.Enzymes which have deoxy-ribonucleic acid
synthesis capability are
I. RNA polymerase I
II. Reverse transcriptase
III. DNA polymerase
(1) II and III only
(2) I and II only
(3) III only
(4) I and III only
Answer ▽ ✅Verified
Answer (1) Reverse transcriptase and DNA polymerase enzymes synthesise the DNA.
Polymerase
AIDS :The word AIDS stands for Acquired Immuno Deficiency Syndrome. This means deficiency of immune system, acquired during the lifetime of an individual indicating that it is not a congenital disease. ‘Syndrome’ means a group of symptoms. AIDS was first reported in 1981 and in the last twenty-five years or so, it has spread all over the world killing more than 25 million persons. AIDS is caused by the Human Immuno deficiency Virus (HIV), a member of a group of viruses called retrovirus, which have an envelope enclosing the RNA genome . Transmission of HIV-infection generally occurs by (a) sexual contact with infected person, (b) by transfusion of contaminated blood and blood products, (c) by sharing infected needles as in the case of intravenous drug abusers and (d) from infected mother to her child through placenta. So, people who are at high risk of getting this infection includes - individuals who have multiple sexual partners, drug addicts who take drugs intravenously, individuals who require repeated blood transfusions and children born to an HIV infected mother.It is important to note that HIV/AIDS is not spread by mere touch or physical contact; it spreads only through body fluids. It is, hence, imperative, for the physical and psychological well-being, that the HIV/AIDS infected persons are not isolated from family and society. There is always a time-lag between the infection and appearance of AIDS symptoms. This period may vary from a few months to many years (usually 5-10 years).After getting into the body of the person, the virus enters into macrophages where RNA genome of the virus replicates to form viral DNA with the help of the enzyme reverse transcriptase. This viral DNA gets incorporated into host cell’s DNA and directs the infected cells to produce virus particles . The macrophages continue to produce virus and in this way acts like a HIV factory. Simultaneously, HIV enters into helper T-lymphocytes (TH), replicates and produce progeny viruses. The progeny viruses released in the blood attack other helper T-lymphocytes. This is repeated leading to a progressive decrease in the number of helper T-lymphocytes in the body of the infected person. During this period, the person suffers from bouts of fever, diarrhoea and weight loss. Due to decrease in the number of helper T lymphocytes, the person starts suffering from infections that could have been otherwise overcome such as those due to bacteria especially Mycobacterium, viruses, fungi and even parasites like Toxoplasma. The patient becomes so immuno-deficient that he/she is unable to protect himself/herself against these infections. A widely used diagnostic test for AIDS is enzyme linked immuno-sorbent assay (ELISA). Treatment of AIDS with anti-retroviral drugs is only partially effective. They can only prolong the life of the patient but cannot prevent death, which is inevitable.
PRINCIPLES OF BIOTECHNOLOGY:Among many, the two core techniques that enabled birth of modern biotechnology are :
(i) Genetic engineering : Techniques to alter the chemistry of genetic material (DNA and RNA),to introduce these into host organisms and thus change the phenotype of the host organism.
(ii) Bioprocess engineering: Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc. Let us now focus on the first instance of the construction of an artificial recombinant DNA molecule. The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid (autonomously replicating circular extra-chromosomal DNA) of Salmonella typhimurium. Stanley Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid which was responsible for conferring antibiotic resistance. The cutting of DNA at specific locations became possible with the discovery of the so-called‘molecular scissors’– restriction enzymes. The cut piece of DNA was then linked with the plasmid DNA. These plasmid DNA act as vectors to transfer the piece of DNA attached to it.The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This makes a new combination of circular autonomously replicating DNA created in vitro and is known as recombinant DNA. When this DNA is transferred into Escherichia coli, a bacterium closely related to Salmonella, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. The ability to multiply copies of antibiotic resistance gene in E. coli was called cloning of antibiotic resistance gene in E. coli. You can hence infer that there are three basic steps in genetically modifying an organism — (i) identification of DNA with desirable genes; (ii) introduction of the identified DNA into the host; (iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.Amplification of Gene of Interest using
PCR :PCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using twosets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase. The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made. Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain active during the high temperature induced denaturation of double stranded DNA. The amplified fragment if desired can now be used to ligate with a vector for further cloning .
Molecular Diagnosis :You know that for effective treatment of a disease, early diagnosis and understanding its pathophysiology is very important. Using conventional methods of diagnosis (serum and urine analysis, etc.) early detection is not possible. Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno-sorbent Assay (ELISA) are some of the techniques that serve the purpose of early diagnosis.A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using autoradiography. The clone having the mutated gene will hence not appear on the photographic film, because the probe will not have complementarity with the mutated gene. ELISA is based on the principle of antigen-antibody interaction. Infection by pathogen can be detected by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesised against the pathogen.
The Machinery and the Enzymes :In living cells, such as E. coli, the process of replication requires a set of catalysts (enzymes). The main enzyme is referred to as DNA-dependent DNA polymerase, since it uses a DNA template to catalyse the polymerisation of deoxynucleotides. These enzymes are highly efficient enzymes as they have to catalyse polymerisation of a large number of nucleotides in a very short time. E. coli that has only 4.6 ×10^6 bp (compare it with human whose diploid content is 6.6 × 10^9 bp), completes the process of replication within 18 minutes; that means the average rate of polymerisation has to be approximately 2000 bp per second. Not only do these polymerases have to be fast, but they also have to catalyse the reaction with high degree of accuracy. Any mistake during replication would result into mutations. Furthermore, energetically replication is a very expensive process. Deoxyribonucleoside triphosphates serve dual purposes. In addition to acting as substrates, they provide energy for polymerisation reaction (the two terminal phosphates in a deoxynucleoside triphosphates are high-energy phosphates, same as in case of ATP).
Transcription Unit :A transcription unit in DNA is defined primarily by the three regions in the DNA: (i) A Promoter (ii) The Structural gene (iii) A Terminator.There is a convention in defining the two strands of the DNA in the structural gene of a transcription unit. Since the two strands have opposite polarity and the DNA-dependent RNA polymerase also catalyse the polymerisation in only one direction, that is, 5'®3' , the strand that has the polarity 3'®5' acts as a template, and is also referred to as template strand. The other strand which has the polarity (5'®3') and the sequence same as RNA (except thymine at the place of uracil), is displaced during transcription. Strangely, this strand (which does not code for anything) is referred to as coding strand. All the reference point while defining a transcription unit is made with coding strand.The promoter and terminator flank the structural gene in a transcription unit. The promoter is said to be located towards 5'-end (upstream) of the structural gene (the reference is made with respect to the polarity of coding strand). It is a DNA sequence that provides binding site for RNA polymerase, and it is the presence of a promoter in a transcription unit that also defines the template and coding strands. By switching its position with terminator, the definition of coding and template strands could be reversed. The terminator is located towards 3'-end (downstream) of the coding strand and it usually defines the end of the process of transcription (Figure 6.9). There are additional regulatory sequences that may be present further upstream or downstream to the promoter.Types of RNA and the process of Transcription In bacteria, there are three major types of RNAs: mRNA (messenger RNA), tRNA (transfer RNA), and rRNA (ribosomal RNA). All three RNAs are needed to synthesise a protein in a cell. The mRNA provides the template, tRNA brings aminoacids and reads the genetic code, and rRNAs play structural and catalytic role during translation. There is single DNA-dependent RNA polymerase that catalyses transcription of all types of RNA in bacteria. RNA polymerase binds to promoter and initiates transcription (Initiation). It uses nucleoside triphosphates as substrateand polymerises in a template depended fashion following the rule of complementarity. It somehow also facilitates opening of the helix and continues elongation. Only a short stretch of RNA remains bound to the enzyme. Once the polymerases reaches the terminator region, the nascent RNA falls off, so also the RNA polymerase. This results in termination of transcription. An intriguing question is that how is the RNA polymerases able to catalyse all the three steps, which are initiation, elongation and termination. The RNA polymerase is only capable of catalysing the process of elongation. It associates transiently with initiation-factor (s) and termination-factor (r) to initiate and terminate the transcription, respectively. Association with these factors alter the specificity of the RNA polymerase to either initiate or terminate (Figure 6.10). In bacteria, since the mRNA does not require any processing to become active, and also since transcription and translation take place in the same compartment (there is no separation of cytosol and nucleus in bacteria), many times the translation can begin much before the mRNA is fully transcribed. Consequently, the transcription and translation can be coupled in bacteria. In eukaryotes, there are two additional complexities – (i) There are at least three RNA polymerases in the nucleus (in addition to the RNA polymerase found in the organelles). There is a clear cut division of labour. The RNA polymerase I transcribes rRNAs(28S, 18S, and 5.8S), whereas the RNA polymerase III is responsible for transcription of tRNA, 5srRNA, and snRNAs (small nuclear RNAs). The RNA polymerase II transcribes precursor of mRNA, the heterogeneous nuclear RNA (hnRNA). (ii) The second complexity is that the primary transcripts contain both the exons and the introns and are non-functional. Hence, it is subjected to a process called splicing where the introns are removed and exons are joined in a defined order. hnRNA undergoes additional processing called as capping and tailing. In capping an unusual nucleotide (methyl guanosine triphosphate) is added to the 5'-end of hnRNA. In tailing, adenylate residues (200-300) are added at 3'-end in a template independent manner. It is the fully processed hnRNA, now called mRNA, that is transported out of the nucleus for translation (Figure 6.11). The significance of such complexities is now beginning to be understood. The split-gene arrangements represent probably an ancient feature of the genome. The presence of introns is reminiscent of antiquity, and the process of splicing represents the dominance of RNA-world. In recent times, the understanding of RNA and RNA-dependent processes in the living system have assumed more importance.
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