Calumpang, Gino I. Flores, Czarriel F. Potencio, Valaine C.

BSED-III/ BioSci106 A February 9, 2012

1. Provide the significance of the following in relation to Recombinant Technology. a. Restriction enzyme Restriction enzymes have the remarkable ability to recognize specific arrangements of DNA base pairsAs and Ts, Gs and Cs. They also have the capacity to act like a molecular scalpel, severing the DNA at exactly the spot where they detect this sequence of genetic letters. Restriction enzymes are a powerful tool because there are thousands of them, and each one acts only on a unique arrangement of As and Ts and Cs and Gs. In other words, a specific enzyme is needed to cut the DNA from the donor genes at a specific site. This enzyme is called a restriction enzyme.The enzyme is used to cut out a piece of DNA that contains one or more desired genes from the donor's DNA. For cloning of DNA, the DNA is cut at specific sites, which are recognized and cleaved by specific enzymes. These enzymes are known as restriction enzymes. These restriction enzymes recognize short sequences of double stranded DNA as targets for cleavage. Different enzymes recognize different but specific sequences, each ranging from 4-8 base pairs. The enzymes are named by a three letter (or four letter) abbreviation that identifies their origin e.g. AluI is derived from Arthrobacter luteus, EcoRI is derived from E.Coli, HpaI is derived from Haemophilus parainfluenzae.

b. Vector A vector is needed to receive the donor DNA. Most frequently, a naturally occurring circular piece of bacterial DNA, called a plasmid, is used for this purpose. Vectors are the vehicles used to carry a foreign DNA sequence into a given host cell. A vector should have a) origin of replication, b) a selectable marker to select the host cells containing the vector from among those which do not have the vector, c) one unique restriction endonuclease recognition site to enable foreign DNA to be inserted into the vector in order to generate a recombinant DNA molecule and, d) it should be relatively small in size. Plasmids as vectors Plasmids are defined as autonomous elements, whose genomes exist in the cell as extrachromosomal units. They are self replicating, circular duplex DNA molecules present in number of copies in a bacterial cell, yeast cell or in organelles in eukaryotic cells. These naturally occurring plasmids have been modified to serve as vectors in the laboratory. pBR322 vectors One of the most commonly used cloning vector in gene cloning procedures is pBR322 (named after Bolivar and Rodriguez who constructed this) derived from E. coli plasmid ColE1. It is 4,362 bp DNA with genes for resistance against two antibiotics- tetracycline and ampicillin. It was constructed after several alterations and modifications in earlier cloning vectors. pUC vectors

The plasmids belonging to pUC series of vectors are 2,700 bp long with ampicillin resistance gene, the origin of replication derived from pBR322 and the lac Z gene derived from E.coli.When the DNA fragments are cloned in this region of pUC, the lac gene is inactivated. Yeast plasmid vectors Special vectors used to introduce DNA segments in yeast cells or a eukaryotic cell is being used to develop genetically engineered yeast cells. E.g. YIp or yeast integrative plasmids which allows transformation by crossing over and have no origin of replication. YEp or yeast episomal plasmids carry 2 micron DNA sequence including the origin of replication and rep gene. These vectors have been widely used to study yeast genome. Shuttle vectors The vectors containing two types of origin of replication which helps them to exist in both eukaryotic cell as well as E.coli are known as shuttle vectors. E.g.Yep vector. Retriever vectors A class of vectors which are used to retrieve specific genes from the normal chromosome of an organism like yeast through recombination. They are very useful in isolation of genes from yeast for molecular experiments like sequencing. Vectors based on bacteriophages Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells. They are used as vectors because they have a linear DNA molecule, which generate two fragments after breakage. These are later joined with foreign DNA to generate chimeric phage particle. The injected DNA is selectively replicated and expressed in the host cell resulting in a number of phages which burst out of the cell (lytic pathway) and further infect the neighbouring cells. E.g. M13 , Lambda ( )

Cosmids as vectors Cosmids are plasmid particles into which specific DNA sequences like cos sites of phage lambda are inserted and they are constructed by combining certain feature of plasmids and the cos sites of phage lambda. The advantage of the using cosmids for cloning is that its efficiency is high to produce a complete genomic library of 10(6)-10(7) clones from only 1 microgrm of insert DNA. Phagemids as vectors Phagemids are prepared artificially by combining features of phages with plasmids. E.g. pBluescript II KS is derived from pUC19 and is 2961 bp long. BAC Vectors BACs or Bacterial Artificial Chromosomes are vectors based on the natural, extra-chromosomal plasmid of E.coli- the fertility or F-plasmid, and are being used in genome sequencing projects. A BAC vector contains

genes for replication and maintenance of the F-factor, a selectable marker and cloning sites and can accommodate up to 300-350 kb of foreign DNA. Plant and animal viruses as vectors A number of plant and animal viruses can also be used as vectors for introducing foreign genes into cells and/or for gene amplification in host cells. Plant viruses like Gemini viruses, cauliflower mosaic virus or CaMV and tobacco mosaic virus /TMV) are three group of viruses that have been used as vectors for cloning of DNA segments. CaMV has a double stranded DNA molecule, 8kbp in size which infects the members of Cruciferae family. Geminiviruses are a group of single stranded DNA plant viruses infecting cassava, maize and other cereals. Animal virus vectors also deliver the foreign genes into the cultured cells which get integrated into the host genome. The expression of foreign genes can also be amplified using the promoters of the virus gene. The cloned genes can be used in gene therapy, for the synthesis of important proteins etc. A vector based on Simian Virus 40 (SV 40) was used in the first cloning experiment involving mammalian cells in 1979. Retroviruses like murine and avian retroviruses are very useful vectors as they are capable of infecting a large variety of cell types and can clone large genes. Herpes virus is a non-integrating large sized virus (150 kb) which is another useful vector which can be used for expression of large intact genes. Artificial chromosome (YAC and MAC) vectors YACs or Yeast Artificial Chromosomes are used as vectors to clone DNA fragments of more than 1Mb in size. These long molecules of DNA can be cloned in yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. These long DNA molecules can be generated and allow construction of comprehensive libraries in microbial hosts. A lot of work is going on to create mammalian artificial chromosomes (MACs) following the isolation of mammalian telomeres and centromere. However YACs have a low cloning efficiency (1000 clones/microgm) DNA as against 106-107 clones/microgm DNA for cosmids) and also it is difficult to recover large amount of pure insert DNA from individual clones.

c. DNA ligase These enzymes form phosphodiester bonds between the adjacent molecules and, covalently links two individual fragments of double stranded DNA. The most commonly used enzyme for cloning experiment is T4 DNA ligase.

d. Host Cell Basically the Host cell is the new chamber where foreign DNA will be inserted.

2. What is a cDNA? How is a cDNA produced? In genetics, complementary DNA (cDNA) is DNA synthesized from a messenger RNA (mRNA) template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists want to express a specific protein in a cell that does not normally expressthat protein (i.e., heterologous expression), they will transfer the cDNA that codes for the protein to the recipient cell. cDNA is also produced by retroviruses (such as HIV-1, HIV-2,Simian Immunodeficiency Virus, etc.) which is integrated into its host's genome to create aprovirus.

cDNA is created from a mature mRNA from a eukaryotic cell with the use of an enzyme known as reverse transcriptase. In eukaryotes, a poly-(A) tail (consisting of a long sequence of adenine nucleotides) distinguishes mRNA from tRNA and rRNAand can therefore be used as a primer site for reverse transcription. This has the problem that not all transcripts, such as those for the histone, encode a poly-A tail. 3. What are the dangers of releasing of a genetically engineered organism in the environment? We would like to quote different comments and answers of experts regarding the release of GEO in the environment.

By taking a stand against genetically engineered foods, Mothers for Natural Law has come out as protectors of values that cannot be denied without placing all of humanity in jeopardy. These include the health and safety of our children and all generations to come; the welfare of the environment; and our fundamental human rights.from Genetically Engineered FoodsGood for us and our Planet? Dr. John Fagan Professor of Molecular Biology, Maharishi University of Management; President, Genetic ID

When genetic engineers disregard the reproductive boundaries set in place by natural law, they run the risk of destroying our genetic encyclopedia, compromising the richness of our natural biodiversity and creating genetic soup. What this means for the future of our ecosystem, no one knows. Dr. John S. Hagelin Professor of Physics, Maharishi University of Management Presidential Candidate, The Natural Law Party

The fact is, it is virtually impossible to even conceive of a testing procedure to assess the health effects of genetically engineered foods when introduced into the food chain, nor is there any valid nutritional or public interest reason for their introduction. Dr. Richard Lacey Professor of Food Safety, Leeds University, UK

Genetic engineering bypasses conventional breeding by using artificially constructed parasitic genetic elements, including viruses, as vectors to carry and smuggle genes into cells. Once inside cells, these vectors slot themselves into the host genome. The insertion of foreign genes into the host genome has long been known to have many harmful and fatal effects including cancer of the organism. Professor Mae Wan-Ho Department of Biology, Open University, UK

In 1983, hundreds of people in Spain died after consuming adulterated rapeseed oil. This adulterated

rapeseed oil was not toxic to rats. Dr. Parke warns that current testing procedures for genetically altered foods including rodent tests do not prove safety for humans. He has suggested a moratorium on the release of genetically engineered organisms, foods, and medicines. Professor Dennis Parke School of Biological Sciences, University of Surrey, UK

Genes encode proteins involved in the control of virtually all biological processes. By transferring genes across species barriers which have existed for aeons between species like humans and sheep we risk breaching natural thresholds against unexpected biological processes. For example, an incorrectly folded form of an ordinary cellular protein can under certain circumstances be replicative and give rise to infectious neurological disease. Dr. Peter Wills Auckland University, New Zealand

Probably the greatest threat from genetically altered crops is the insertion of modified virus and insect virus genes into crops. It has been shown in the laboratory that genetic recombination will create highly virulent new viruses from such constructions. Certainly the widely used cauliflower mosaic virus is a potentially dangerous gene. It is a pararetrovirus meaning that it multiplies by making DNA from RNA messages. It is very similar to the Hepatitis B virus and related to HIV. Modified viruses could cause famine by destroying crops or cause human and animal diseases of tremendous power. Dr. Joseph Cummins Professor Emeritus of Genetics, University of Western Ontario

We see this as a multi-million dollar problem. In Europe, there is already a big problem with gene flow between wild beet and cultivated beet. Oil-seed rape (canola) also has close relatives and is going to cause problems in the future. One would expect that the kind of genes that are now being engineered are going to be the ones that have a higher potentiality for causing trouble. Dr. Norman Ellstrand Professor of Genetics, University of California

The generation of genetically engineered plants and animals involves the random integration of artificial combinations of genetic material from unrelated species into the DNA of the host organism. This procedure results in disruption of the genetic blueprint of the organism with totally unpredictable consequences. The unexpected production of toxic substances has now been observed in genetically engineered bacteria, yeast, plants, and animals with the problem remaining undetected until a major health hazard has arisen. Moreover, genetically engineered food or enzymatic food processing agents may produce an immediate effect or it could take years for full toxicity to come to light.

Dr. Michael Antoniou Senior Lecturer in Molecpar Pathology, London, UK

Recombinant DNA technology [genetic engineering] faces our society with problems unprecedented not only in the history of science, but of life on the Earth. It places in human hands the capacity to redesign living organisms, the products of some three billion years of evolution. Such intervention must not be confused with previous intrusions upon the natural order of living organisms; animal and plant breeding, for example; or the artificial induction of mutations, as with X-rays. All such earlier procedures worked within single or closely related species. The nub of the new technology is to move genes back and forth, not only across species lines, but across any boundaries that now divide living organisms. The respts will be essentially new organisms, self-perpetuating and hence permanent. Once created, they cannot be recalled. Up to now, living organisms have evolved very slowly, and new forms have had plenty of time to settle in. Now whole proteins will be transposed overnight into wholly new associations, with consequences no one can foretell, either for the host organism, or their neighbors. It is all too big and is happening too fast. So this, the central problem, remains almost unconsidered. It presents probably the largest ethical problem that science has ever had to face. Our morality up to now has been to go ahead without restriction to learn all that we can about nature. Restructuring nature was not part of the bargain. For going ahead in this direction may be not only unwise, but dangerous. Potentially, it could breed new animal and plant diseases, new sources of cancer, novel epidemics. Dr. George Wald Nobel Laureate in Medicine, 1967 Higgins Professor of Biology, Harvard University. From The Case Against Genetic Engineering by George Wald in The Recombinant DNA Debate, Jackson and Stich, eds. P. 127-128 (Reprinted from The Sciences, Sept./Oct. 1976 issue)

The truth is that some scientists are wholeheartedly against genetic engineering and some are wholeheartedly for it. In this situation the only scientific solution is to foster public scientific debate and to delay application until all fundamental questions are resolved. Corporations, however, have a vested interest in speedy application. They are not willing to wait and are attempting to gather the support of the public through extensive marketing campaigns. But there is a vast discrepancy between biotech claims and the simple facts.

References: http://wiki.answers.com/Q/Role_of_restriction_enzymes_and_DNA_ligase_in_creating_recombinant_ DNA http://www.scienceclarified.com/scitech/Genetics/Genetic-Engineering.html#b#ixzz1lr6y3Gil http://www.biotechnology4u.com/basic_concepts_recombinant_dna_technology.html http://en.wikipedia.org/wiki/CDNA_library http://en.wikipedia.org/wiki/Complementary_DNA#Synthesis