ITALIANA PRINCIPATO

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1 I SATELLITI DI SCIENZE NATURALI I CLIL MARINA PORTA PIA CLARA PAFUNDI CLIL BIOTECHNOLOGY PRINCIPATO RICERCA E DIDATTICA DELLE SCIENZE PER LA SCUOLA ITALIANA

2 I SATELLITI DI SCIENZE NATURALI I CLIL MARINA PORTA PIA CLARA PAFUNDI BIOTECHNOLOGY CLIL PRINCIPATO RICERCA E DIDATTICA DELLE SCIENZE PER LA SCUOLA ITALIANA

3 Direzione editoriale: Franco Menin Coordinamento: Marinella Torri, Alessandra Brunetti Redazione: Marina Mansi Progetto graico e copertina: Giuseppina Vailati Canta Ricerca iconograica: Marina Mansi Impaginazione: Fabio Bergamaschi, Studio In.pagina Disegni: Daniele Gianni Le pagine espositive sono un libero adattamento di parti dell opera I satelliti di scienze naturali Biotecnologie di Maria Vezzoli e Claudio Vicari, edita dalla Casa editrice Principato nel Si ringrazia la professoressa Alexandra McLennan per la consulenza e la revisione linguistica di tutti i materiali. Ha collaborato alla redazione del testo Martina Mirabella. Immagine di copertina: Shutterstock I materiali reperibili nel sito o allegati all edizione digitale sono messi a disposizione per un uso esclusivamente didattico. All atto della pubblicazione la casa editrice ha provveduto a controllare la correttezza degli indirizzi web ai quali si rimanda nel volume; non si assume alcuna responsabilità sulle variazioni che siano potute o possano intervenire successivamente. Per le riproduzioni di testi e immagini appartenenti a terzi, inseriti in quest opera, l editore è a disposizione degli aventi diritto non potuti reperire, nonché per eventuali non volute omissioni e/o errori di attribuzione nei riferimenti. ISBN (I CLIL Biotechnology) ISBN (sola versione digitale) Prima edizione: marzo 2015 Ristampe VI V IV III II I * Printed in Italy 2015 Proprietà letteraria riservata. È vietata la riproduzione, anche parziale, con qualsiasi mezzo effettuata, compresa la fotocopia, anche ad uso interno o didattico, non autorizzata. Le fotocopie per uso personale del lettore possono essere effettuate nei limiti del 15% di ciascun volume dietro pagamento alla SIAE del compenso previsto dall art. 68, commi 4 e 5, della legge 22 aprile 1941 n Le riproduzioni per inalità di carattere professionale, economico o commerciale o comunque per uso diverso da quello personale, possono essere effettuate a seguito di speciica autorizzazione rilasciata da EDISER (Centro licenze e autorizzazioni per le riproduzioni editoriali), corso di Porta Romana 108, Milano, e sito web Casa Editrice G. Principato S.p.A. Via G.B. Fauché Milano La casa editrice attua procedure idonee ad assicurare la qualità nel processo di progettazione, realizzazione e distribuzione dei prodotti editoriali. Stampa: SEBEGRAF - Arese (Mi)

4 i CLIL Un apprendimento integrato di lingua e contenuti La metodologia di insegnamento CLIL, Content and Language Integrated Learning, creata nel 1994 da David Marsh e Anne Maljers, rappresenta un nuovo approccio metodologico in ampia espansione nelle scuole italiane ed europee, con lo scopo di promuovere l insegnamento di una disciplina in una lingua comunitaria, solitamente la lingua inglese. Gli obiettivi del CLIL Il CLIL facilita la comunicazione nella seconda lingua, permettendo allo studente di esercitare le sue abilità su contenuti disciplinari, piuttosto che solo e unicamente sulle strutture grammaticali. Le quattro C Il metodo CLIL permette di lavorare su più piani contemporaneamente, le cosiddette 4 C : Cognition, Content, Culture, Communication. L approccio integrato e facilitato (scaffolding) favorisce la costruzione di un personale sapere globale e il conseguimento di specifiche competenze linguistiche e disciplinari. I CLIL di scienze naturali Nei CLIL di scienze naturali i contenuti sono segmentati per facilitarne la comprensione e accompagnati da numerose e diverse tipologie di esercizi, ideate non solo per valutare l acquisizione delle prestazioni previste (reading, writing, defining, completing, checking, reconstructing, filling, matching), ma anche per permettere agli studenti di raggiungere una performance adeguata alle personali capacità e ai diversi stili cognitivi. 3

5 I SATELLITI DI SCIENZE NATURALI I CLIL BIOTECHNOLOGY è proposto anche in versione ebook+, un nuovo modo per studiare i contenuti utilizzando diversi dispositivi multimediali, come computer e tablet. I contenuti digitali integrativi di questo volume sono disponibili sul sito L ebook+ è multimediale L ebook+ arricchisce il libro di testo digitale con contributi audio, video, oggetti interattivi, gallerie fotografiche e link a risorse esterne. L ebook+ è interattivo L ebook+ integra esercizi direttamente sul testo digitale per un immediata verifica dell apprendimento. L ebook+ è coinvolgente L ebook+ aiuta gli studenti a comprendere e approfondire i contenuti, rendendo l apprendimento più attivo e divertente. Nelle pagine sono inserite delle icone che indicano la presenza e il tipo di contributi disponibili sul libro di testo. È sufficiente cliccarle per accedere ai materiali didattici aggiuntivi: foto, video, esercizi, link e oggetti interattivi. Audio Integra il testo con la versione audio dei contenui. Approfondimenti Rimanda ad altre sezioni del libro digitale che contengono materiali per l approfondimento. Link Rimanda a siti e a pagine web esterne. Test Ofre esercizi, test a risposta mulipla o veriiche di apprendimento. La correzione è immediata e permete di individuare e ripassare in autonomia i contenui non assimilai. Video Integra il testo con ilmai e animazioni. HTML 5 Propone oggei interativi che integrano immagini e tesi e ofrono un nuovo modo di studiare e approfondire i contenui. Fotogallery Aggiunge fotograie, disegni e altro materiale graico al testo. Può essere una singola immagine o una gallery di immagini. 4

6 Recombinant DNA and chapter1 genetic engineering 1.1 What is biotechnology? 7 BEFORE READING 7 WHILE READING 8 Is biotechnology an ex novo science or the result of the ongoing research? 8 An important discovery: living microorganisms are at the core of the fermentation process 8 The transition from traditional to innovative biotechnology 9 AFTER READING Recombinant DNA 12 BEFORE READING 12 WHILE READING 14 Recombinant DNA technology 14 Restriction enzymes 14 How do we get recombination? 16 Further exploitations: cloning 17 AFTER READING Biotechnology: the instruments 21 BEFORE READING 21 WHILE READING 22 DNA gel electrophoresis 22 Gel electrophoresis in practice 23 Polymerase chain reaction (PCR) 24 PCR: the three steps 26 AFTER READING Genetic engineering and GMOs 30 BEFORE READING 30 WHILE READING 30 Vectors introduce new DNA into host cells 30 Genetically Modiied Organisms (GMOs) 32 AFTER READING 34 Final test chapter 1 36 Contents Genome study chapter2 and bioinformatics 2.1 Genome sequencing: the methods 39 BEFORE READING 39 WHILE READING 39 Biotechnology in real life 39 Sequencing methodologies 40 Sanger method 40 Shotgun sequencing 41 AFTER READING Genomic libraries 45 BEFORE READING 45 WHILE READING 46 Genomic libraries contain collections of DNA fragments 46 cdna libraries are built starting from mrna transcripts 48 AFTER READING Genome study: probes, microarrays and chips 51 BEFORE READING 51 WHILE READING 51 Developments in the genome study 51 AFTER READING The Human Genome Project (HGP) 55 BEFORE READING 55 WHILE READING 55 How the Human Genome Project was born 55 HGP results 56 The importance of junk DNA 57 AFTER READING Bioinformatics: a growing field of research 60 BEFORE READING 60 WHILE READING 60 The birth of Bioinformatics 60 Bioinformatics and evolutionary biology 61 RNA interference 62 AFTER READING The future: the omics 65 BEFORE READING 65 WHILE READING 65 From the HGP to the omics era 65 How can we understand the proteome? 66 AFTER READING 68 Final test chapter 2 70 Index 72 5

7 Recombinant DNA and genetic engineering 1.1 What is biotechnology? 1.2 Recombinant DNA 1.3 Biotechnology: the instruments 1.4 Genetic engineering and GMOs 1 chapter 6

8 chapter 1 ReCoMbiNANT DNA AND GeNeTiC engineering 1.1 What is biotechnology? BEFORE READING 1 Work in group and agree on a deinition for the word biotechnology. 2 Match the words in column A with their deinitions in column B. 3 Solve the crossword below. A a DNA 1 Specialized eukaryotic cell structures delimited by a membrane. b Enzyme 2 A low-energy attractive force between hydrogen and another element. c Chromosome 3 A nucleic acid existing in diferent forms. d Bacteria 4 Nucleic acid with heritable, genetic material. e Organelles 5 Protein which catalyzes speciic cell reactions. f RNA 6 A threadlike structure within the cell, placed either in the nucleus or in the cytoplasm which bears genetic material. g Replication 7 Prokaryotic microorganisms. h Hydrogen bonds 8 The fundamental, physical and functional units of heritability. i Genes 9 The process of producing an exact copy of DNA. j Genetics 10 Study of both the genotype and phenotype of parent organisms in order to produce a hybrid with desirable features from the parents. k Artiicial selection 11 The science which studies heritability. B 7 Down 2. Study of both genotype and phenotype of parent organisms to produce a hybrid with desirable features found in their parents. (2 Words) 3. Protein which catalyzes specific cell reactions. 4. The science which studies heritability. 6. Specialized eukaryotic cell structures delimited by membrane. 10. A nucleic acid existing in different forms Across 1. Nucleic acid with the function of heritable genetic material. 5. The process of producing an exact copy of DNA. 7. The fundamental, physical and functional unit of heritability. 8. Prokaryote microorganisms. 9. A threadlike structure within the cell, placed either in the nucleus or in the cytoplasm, that bears the genetic material. 11. Low-energy attractive forces between hydrogen and another element. (2 Words)

9 chapter1 ReCoMbiNANT DNA AND GeNeTiC engineering 1.1 What is biotechnology? WHILE READING 4 Read the text carefully. Underline any words you don t understand and look them up in the dictionary. Is biotechnology an ex novo science or the result of ongoing research? Biotechnology can broadly be deined as any technology which uses either living organisms or puriied sub-cellular components in order to obtain large quantities of useful products; improve the characteristics of plants and animals; or develop useful microorganisms for speciic uses. Although the term biotechnology is a relatively new one, it has ancient even prehistoric origins. Humans began turning milk into yogurt and cheese, and producing beer, wine and bread thousands of years ago 1. Our ancestors did not understand the mechanisms underlying these changes (rising, fermentation, etc.) and hence could not have realized that living organisms were at the core of these processes. 1 Ancient Egypts and beer An Egyptian funerary stele, dating back to 1350 B.C., depicts a man tasting beer. An important discovery: living microorganisms are at the core of the fermentation process In 1861 Louis Pasteur understood and described these common and yet mysterious events. He identiied the organisms responsible for the changes involved in things such as beer brewing and the fermentation of milk 2. Pasteur can be seen as the father of biotechnology. He laid the foundations for the fermentative processes which form the basis of the bio-industry. These processes use pure cultures of microorganisms to produce food, drink and other useful products. 2 Yogurt Yogurt is the product of milk fermented by speciic bacteria. 8

10 The transition from traditional to innovative biotechnology The transition from the traditional to the innovative stages of biotechnology is linked to the selection and characterization of strains of microorganisms and to the development of technologies for the cultivation and optimization of production 3. However, innovative biotechnology is only clearly deined by the use of recombinant DNA technology (genetic engineering). Modern biotechnology is based on a series of advances made in molecular genetics between 1950 and The conluence of the methods from genetics and molecular biology led to the development of genetic engineering around the 1980s. Over the past thirty years, genetic engineering has specialized to include a range of experimental methods to isolate, characterize and manipulate genes. Genes, made of DNA, contain the instructions to produce either functional or structural speciic proteins. The ability to locate, transfer and modify genes is an important feature of innovative biotechnology. 3 Small spelt, Triticum monococcum, grown in the Neolitic, is today used for the genetic improvement of the other species of spelt. 9

11 chapter1 ReCoMbiNANT DNA AND GeNeTiC engineering 1.1 What is biotechnology? AFTER READING READING COMPREHENSION 5 Read the text on biotechnology. Mark the following statements as true (T) or false (F) and correct the false statements. 1 Biotechnology can functionally be deined as any technology based on the information contained in DNA. T F 2 Modern biotechnology uses non-living organisms to isolate genes. T F 3 In 1700, Pasteur discovered the microorganisms responsible for the fermentation process. 4 The transition from traditional to innovative biotechnologies is based only on the selection of the strains of microorganisms to be used. 5 Genetic engineering includes several methods in order to isolate, characterize and manipulate genes. T F T F T F EXPANDING VOCABULARY 6 Fill in the gaps with the words below. engineering biotechnology methods manipulate biology isolate experimental biology specialized genes information 1980s proteins Modern is based on a series of advances made in molecular between 1950 and The conluence of the from genetics and molecular led to the development of genetic around the. Over the past thirty years genetic engineering has to include a range of methods to isolate, characterize and genes. Genes, made of DNA, contain the to produce either functional or structural speciic. The ability to, transfer and modify is an important feature of innovative biotechnology. CONSOLIDATING KNOWLEDGE 7 Answer the following questions. a. Which organism is responsible for fermentation? b. Why can t we consider biotechnology a recent innovation? 10

12 APPLYING NEW KNOWLEDGE 8 Create a timeline in which you place at least ive signiicant events in the discovery of DNA between the 17 th and 20 th centuries. Use the appropriate software (e.g. SimpleMind software) to build your timeline as a conceptual map. 9 Read the following table and answer the questions. Biotechnology through time Before Christ In Mesopotamia, animals are frequently crossed with one another (artificial selection) in order to improve livestock. Fermentation is first done with the use of yeast to produce beer, wine and bread Anton van Leeuwenhoek discovers the existence of microorganisms thanks to the microscope. XIX century Louis Pasteur identifies the role of microorganisms. He is considered the founder of microbiology. In 1865 Gregor Mendel formulates the three fundamental laws of inheritance Karl Ereky, a Hungarian agronomist, uses the term biotechnology for the first time. In 1950, the first generation of whole plants is grown from in vitro culture Identification of the DNA double helix, responsible for the transmission of genetic information Birth of modern biotechnology Birth of genetic engineering. In 1983, Kary B. Mullis creates the polymerase chain reaction (PCR) technique, a revolution in the world of biotechnology First cloning of a mammal, Dolly the sheep, using adult sheep DNA Completion of the Human Genome Project. a. Who used the term biotechnology for the irst time? b. When was the Human Genome Project completed? c. When was fermentation irst done? d. Which animal was cloned irst? 10 Using the information in the table, try to write a short text about the origins and development of biotechnology. (You may start like this: Biotechnology is a very ancient science. In fact, even before Christ, ) 11

13 chapter 1 ReCoMbiNANT DNA AND GeNeTiC engineering 1.2 Recombinant DNA BEFORE READING 1 Work in group and agree on a deinition for the word recombination. 2 Match the words in column A with their deinitions in column B. A a Genome 1 Organisms which spend a signiicant portion of their life in or on a host organism, causing it harm even if not immediately killing it. b Vector 2 A gram-negative bacillus abounding in the intestine of endotherms commonly used as a model organism. c DNA Ligase 3 The ability of a microorganism to withstand the efects of an antibiotic. d Parasites 4 A DNA or RNA sequence that reads the same in both directions. e Codon 5 The physical appearance or biochemical characteristic of an organism as a result of the interaction of its genotype and the environment. f Palindrome 6 An organism s complete set of DNA, including all of its genes. g Escherichia coli 7 An enzyme which joins DNA strands together by forming a phosphodiester bond. h Phenotype 8 A triplet in mrna which forms a base-pair with the corresponding anticodon of a trna molecule. i Antibiotic resistance 9 A plasmid or viral chromosome into whose genome a fragment of foreign DNA is inserted. B endotherms organisms/animals that maintain a constant body temperature independently of the environment Electron microscope image of Escherichia coli 12

14 3 Solve the crossword below. Across 5. A nucleic acid present in all living cells, which is generally single stranded (double stranded in some viruses) and plays a crucial role in transferring information from DNA to the protein synthesis system of the cell. 7. The ability of a microorganism to withstand the effects of an antibiotic. (2 Words) 9. The physical appearance or biochemical characteristic of an organism as a result of the interaction of its genotype and the environment. 10. A plasmid or viral chromosome into whose genome a fragment of foreign DNA is inserted Down 1. A gram-negative bacillus which abounds in the intestine of endotherms. It is commonly used as a model organism. (2 Words) 2. An enzyme which closes either nicks or discontinuities in one strand of double stranded DNA by creating an ester bond between adjacent 3 OH and 5 PO4 ends on the same strand. (2 Words) 3. A DNA or RNA sequence that reads the same in both directions. 4. A triplet of nucleotide bases in transfer RNA that identifies the amino acid carried and which binds to a complementary codon on messenger RNA during protein synthesis at a ribosome. 6. Organisms which spend a significant portion of their life either in or on a host organism, causing it harm even if not immediately killing it. 8. An organism s complete set of DNA, including all of its genes. 13

15 chapter1 ReCoMbiNANT DNA AND GeNeTiC engineering 1.2 Recombinant DNA WHILE READING 4 Read the text carefully. Underline any words you don t understand and look them up in the dictionary. Recombinant DNA technology Recombinant DNA technology is a laboratory technique in which short sequences of DNA are isolated and cut in order to transfer and insert them into the genome of other cells, in turn modifying one or more genes. This technology allows for the speciic modiication of a particular gene or characteristic. Moreover, current methods allow for the transfer of DNA among individuals of species which are often very different from one another (e.g., the transfer of a bacterial gene to a plant or the introduction of a eukaryotic gene into a bacterium). In practice, recombinant DNA technology is very complex but conceptually it is based on very simple criteria: (a) identiication of the gene; (b) the cutting and isolation of the gene from the DNA molecule; (c) cohesion of the gene with a DNA vector and (d) transfer of the gene to a recipient cell. This allows for a range of possibilities, from a simple genetic improvement in the recipient individual (e.g., increased resistance to parasites) to the cloning of an introduced gene in a host cell. This latter process creates a cell which in a sense becomes a factory producing useful molecules. Restriction enzymes DNA recombination relies on cutting DNA, an operation which requires speciic enzymes (restriction enzymes and DNA ligases). At the end of the 1960s, it was found that some bacteria defended themselves from virus infection by producing special enzymes, namely restriction enzymes, which cut the extraneous DNA molecules and reduced them into smaller, non-infectious, fragments 1. These enzymes break the backbone of the DNA, the phosphodiester bond, between two subsequent nucleotides. Nowadays we know several restriction enzymes, each with a speciic recognition sequence (4-6 base pairs), known as the restriction site. Once restriction enzymes have completed the cut, two types of 1 Enzymes acting as scissors Bacteria defend themselves from the attack of viruses (bacteriophages) thanks to the action of restriction enzymes. 1 A bacteriophage introduces its genetic material into the bacterial cell. 2 A restriction enzyme cuts viral DNA at the level of speciic sequences of nucleotides. 3 Other enzymes further degrade viral DNA into even smaller fragments. 4 Bacterial DNA is protected by the restriction enzymes attack thanks to methyl groups on the restriction site of the enzyme. 14

16 single-stranded ends can be obtained, each containing a sequence of bases able to bind to another one for complementarity 2: - blunt ends, when the two helices are interrupted in the centre of the restriction site: A-A T-T T-T A-A sticky ends, when the two helices are cut asymmetrically, resulting in one longer than the other: G A-A-T-T-C C-T-T-A-A G Note that, reading these sequences in the 5-3 direction, they are palindrome. Restriction enzymes never cut the DNA of their producing cell. Indeed, bacteria protect their DNA through methylation, the addition of a methyl group (-CH 3 ) to every restriction site on the bacterial DNA. Today, there are hundreds of restriction enzymes and once isolated, extracted and puriied from various organisms, they are used in the laboratory as biochemical scissors. They are so called because when incubated with the DNA of an organism in a test tube, they are able to cut that organism s DNA at each point where a restriction site is present. Thus, the same DNA sample can be cut by more than one enzyme, each recognizing a different restriction site. In this way molecular biologists can blunt end the simplest DNA end of a double stranded molecule. Both strands terminate in a base pair sticky end long overhangs, stretches of unpaired nucleotides in the end of a DNA molecule. These unpaired nucleotides can be in either strand, creating either 3 or 5 overhangs. These overhangs are in most cases palindromic. These ends are called sticky since they are easily joined back together by a ligase 2 How recombinant DNA originates The restriction enzyme recognizes the palindromic sequences and cuts them asymmetrically (red arrows), thus producing fragments with sticky ends. Such fragments pair with the complementary bases and hydrogen bonds form, linking the two strands of DNA. The result is a recombinant DNA molecule. Restriction sites of EcoRI in two different DNA molecules DNA 5' CGATCCAGGAATTCATCCAGCC AG A CAGCC CC 3' 3' GCTAGGTCCTTAAGTAGGTCGG TC GTCGG GG 5' 5' AGGCTCTAGAATTCTTCTAGCT TAG TTC TAGCT 3' 3' TCCGAGATCTTAAGAAGATCGA GAT ATC AAG GATC ATCGA 5' Fragments with sticky ends CGATCCAGG AGG GCTAGGTCCTTAA TCC AATTCATCCAGCC ATCCAG CAGCC CC GTAGGTCGG GTCGG GG AGGCTCTAG TAG TCCGAGATCTTAA ATC AATTCTTCTAGCT T TAGCT GAAGATCGA ATCGA CGATCCAGGAATTCTTCTAGCT AG TTC TAGCT GCTAGGTCCTTAAGAAGATCGA TCC AAGATC ATCGA Recombinant DNA 15

17 chapter1 ReCoMbiNANT DNA AND GeNeTiC engineering 1.2 Recombinant DNA choose among different sites and cut a DNA fragment with surgical accuracy Table 1. The obtained fragments of DNA are called restriction fragments. Since the recognizing sequences do not occur at regular intervals, restriction fragments have different lengths. This allows them to be distinguished, both by the determination of the number of single fragments and their molecular size and by the identiication and puriication of fragments of particular interest. As we will see in the next paragraph, gel electrophoresis is one method used to either separate or purify DNA fragments. Table 1 Some restriction enzymes, the original organism and the recognized sequence Enzymes Original organism Recognized sequence EcoRI Escherichia coli 5 GAATTC3 3 CTTAAG5 EcoRV Escherichia coli 5 GATATC3 3 CTATAG5 BamHI Bacillus amyloliquefaciens 5 GGATCC3 3 CCTAGG5 HindIII Haemophilus inluenzae 5 AAGCTT3 3 TTCGAA5 How do we get recombination? During DNA duplication, Okazaki fragments are joined together by enzymes called DNA ligases. Once biologists were able to isolate these enzymes, they realized that they could be useful in welding together any two DNA sequences. In 1973, Stanley Cohen and Herbert Boyer irst cut two plasmids of Escherichia coli (both containing a gene for antibiotic resistance) with restriction enzymes and then joined these with a DNA ligase. Once the resulting plasmid was inserted into Escherichia coli cells they became resistant to both antibiotics. The era of recombinant DNA was born 3! The oblique cut created by most restriction enzymes on palindrome restriction sites on the DNA strand is fundamental in obtaining recombinant DNA. Between the two types of cut, sticky ends are the most suitable to produce recombinant DNA. This is because they can be easily linked through hydrogen bonds between the complementary bases of other DNA sequences (e.g., that of a plasmid) cut by the same restriction enzyme and regardless of their origin. DNA ligases stabilize the bonds between adjacent fragments joining the 5 end of one of them to the 3 end of the next one. Some DNA ligases are also able to combine restriction enzymes with blunt ends using slightly different mechanisms. Note that DNA ligases are very sensitive to temperature (which greatly affects hydrogen bonds) and require ATP to operate. Using these enzymes, molecular biologists are able to produce recombinant DNA from a variety of sources. 3 Plasmids Circular DNA molecule of the plasmids of a bacteria, containing a few useful genes, none of which are indispensable to the cell s life. plasmid bacterial chromosome Okazaki fragments the simplest DNA end of a double stranded molecule. Both strands terminate in a base pair ATP (Adenosine TriPhosphate) substance present in all living cells which provides energy for many metabolic processes 16

18 Further exploitations: cloning Nature has provided molecular biologists with the tools for their toolbox, but the extraordinary variety of life offers biotechnology much more than this. Often, a function lacking in a certain species can be found in another species. Finding a methodology is often suficient to allow for the insertion of an exogenous gene of interest into the genome of the species with the deicient function. Nature even provides the appropriate organisms to do this: viruses. The most common application of recombinant DNA in biotechnology is the cloning (i.e., the production of many copies) of a particular gene in order to either analyse or obtain large quantities of its protein product 4. In the latter case, recombinant DNA must either be inserted or transfected into host cells. These cells are considered transgenic after this modiication. The choice of the host cell is extremely important for the attempt to be successful. Host cells can be either prokaryotic or eukaryotic. Once the host species have been chosen, recombinant DNA is brought into contact with a population of host cells and, under appropriate conditions, it is inserted into some of them. Early successes in recombinant DNA technology were achieved using bacteria as they are easy to grow and manipulate in the laboratory. Additionally, their molecular biology (e.g., Escherichia coli) is widely understood and there are several genetic markers available to identify the cells hosting the recombinant DNA. Moreover, bacteria contain plasmids, small, circular chromosomes easily manipulated to carry recombinant DNA into a cell. However, bacteria are not ideal for the study and the expression of eukaryotic genes. There is too much of a difference between the transcription and translation of genomes in prokaryotes and eukaryotes and these functions are often regulated by signals contained within the DNA itself. It is better, therefore, to choose a eukaryotic host, the most common of which are yeasts (like Saccharomyces cerevisiae) because they have a short cell cycle (just a few hours); a relatively small genome (approximately 12 million base pairs and 6000 genes); and they are easily cultivated in the laboratory. Yeasts share many basic features with eukaryotes except for those involved in the multi-cellular state. These latter features require their DNA to be carried inside a vector and the gene to be transferred into either an animal or plant genome. For example, some human proteins must be chemically modiied in order to operate after translation. As all the host cells duplicate themselves, the researcher must be able to recognize which cells actually contain the sequence to be cloned. A common method used to detect cells containing recombinant DNA is to mark the inserted sequence with reporter genes. These are genes whose phenotype is easy to observe and hence can serve as a genetic marker for the sequences of interest. 4 A researcher observes culture plates on which genetically modiied plants are growing transfected inserted into a cell of a bacterial plasmid which contains a foreign virus or genetic material prokaryotic single-celled organism which lacks a membrane-bound nucleus, mitochondria, or any other membrane-bound organelles eukaryotic any organism whose cells contain a nucleus and other structures (organelles) enclosed within membranes 17

19 chapter1 ReCoMbiNANT DNA AND GeNeTiC engineering 1.2 Recombinant DNA GENIC CLONING THROUGH PLASMIDS Escherichia coli bacteria plasmid bacterial chromosome cell containing the gene to be cloned 1 The plasmid and the DNA of the cell are isolated and then cut with the same restriction enzyme. 2 The DNA fragment to be cloned is inserted into the plasmid. 2 1 gene to be cloned gene to be cloned 3 The recombinant plasmid is inserted into the bacterium. 4 The bacterium in culture quickly replicates into great numbers and lots of recombinant plasmid copies are obtained. 5 The genes or the proteins of the cloned bacterium are isolated. recombinant plasmid 3 gene to be cloned 6 6 The genes are inserted into other organisms; e.g. into plants to increase their resistance to diseases or cold, or into bacteria to allow them to eliminate toxic waste. recombinant bacterium bacteria clones The proteins produced have useful applications. In medicine they are used for different therapies: against strokes (by helping to dissolve blood clots); keeping diabetes under control (insulin); and in treating children who do not produce enough growth hormones. 18

20 AFTER READING READING COMPREHENSION 5 Read the text on recombinant DNA. Mark the following statements as true (T) or false (F) and correct the false statements. 1 It is possible to transfer genes from prokaryotic to eukaryotic cells. T F 2 Restriction enzymes recognize a speciic DNA sequence. T F 3 Restriction enzymes also cut bacterial DNA. T F 4 DNA ligases use the same mechanism to join both blunt ends and sticky ends. T F 5 The reporter gene is generally used to mark DNA sequences. T F EXPANDING VOCABULARY 6 Fill in the gaps with the words below. species recombinant host cloning prokaryotic protein population inserted transgenic The most common application of DNA in biotechnology is the (i.e., the production of many copies) of a particular gene in order to either analyse or obtain large quantities of its product. In the latter case, recombinant DNA must be either or transfected into host cells. These cells are considered after this modiication. The choice of the cell is extremely important in order for the attempt to be successful. Host cells can be either or eukaryotic. Once the host have been chosen, recombinant DNA is brought into contact with a of host cells and, under appropriate conditions, it is inserted into some of them. CONSOLIDATING KNOWLEDGE 7 Answer the following questions. a. What is the major diference between genetic recombination in prokaryotes and eukaryotes? 19

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