Molecular and ellular Biology - Essay Example

? Molecular and cellular Biology Essay Questions 12/04 Question 2 Genetic Engineering: Genetic engineering is one of the most advanced technologies that is being used these days. It is a laborious process and requires extensive research. Genetic engineering is a process in which gene is taken from one organism and incorporated into other. Genes may be incorporated into bacteria, plants or animals. In this process recombinant DNA (rDNA) technology is used to incorporate gene of interest into organisms and that organism in which gene is inserted is said to be genetically engineered. Through this process off springs with unique characters are produced. Recombinant DNA is also referred to as Chimera. By the combination of two different strands of DNA a new strand of DNA is obtained. The first step in genetic engineering involved the production of hormone insulin which is important and helps the cells to properly absorb sugar. In recombinant DNA technology recombinant DNA is made by splicing a small fragment of a foreign DNA into a small molecule that can replicate on its own such as bacterial plasmid as a result a clone of inserted DNA can be obtained. AN organism that receives a foreign DNA is called transgenic organism and these organisms can be used in research or in commercial applications such as production of human insulin in transgenic bacteria (which receives genes from human responsible for the production of insulin). This application gives the idea that how important DNA recombinant technology is.(Recombinant DNA technology). Two classes of enzymes play an important role in DNA recombinant technology. Restriction endonucleases: They recognize specific sites on DNA and cleave DNA at that site into smaller fragments. DNA ligases: They link the foreign DNA with the vector DNA. Cloning of DNA involves the following steps: 1: First step in making recombinant DNA is to isolate donor and vector DNA. 2: Cloning vector is cleaved with restriction endonucleases. 3: gene of interest is obtained by cleaving chromosome of foreign DNA with the same restriction endonuclease into smaller fragments. 4: Fragments are ligated to the cloning vector by DNA ligase as a result a recombinant vector is obtained. 5: This recombinant DNA is introduced into the host cell where it replicates and produces copies of recombinant DNA. (Lehninger, Principles of Biochemistry fourth edition). Recombinant DNA technology in the synthesis of insulin: Insulin is an important hormone which regulates the storage of carbohydrates in the body. It is secreted by the beta cells present in the pancreas islets of langerhans. Insulin is a small protein and is composed of two polypeptide chains which are linked together by disulfide bonds. When blood glucose level is high insulin is secreted into the blood stream and removes excess of glucose from the body. Human insulin is the only known protein which can be produced in bacteria by DNA recombinant technology in a way that its structure and composition resembles the original molecule. In diabetes patients problems are associated with the production of insulin due to which sugar increases to dangerous levels which could be life threatening. These patients are treated with insulin which is produced in another organism. Although insulin can be produced in animals whose insulin resembles human insulin in terms of composition with minor variations like bovine and porcine insulin but, it was observed that when bovine and porcine insulin were injected into the patient’s body antibodies were produced against them thereby, neutralizing it’s action and producing inflammatory response at the site of injection. These problems suggest that a suitable vector like E.coli could be used to produce insulin. E.coli is a common inhabitant of the human digestive tract and is a key organism used in the production of insulin by genetic engineering. Following are the steps involved in the production of insulin from a bacterial host. 1: Isolate gene: The gene that produces insulin in human is isolated and is copied to produce many insulin genes. 2: Prepare target DNA: A circular piece of DNA known as plasmid is taken from a bacterial cell. In order to incorporate gene of interest (the one responsible to produce insulin in humans) plasmid must first be cleaved. Special proteins called restriction endonucleases are used to cleave plasmid at a specific site. 3: Insert DNA into plasmid: Genes for producing insulin is incorporated at the site where plasmid is cleaved. Fragments are joined together by DNA ligase and a recombinant vector is obtained. 4: Insert plasmid back into cell: The bacterial plasmid containing genes for the production of human insulin is now inserted into a bacterial cell. A very small needle is used to insert plasmid through the bacterial membrane. 5: Plasmid multiply: Once the plasmid enters inside the bacterial cell it needs nutrients in order to survive and divide. Bacterial cell turn on the insulin producing gene and insulin is produced. When bacterial cell divides the insulin gene also reproduces and many copies of recombinant DNA are produced. By using recombinant DNA technology millions of diabetic patients can be cured from the insulin produced in bacterial cells.(Recombinant DNA: Example using insulin). Problems associated with gene expression: Although human proteins have been successfully produced using a bacterial cell. There are some issues related to gene expression in bacterial host, some of them are: Since animal cells have complex growth requirements as compared to bacterial cells which requires simple nutrients. So, there is a possibility that proteins of human origin may not be produced in a bacterium due to complex nutritional requirements. In Eukaryotic cells when proteins are produced they are inactive and needs certain modifications to become active. This modification cannot occur in bacterial cells and is stored as proinsulin (inactive form). It becomes active only when the body is in need. In bacterial cells inactive insulin is produced rather than active insulin. E.coli produces enzymes that rapidly degrade foreign proteins such as insulin. Once insulin is produced within the bacterial cell it may be degraded by protease enzyme. To overcome this problem a mutant strain devoid of enzyme protease should be used. Insulin may be toxic to bacterial cells so a suitable bacterium should be used which can efficiently produce insulin. (Biotechnology: Demystifying the concepts p 138-168) Ethical issues: Many people do not understand the importance of genetic engineering and raise certain issues which create hindrances to utilize the benefits of this advanced technology. Since most of the insulin is derived by altering either bacterial DNA or animal DNA it may cause allergic reactions in human which is why people don’t like the idea of taking insulin from bacterial or animal origin. There are some religious issues as well like people of Jewish faith would not take insulin derived from pork and those of hindu faith rejected insulin derived from beef. Animal rights issues are also involved as initially insulin was obtained from pancreas of pigs and cows after they were slaughtered. Genetic engineering is an important and valuable technique used today. It has many advantages like it can be used to produce better quality crops which are resistant to drought and heat. By using recombinant DNA technology recombinant vaccines can be obtained which is a great achievement in the field of medicine as it helps to cure many diseases like Hepatitis, sickle cell anemia, cystic fibrosis etc. Because of its uncountable benefits this technology will be widely used in future. Question 4 a TRANCRIPTION FACTORS: Transcription Factors are regulatory proteins which help in the transcriptions of DNA by binding to specific sequences or sites on DNA. Classification of Transcription Factor depends on three dimensional protein structure which includes helix turn helix, helix loop helix and zinc finger proteins. These structures make the Transcription Factor specific and because of this specificity they can bind only on particular sites of DNA. Transcription Factors are the most important component of gene regulation in both prokaryotes and eukaryotes. However in Eukaryotes Transcription Factor regulates gene expression in a co0ordinated manner and is said to be a combinatorial that is it requires the interaction of multiple proteins as compared to prokaryotes in which regulation of gene expression is much more simpler and requires only a single protein. These Transcription Factors recognize TATA and CCAAT sites which are the components of RNA polymerase II system. Transcription is activated by the combined action of several regulatory proteins which binds at a specific region of promoter. The TATA box is located 20-30 basepairs upstream of transcription initiation site. This is the specific region which determines from where RNA synthesis will begin and which of these two DNA strands is to be transcribed. (Corden et al-1980). Other regulatory sequences like enhancers, silencers etc are also important to carry out efficient transcription. Transcription Factors have shown to promote accurate initiation of transcription. A variety of transcription Factor has been identified that initiated transcription by acting on the promoter region in Eukaryotes. In Eukaryotes transcription protein encoding genes requires RNA polymerase II an five general Transcription Factor TFIIA, TFIIB, TFIID, TFIIE and TFIIF. (i) TFIIA: It is a Transcription Factor which interacts with both TBP and TBP associated factors. It has three subunit complexes in eukaryotes with sizes of 12000, 19000, and 35000 respectively. It is not completely understood how TFIIA helps in gene regulation or transcription. Transcription starts at TATA sites when TFIIA alters the structure of TBP (TATA BINDING PROTEIN) which enhances the ability of TBP to detect specific TATA elements and associate with it. There are certain negative factors which prevents the formation of initial complexes by binding to TBP so,TFIIA has to compete with these negative factors in order to bind TBP.The main role of TFIIA is to stabilize the binding of TFIIB and TBP to the promoter region. (ii) TFIIB: It has a single subunit with a molecular size of about 35000.TFIIB is the key component in transcription and plays two important roles. It can bind directly with the TATA-binding protein (TBP) and also helps RNA polymerase II to form the initiation complex.TFIIB consists of a zinc binding domain and an imperfect duplication of 70 amino acids. The TFIIF and polymerase II. It has been observed that two highly conserved amino acids are present with in TFIIB and these are involved in the interaction between TFIIB and TBP. TFIIB may be involved in the separation of DNA strands at the initiation site IF, somehow the two highly conserved amino acid residues are mutated it will result in a weak interaction of TBP with TFIIB.It is not understood that TBP-TFIIB binds on one side of the DNA helix or TBP binds on one side and TFIIB on other. (iii) TFIID: Like all other transcription factors TFIID is present within the nucleus and helps RNA polymerase to recognize the site of transcription initiation by binding itself to the TATA box present on DNA.It also recognizes the TATA site through TATA binding protein.TFIID has a horse shoe shape. TBP protein binds at the top of the central cavity of TFIID unlike TFIIA and TFIIB which binds on the opposite sides of TBP. Main function of TFIID is that it can bind specifically to the TATA box is the promoter region. (iv) TFIIE: It has two subunits with a molecular size of 34,000 and 57000 Daltons respectively. It helps both in the initiation of transcription and promoter escape. It helps both in the initiation of transcription and promoter escape. It has been observed from experiments that TFIIE enters the pre-initiation complex after RNA polymerase II and interacts with RNA polymerase II and other TF like TFIIB and TFIIF (orphanides et al, 1996, Roeder, 1996).It helps in the recruitment of TFIIH and has ATPase and helicase activities.TFIIE binds with TFIIH to create a closed complex. Among the two subunits of TFIIE subunit interacts with the promoter DNA at the initiation site and subunit B interacts near TATA box. (v) TFIIF: It has 2 subunits with a molecular size of 30,000 and 74,000 Daltons respectively .It binds directly to RNA polymerase II and binds to TFIIB and prevents the binding of Pol II to non specific DNA sequences. It promotes transcription elongation by associating with elongating polymerase. TFIIF is important in a way as it controls the activity of RNA polymerase II both in the initiation and elongation phase of transcription.TFIIF promotes transcription initiation and elongation phase of transcription.TFIIF promotes transcription initiation by helping TFIIB to recruit Poly II into the preinitiation complex. TFIIF is not an essential component for initation.however it can efficiently activate the catalytic rate of transcribing polymerase II. Table 26-1 from lehninger Question 4 b Transcription: To understand the process of transcription in prokaryotes the differences in transcription between prokaryotes and eukaryotes must be taken into consideration. Although process of transcription is same in both but, it is much more complicated in eukaryotes. Some differences are as under: In eukaryotes transcription and translation occurs in separate cellular compartments while in prokaryotes transcription and translation occurs in the cytoplasm. Eukaryotic RNA polymerase II is made up of 12 subunits whereas; prokaryotic RNAP is made up of fewer subunits. (Subunit difference) In eukaryotes transcription initiation requires a number of transcription factors while in prokaryotes a single polypeptide is enough to do the job. In prokaryotes RNA polymerase have easy access to DNA while in eukaryotes DNA is wrapped around proteins called histones and form nucleosomes so, RNA polymerase cannot interact directly with eukaryotic DNA. mRNA that is produced as a result of transcription is not modified in prokaryotes while in eukaryotes mRNA is modified by RNA splicing. mRNAs are monocistronic in eukaryotes which means that one gene is required to produce one protein. Prokaryotes have polycistronic mRNA. Eukaryotes require multiple RNA polymerases for transcription whereas prokaryotes needs only single RNA polymerase. In eukaryotes transcription initiation requires ATP whereas prokaryote does not need ATP for transcription. (Eukaryotes vs prokaryotes transcription) Principles of gene regulation: These differences suggests that the process of transcription is much more complicated in eukaryotes as compared to prokaryotes. To get a better idea of what transcription is like in prokaryotes E.coli is a good example. The chromosome of E.coli is circular and has millions of base pairs. The chromosome replicates in a bidirectional method. E.coli has the ability to synthesize about more than 1700 enzymes therefore it possess genes required for the synthesis of such large no of mRNAs. For transcription to start RNA polymerase binds to the promoter region of DNA. A gene that codes for a specific protein is known as structural gene. If the product of gene increases in concentration due to a particular signal they are referred to as inducible and the process by which gene products increases is called induction. For example in conditions when DNA damage occurs a signal is sent as a result of which certain genes are expressed which produce enzymes required in DNA repair. On the other hand if gene products decrease in concentration in response to a signal they are referred to as repressible and the process by which gene product decreases is called repression. For example in bacteria if tryptophan is produced more than it is required a stop signal is sent which tells the genes to stop producing enzymes that catalyze tryptophan biosynthesis.(Principles of biochemistry, Lehninger fourth edition, page 1082) Principles of gene regulation in E.coli can be best explained by the operon model which was proposed by Fancois Jacob and Jacques Monod. Operons are a group of genes which are responsible to produce proteins that are needed by the cell. Genes present in operons are of two different types. 1: Structural genes: They code for those proteins that carry out the normal functions of cell. Example includes proteins that are required for the breakdown of sugar. 2: Regulatory genes: They code for those proteins that regulate other genes. Operons are only present in prokaryotes and absent in eukaryotes. Overall structure of operon comprises of an operator, promoter, regulatory and structural genes. Three types of proteins regulate transcription initiation. i) Specificity factors: It alters the specificity of RNA polymerase. ii) Repressors: It blocks RNA polymerase-promoter interaction. iii) Activators: It enhances RNA polymerase-promoter interaction. If repressors are present they bind to specific sites on DNA called operators thereby obstructing the promoter as a result of which transcription does not proceed. This regulation by repressor protein which blocks transcription is known as negative regulation. To initiate transcription repressor must be removed. Activators function opposite to repressors, they bind to DNA and enhance the activity of RNA polymerase at promoter region which results in efficient transcription. This is known as positive regulation.( Principles of biochemistry, Lehninger fourth edition, page 1083-1084) Figure 28-4 from lehninger Principles of prokaryotic gene expression were first defined by studies of lactose metabolism in E.coli which utilizes lactose as a sole source of carbon. The lac operon: Lactose is the main sugar present in milk. If lactose is present in E.coli needs three enzymes to utilize it which are ?-galactosidase (Z), galactosidase permease (Y) and thiogalactosidase transacetylase (A). In normal condition the genes responsible to produce these enzymes do not function because in that case a repressor protein is bound to the DNA promoter region which prevents transcription. These enzymes are produced only when there is a need but, repressor protein must first be removed for transcription to proceed. Repressor binds to the operator site which is located near the promoter. The promoter is that region of DNA which allows the binding of RNA polymerase. The entire unit including cluster of genes, promoter and operator that function together in regulation is called an Operon. Lac operon is an example of an inducible operon, lac operon consists of one regulatory gene the I gene which codes for lac repressor and three structural genes Z, Y and A which codes for three enzymes. In the absence of lactose the lac operon genes are repressed and lac repressor binds to the operator region and blocks the binding of RNA polymerase with the promoter as a result transcription is blocked. If mutation occurs in I gene lac repressor would not be able to bind with the operator thus allowing RNA polymerase-promoter interaction and initiates transcription. The lac operon is repressed even in the presence of lactose if glucose is also present because the organism will not waste its energy in utilizing complex molecule is much simpler form is available. This repression will continue until all the glucose has been utilized. The lac operon repression under these conditions is termed as catabolic repression. In the presence of lactose lac operon is reduced. An inducer (a molecular signals) binds to a specific site on a repressor causing a change in its structure as a result of which repressor detaches from the operator and gives a chance for RNA polymerase to bind with the promoter region and continue transcription. The inducer molecule is allolactose and not lactose. This is because when lactose enters into E.coli cell it is converted into allolactose by ?-galactosidase. The repressor molecule dissociates from operator after binding to allolactose which allows the expression of the lac operon genes. Both repression and induction are examples of negative control. (Principles of biochemistry, Lehninger fourth edition, page 1085-1087) Figure 28.-7 9(a) and (b) Work cited: 1. NELSON, DAVID L., & COX, MICHAEL M. (2004). Lehninger Principles of Biochemistry, Fourth Edition + Lecture Notebook. W H Freeman & Co. 2. Recombinant DNA : Example using insulin, genetic engineering http://www.iptv.org/exploremore/ge/what/insulin.cfm 3. Recombinant DNA technology http://www.immuneweb.com/Genetic%20Analysis/ch12.pdf 4. BOURGAIZE, D., JEWELL, T. R., & BUISER, R. G. (2000). Biotechnology: demystifying the concepts. San Francisco, Benjamin/Cummings. http://www.scribd.com/doc/20653295/Leture-3-Cloning-and-Expression-of-Insulin-Gene-in-Bacteria Read More