Microbiology Final - Lesson Objectives

What is the role of the promoter, terminator, and mRNA in transcription?

A gene is composed of the promoter region, coding region and the terminator. The promotor is the region where the RNA polymerase can identify and bind to. In order to transcribe a segment of a genome it is highly important that the RNA polymerase specifically binds to these promoters. Once the RNA polymerase binds to the promotor region, it starts transcribing coding part of the gene that codes for a specific protein in the form of a messenger RNA (mRNA). When the RNA polymerase reaches the poly adenine tail of a gene, the transcription of the gene stops or terminates. The poly A region is called the terminator region. This type of transcription termination is known as rho independent termination. In the rho dependant termination once the RNA polymerase reaches the poly A tail a protein named rho attaches to the RNA polymerase and removes the newly synthesized RNA from the DNA. Thus mRNA is translated to a protein.

Define operon.

A set of operator and promoter sites and the structural genes they control Gene is a part of a genetic material that codes for a specific protein. An operon is a cluster of genes that codes for a set of proteins or enzymes which would be regulated through many mechanisms. The structural genes are controlled by regulatory gene, operator, promoter and RNA polymerase. These genes are transcribed into mRNA strand and translated in the cytoplasm. They further undergo trans-splicing to form monocisstronic mRNA that are translated.

A. A mutagen that is incorporated into DNA in place of a normal base. B. A mutagen that causes the formation of highly reactive ions. C. A mutagen that alters the adenine so that its base-pairs with cytosine. D. A mutagen that causes insertions. E. A mutagen that causes the formation of pyrimidine.

A. Nucleoside analog B. Ionizing radiation C. Base-pair mutagen D. Frameshift mutagen E. Nonionizing radiation

Of what value is the plant pathogen Agrobacterium?

Agrobacterium a plant pathogen which is capable of inducing crown gall in the plants is characterized by the presence of the Ti Plasmid (Tumor inducing plasmid). These Ti plasmids are capable of infecting the plant cells and in the process transfer the Ti plasmid into the plant system. The Ti plasmid induces the formation of a tumor in specific locations of a plant and these locations starts synthesizing chemicals that attract more numbers of the microbe, Agrobacterium microbe. With the advent of rDNA technology, with the help of any manipulations in the Ti-plasmid of the Agrobacterium, a foreign DNA could be incorporated and thus can be made very well established in the plant cell. The Ti-plasmid that enters the plant cell integrates in the plant genome producing the product of the gene of interest. New hybrid varieties of plants that are resistant towards the herbicides are produced. While the weeds are still sensitive to the herbicides, the resultant will be the killing of the weeds which is the greatest achievement of the recombinant DNA technology aided by the service of Agrobacterium.

Outline genetic engineering with Agrobacterium.

Agrobacterium is a bacterium that could be used in the genetic manipulation of a plant cell. The Ti-plasmid of the Agrobacterium tumifaciens is the major component involved in tumor formation in plants. These Ti plasmids (tumor inducing) would be transferred to the plant cells when the plant is infected with the Agrobacterium strains. These plasmids gets themselves integrated with the plant genome expressing the products that would induce the attraction of more Agrobacterium around the infected root. The exudate that comes out of the root as a result of infection attracts more microbes, which further infects more plant cells. The foreign DNA which is incorporated into the Ti-plasmid would be transferred into the cells of Agrobacterium tumifaciens. Now, these cells are recombinant cells carrying the new plasmid containing the foreign DNA in it. When this bacterium infects a plant cell, it would be having the Ti-plasmid expressing the foreign DNA.

What is the principle behind the Ames test?

Ames test is used to check the carcinogenicity of a chemical utilizing the services of microbes (auxotrophs of Salmonella that have lost its ability in synthesizing histidine (amino acid)). The principle behind Ames is that under the influence of the mutagen the auxotrophic strain reverts back to histidine synthesizing strains. The degree of mutation is directly proportional to the number of colonies arising after exposing the microbial strains to the potent mutagen.

Identify the purpose of and outline the procedure for the Ames test.

Ames test is used to identify the specific chemical mutagen whether it is a probable carcinogen or not. The principle behind the Ames test is that, the chemical carcinogen reverts the histidine auxotrophic mutant of the microbe to a prototroph which would colonise in the minimal media that is devoid of the amino acid. In this test microbe used is histidine auxotrophic cell of Salmonella sp., which would be plated in a minimal media which is treated with the given chemical mutagen. Thus if a colony of the microbe appears in the plate it exemplifies the carcinogenic nature of the chemical tested. The degree of mutagenicity of the chemical would normally be calculated through the number of microbial colonies that arise in the plate incubated after the microbe is converted to a prototroph.

How would you isolate an antibiotic-resistance bacterium? An antibiotic-sensitive bacterium?

An antibiotic resistant bacterium could be isolated by plating the microbes in the medium containing the desired antibiotic. The resistant strains of the microbes grow without any inhibition. In the case of an antibiotic sensitive bacterium, the isolation should be done using the replica plating technique. The microbial strains should be plated on a master plate, using nylon filter the microbes present in the master plate would be transferred to plates amended with the antibiotic against which the microbe is sensitive and other plate without any antibiotic. The colony that arises in the plate without any antibiotic should be compared with that of the plate containing medium amended with antibiotic.

Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, and gene products.

Antibiotic-resistance genes - Blue white screening could be used in selecting a recombinant clone using the antibiotic resistant genes. The colonies that form blue colonies are the recombinant ones, and those that form the white colonies are the non-recombinant forms of microbial strains. DNA probes - Colony hybridization could be used to identify a cloned gene of interest using DNA probes. Southern blotting, nuclear hybridization, and microarray techniques work with this principle. Gene products - With the help of western blotting technique, a specific gene product (a particular protein) could be identified, thereby identifying a clone.

Differentiate biotechnology and rDNA technology.

Biotechnology helps in utilization of microbial cell or cell components to make a specific product, whereas recombinant DNA technology helps in the addition of a specific gene of interest producing a commercial product in a microbial cell that was not a part of the cell before. Biotechnology helps us to study the properties of the microbes and cells, whereas the recombinant DNA technology helps to manipulate the genome of the microbe by incorporating the gene of interest into it. Using the rDNA technology, it is possible to produce required products in huge amounts using the microbes. The rDNA technology is used to amplify the gene of interest to study it. Thus, it is a part of biotechnology.

Compare and contrast biotechnology, genetic modification, and recombinant DNA technology.

Biotechnology is an interdisciplinary arena, which helps in the production of specified product or cell components using microorganisms. The exploitation of microbes for the continual production of the specific gene product is through biotechnology, whereas any possible modification in the gene of interest through mutation for inducing an enhanced production of the product, produced by the gene of interest is termed recombinant DNA technology. In the recombinant DNA technology, it is also possible for us to insert new genes, and expect the product in large quantities, to be utilized by the human population. Genetic modifications can be done using the recombinant DNA technology, which is also called as genetic engineering.

What is the mechanism by which the presence of glucose inhibits the lac operon?

Catabolic repression by the catabolic activator protein binds to lac operator region in presence of glucose. Catabolic repression allows bacteria to adapt quickly to a preferred carbon and energy source first. This is usually achieved through inhibition of synthesis of enzymes involved in catabolism of carbon sources other than the preferred one.

Differentiate cDNA from synthetic DNA.

Complementary DNA (cDNA) is prepared using the processed mRNA in a eukaryote that could actually code for a particular protein. The mRNA is converted to a single stranded DNA with the help of reverse transcriptase. The single stranded DNA would be converted to a double stranded molecule through DNA polymerase. Thus, a double stranded DNA molecule that actually codes for a particular protein is built. On the other hand, a synthetic DNA is built manually. These are short DNA sequences that could be attributed to develop a particular product, when it is introduced into a cell. The synthetic DNA is completely synthesized outside the cell, which does not have any connectivity with that of the living organism.

Why is the base pairing in DNA important?

DNA base pairing is very important in order to maintain stability to the double helix of the molecule. There is a greater stability in the molecule because it could be accommodated within the system with its gene both actively and dormantly. If there is any mismatching of base pairs then there would be malfunction in the product released through genetic manipulation.

Describe how DNA serves as genetic information

DNA codes for the entire morphological and physiological responses of the microbial cell. On multiplication the cell divides through fission. The nuclear material multiplies and forms 2 copies of the DNA in the same cell. Out of which one moves to the newly formed cell or divided cell and the other part remains with the same cell.

Briefly describe the components of DNA, and explain its functional relationship to RNA and protein.

DNA consists of a strand of alternating sugars (deoxyribose) and phosphate groups with a nitrogenous base attached to each sugar. The nitrogen bases include adenine, guanine, thymine and cytosine. The DNA exists in a cell as a double helix, where two DNA strands are held together by hydrogen bonds between the alternating nitrogen bases. Adenine always pairs with thymine with two hydrogen bonds while guanine pairs with three hydrogen bonds to cytosine. The sequence of nitrogen base in DNA holds the genetic information of the cell. This information is passed on to next generation by replication. The specific proteins are synthesized from this genetic information. This sequence of nucleotides on DNA forms the basis of RNA and thereby protein synthesis in the cell

Describe DNA replication, including the functions of DNA gyrase, DNA ligase, and DNA polymerase.

DNA replication is a process in which a single molecule of the double stranded helical DNA gets multiplied to two. During the initiation of DNA replication the enzyme gyrase helps in the unwinding of the DNA molecule and helps in the smooth traversing of the replication fork in one direction from 3' - 5' direction for the DNA polymerase which helps in the addition of the nucleotides for the newly synthesizing DNA. DNA polymerase can only add nucleotides in a 3' - 5' hence in a replication fork the strand (leading strand) that moves in the same direction as that of the polymerase would be synthesized without any problem. The lagging strand is synthesized in the opposite direction from 5' - 3' direction this is because of two reasons: 1) The replication fork keeps on moving in one direction. 2) The action of DNA polymerase is in 3' - 5' direction. The fragments that were formed in the lagging strand are termed the okazaki fragments. These fragments would be ligated or joined by DNA ligase.

Describe the process of DNA replication.

DNA replication starts with the enzyme gyrase (topoisomerase) which acts on the DNA molecule by removing the negative super coiling of the molecule. Once the negative super coiling is done, the enzyme helicase acts on DNA thus separating the double stranded molecule thereby enabling the DNA polymerase to attach and start its polymerization effect. The double stranded DNA molecule is separated by an enzyme polymerase which initiates polymerization reaction by adding nucleotides to the already attached primer (small DNA fragment). The DNA polymerase acts on the nucleotides in the 3' end of the newly growing strand. DNA polymerase acts in the 5'-3' direction. This enzyme adds nucleotides towards 5'-3' in the leading strand. The replication fork moves throughout the length of the DNA molecule by the synthesis of the DNA fragments which are dis-continuous and termed as okazaki fragments. The discontinuous okazaki fragments would be joined by the action of ligase enzyme. Thus once the replication fork traverses throughout the DNA molecule two copies of the genetic material would be formed.

Natural selection means that the environment favors survival of some genotypes. From where does diversity of genotypes come?

Diversity of genotypes comes as a result of horizontal transfer of genes which include different methodologies i.e., transformation, conjugation, transduction etc. When a particular gene of interest is present in a microbe (in a plasmid) that is capable of establishing the growth of a microbe in a particular area. The plasmids are transferred to the microbe through transformation, and the plasmid could be entertained for a possible transfer to other microbes of the same group or compatible group of microbes enabling the character to its counterpart as well.

What types of cells are used for cloning rDNA?

E.coli is the basic prokaryote that is used for cloning of rDNA mainly because it multiplies at a faster rate. Also since the genetic mapping of the organism and its operon system were studied thoroughly, it forms the apt microbe for genetic manipulation. Though its ability of producing endotoxins and its inability in secreting the product makes it tougher for purifying the product of interest, it is still the preferred microbe for genetic recombination. Saccharomyces cerevisiae is the eukaryotic microbe (yeast) that is capable of harboring the plasmid forms another cell for the expression of the specified gene. But the only factor is that, before the cell is transformed, the cell should be made devoid of its existing cell wall. Since it continuously secretes the product, it is termed as the eukaryotic workhorse of Biotechnology. Plant cell, mammalian cell also could be used for the expression of foreign DNA. Mammalian cells are used when the products of medical importance are extracted reducing the possibility of a potent endotoxin when E.coli would be used. To incorporate a foreign DNA into a mammalian cell, the DNA should first be incorporated into a plasmid and be transformed into a prokaryote. This prokaryote could be used to make multiple copies of the plasmid, which is incorporated into the mammalian cells which secretes the same product with minimum possible toxins. The same is the case of a plant as well. If a plant is to be used for the synthesis of a specific product, the plasmid should be incorporated by the help of bacteria forming clones of the plasmid. Through plants, the cost of production reduces to a considerable range and the possible infection by the mammalian pathogens reduces, thus improving the overall purity of the product.

List one advantage of modifying each of the following: E. coli, Saccharomyces cerevisiae, mammalian cells, and plant cells.

E.coli is the most sorted out microorganism that could be manipulated very easily. Owing to its very short replication time, the recombinant gene product could be expressed at the earliest. Saccharomyces cerevisiae is a eukaryote that would be used for expressing rDNA. It could accommodate larger DNA fragments than E.coli. It is called as a protein factory of rDNA, since they can synthesize the gene product continuously, thus being called as a workhorse of eukaryotes. Mammalian cells are the best suited forms of cells that could be used for expressing products of medical use in human beings. The plant cells on genetic modification could produce products at a relatively cheaper cost compared to all the other techniques.

List at least two examples of the use of rDNA in medicine and in agriculture.

Examples of rDNA in medicine: 1. Monoclonal antibodies are formed by the somatic fusion of HGPRT-, myeloma TK- cells , HGPRT+, TK+ B cells to monoclonal antibodies specific to one antigenic determinant or epitope. Monoclonal antibodies are used for disease diagnosis, aids in therapy for cancer and transplant rejection. 2. Hepatitis-B vaccine is produced by introducing plasmid containing hepatitis virus envelope coding gene into Sacharomyces cerevisiae to produce viral coat protein that acts as a vaccine to act against hepatitis-B infection. Examples of rDNA in agriculture 1. Rhizobium meliloti is a nitrogen fixing bacteria. Its nitrogen fixing ability is enhanced using genetic engineering. 2. Bt cotton is produced by introducing toxin producing gene from Bacillus thuringenesis into cotton plant itself. Thus such cotton plant itself will prevent growth of boll worms.

How does feedback inhibition differ from repression?

Feedback inhibition differs from repression because feedback inhibition stops the action of preexisting enzymes.

What is gene silencing?

Gene silencing is a natural process that occurs after transcription in a eukaryotic cell. After transcription, the transcribed product codes for both coding regions (exons) and non-coding regions (introns). During processing, the coding region alone will be retained in the transcript whereas the introns will be removed through the splisosomes that are mediated by the RNA molecules. The determinations of the coding and non-coding regions are done only with the help of miRNA (micro RNA). These miRNA undergoes processing forming small interfering RNAs (siRNA). It is this molecule that bonds with the primarily transcribed product. When the siRNA bonds with a specific region of the transcript, it gets denatured using the RNA dependent silencing complex. The bond formation will be in about 6 base pairs. The siRNA will be around 22 base pairs in length. This is the technique by which the eukaryotic system expresses different products at different locations in their cell, though all the organelles contain the same genomic blue print.

Compare the mechanisms of genetic recombination in bacteria.

Genetic recombination occurs in vertical as well as in horizontal transfer of genetic material. In both the cases the recombination concept involved would be the same but the mode in which the DNA moves into the cell in the case of a horizontal state alone changes. In case of a vertical transfer the DNA molecule during replication finds a homological sequence along the side of the new growing DNA strand where the exchange takes place. The microbes formed would be a genetically modified microbe but in a natural process process the two homologically similar strands are stranded opposite each other. The protein Rec A binds to the homologous region of one strand in DNA molecule catalyzes the joining of two strands. As a result the recipient chromosome contains the new DNA where the gap formed between would be rectified by the enzymes polymerase and ligase. The donor gene would be destroyed and the recipient would contain the new gene sequences. In case of horizontal transfer the same concept occurs between a donor and the recipient DNA but the mode in which the donor enters the recipient cell differs. There are three basis methods through which the donor DNA can enter the recipient i.e., through transformation, conjugation and through transduction.

Define genetics, genome, chromosome, gene, genetic code, genotype, phenotype, and genomics.

Genetics: is the science of heredity Chromosome: structures containing DNA that physically carry hereditary information Genes: segments of DNA that code for function products Genetic code: the set of rules that determines how a nucleotide sequence is converted into the amino acid sequence of a protein Genotype: is an organisms genetic make up (represents the potential properties) Phenotype: refers to actual, expressed properties (manifestation of genotype) • The study in relation to the inheritance of characters of microbes from one generation to the next generation is known as microbial genetics. • Genome is known as the genetic material of the organism. The genome of a prokaryotic microbe is a double-stranded DNA molecule. Genome is the primary blueprint that is responsible of the response that a microbe or a living being gives to the external stimulus. • Genome is formed of numerous genes. Genes are responsible for an individual response. The degradation of lactose on addition of the sugar to the microbial biomass is due to the action of the lac operon or lac gene cluster. • Genetic codes the basics of a gene. The genetic code is a triplet code. These triplets when transcribes in turn forms a messenger RNA that is paves way for the production of specific proteins. These triplet code or genetic code, codes for individual amino acid. • Genotype is the genetic map of the organism usually under reference with a specific character. • Phenotype on the other hand, depicts the external morphological appearance of the microbe or in simple terms any observable changes in the structure is covered by phenotype. • Genomics is a part of genetics that studies about the genomes of the microbes.

Discuss how genetic mutation and recombination provide material for natural selection to act upon.

Genome is modified on the changes in the environment in which it thrives upon. Based on the changes in the environment specific gene sequences in the genome of an organism changes. This helps to withstand the troubles in the external environment. This property that is inherited as a result of the change in the environment would be vertically transferred to the other generations to its progeny. This means that the progeny has already been acclimatized to the external environment with its set of genes that helps it maintain its normal metabolic activity. For example a microbe that grows in a normal environment when it is transferred to a hot geysers (can survive for a brief period by producing the heat shock proteins) by transferring them step by step through differing increasing temperature ranges over a very long period of time, the microbe tends to thrive normally exhibiting its metabolic activity by the increased synthesis of heat shock proteins.

What is the purpose of a genomic library?

Genomic library helps in the storage of specific genes that are present in a genome. These genomic libraries would be developed using different techniques and one among them is with the development of cDNA molecules. In prokaryotes, the DNA of the organism is isolated, and restricted with an enzyme. The fragments are ligated with a vector and are cloned into a microbe. The microbe that gets ligated fragment (known as a result of the expression of gene) is stored. Similarly, all the clones of microbes carrying the gene fragments are stored as such to prevent the preserved gene from getting extinct. In eukaryotes, the process of developing a DNA library is different, since the genes are interspersed with introns. In such a case, the processed transcript (mRNA) with the help of a reverse transcription PCR would be converted to a DNA fragment, which would be transformed into a bacterial cell and the clone would be preserved.

Discuss the value of genome projects.

Genomic project has helped us in understanding the genetic details of human. Only after the human genome project, it came to know that more than 98% of gene was found to produce the miRNAs and only about 2% codes for the protein products. The human genome project also helps us to know the details of gene expression, as well as, the gene or part of the DNA that is involved in DNA splicing or silencing. Any possible gene therapy could be used for treating any disease with the help of gene silencing techniques.

Give a clinical application of genomics.

Genomics is a concept by which the entire behavioral pattern of a microbe is studied. The development of microbial resistance to existing and used antibiotics has become a serious concern for the doctors in having a control over the disease. With the aid of genetics it could be very well noted that the genetic blue print and the change in the genome necessitating a possible change and its altered behavior would be clearly found out. Such studies are highly important in putting an end to the pathogens and many other clinical complications. To study West Nile Virus.

Differentiate horizontal and vertical gene transfer.

Horizontal transfer refers to the artificially induced genetic transfer including transformation, conjugation and transduction whereas vertical transfer refers to the transfer of genetic material from the one generation to another through general replication. Vertical transfer occurs naturally whereas in the case of horizontal transfer, it is not imperative the transfer has to be natural, it could be induced as well. Horizontal gene transfer occurs between bacteria in several ways like conjugation, transformation, transduction etc and vertical gene transfer occurs when genes are transferred from an organism to offspring. The microbial strain improvises a lot under horizontal gene transfer. Thus the microbe would incorporate with new sets of genes in its system.

How does RNAi "silence" a gene?

In RNAi (RNA interference), the specific siRNA (RNA silencer) binds to its complementary mRNA forming double stranded RNA. This RNA cannot act as template for protein synthesis since it is double stranded and it will soon be degraded due to short half-life of mRNA. Thus the action of RNA is interfered or silenced.

Compare protein synthesis in prokaryotes and eukaryotes

In a prokaryote the nuclear material is not packaged in a nuclear membrane whereas in a eukaryotic system the nuclear material is enclosed within a well established nuclear membrane. In a eukaryote the DNA would be interspersed with non-coding regions (introns) which need to be removed before it is translated. Hence in a eukaryote the mRNA transcript once formed undergoes processing in the form of splicing by removing the introns. These are the two main aspects that differentiate the transcription between a prokaryote and a eukaryote.

Contrast the 5 ways of putting DNA into a cell.

In order to develop a genetically modified microbial strain, the gene should be added to the vector outside the cell and should be sent inside for the production of the gene product. There are different methods by which the vector is sent into a microbial system. They are transformation, electroporation, protoplast fusion, gene gun technique, and microinjection. • Transformation is done by subjecting a recipient cell to alternate cool and hot conditions in a calcium chloride solution. On repeating the process for many times, the calcium chloride crystals formed during the cooling period leaves a pore when the temperature is raised. This pore could be used for the transportation of the vector (plasmid) in to the recipient cell. After transformation, the recipient cell is termed as competent cell. • Electroporation is a process, which is similar to that of transformation, but in this process electric current is used to form pores in the microbial cell that could be used for transformation. • Protoplast fusion is a process by which the microbial cell containing the plasmid or the donor DNA, and the cell that doesn't have them will be fused. The probability of protoplast fusion is low, but with the introduction of polyethylene glycol (PEG) it increases. In the hybrid cell, the DNA obtained from both the cells could undergo natural recombination. • In gene gun technique, microscopic particles like gold are coated with the DNA. These are sent into the plant cell through a burst of helium. • In microinjection, the DNA is introduced directly into the animal cell by punching the animal cell with the help of a glass pipette (micro diameter).

Describe five ways of getting DNA into a cell.

In order to develop a genetically modified microbial strain, the gene should be added to the vector outside the cell and should be sent inside for the production of the gene product. There are different methods by which the vector is sent into a microbial system. They are transformation, electroporation, protoplast fusion, gene gun technique, and microinjection. • Transformation is done by subjecting a recipient cell to alternate cool and hot conditions in a calcium chloride solution. On repeating the process for many times, the calcium chloride crystals formed during the cooling period leaves a pore when the temperature is raised. This pore could be used for the transportation of the vector (plasmid) in to the recipient cell. After transformation, the recipient cell is termed as competent cell. • Electroporation is a process, which is similar to that of transformation, but in this process electric current is used to form pores in the microbial cell that could be used for transformation. • Protoplast fusion is a process by which the microbial cell containing the plasmid or the donor DNA, and the cell that doesn't have them will be fused. The probability of protoplast fusion is low, but with the introduction of polyethylene glycol (PEG) it increases. In the hybrid cell, the DNA obtained from both the cells could undergo natural recombination. • In gene gun technique, microscopic particles like gold are coated with the DNA. These are sent into the plant cell through a burst of helium. • In microinjection, the DNA is introduced directly into the animal cell by punching the animal cell with the help of a glass pipette (micro diameter).

How does mRNA production in eukaryotes differ from the process in prokaryotes?

In prokaryotes the mRNA synthesized could be translated immediately before the transcription is completed. But in eukaryotes this process is not possible, since in eukaryotes the genetic material is present inside a well enclosed nuclear membrane. The transcribed mRNA would be interspersed with introns (non-coding regions) apart from the coding regions (Exons). The introns are removed from the transcribed mRNA by a process called splicing occurs inside the nucleus. The spliced product comes out of the nuclear membrane and reaches the cytoplasm where it is translated as the amino acid pool and other accessories of building the protein factory is present in the cytoplasm. These are the differences between a prokaryotic and a eukaryotic transcription.

Restriction enzymes were first discovered with the observation that phage DNA is destroyed in a host cell.

In the host cell observed, phage DNA is destroyed and denatured. This led to the idea that there must be some enzymes that are specific against foreign DNA. Host DNA or phage DNA has no difference in chemical structure, they only vary in sequence. Hence, these are identified as cleaving proteins, called nucleases, which act at specific sequences of DNA.

Describe how a genomic library is made.

In the process of developing a genomic library in prokaryotes, the DNA of the organism is isolated from the microbe, and restricted with an enzyme. The fragments are ligated with a vector and are cloned into a microbe. The microbe that gets ligated fragment (known as a result of the expression of gene) is stored. Similarly, all the clones of microbes carrying the gene fragments are stored as such to prevent the preserved gene from getting extinct. In eukaryotes, the process of developing a DNA library is different, since the genes are interspersed with introns. In such a case, the processed transcript (mRNA) with the help of a reverse transcription PCR would be converted to a DNA fragment, which would be transformed into a bacterial cell and the clone would be preserved.

What is the mechanism by which latose controls the lac operon?

Lactose is an inducer molecule that binds to lac repressor and thereby makes the lac operator region free for transcription to proceed.

Identify 2 advantages and 2 problems associated with genetically modified organisms.

Microbes, in general are useful in many ways. Only with the strides in the field of microbiology, the features of microbes are known. With the ultimately achieved knowledge about these microbes, scientists started manipulating the genetic blue prints of these known microbes in producing products of high economic value. Though these manipulated microbes are of great importance, it should be noted that the level of manipulations and the after effect of these changes over a period of time would be known only after many generations. Hence, it could be noted that there are both advantages and disadvantages in the utilization of genetically modified microbes. They are as follows: Advantages: 1. The microbial cell that is capable of a faster multiplication rate is used in genetic recombination techniques for an effective and a faster production of a desired product of interest that was not a part of the microbial community (for example: microbes were used for the production of human insulin even though insulin is generally not produced in the microbial community). 2. The genetically modified agriculture products with high protein content and resistance against the weedicides which normally is not evident in plants are possibly produced by the recombinant cells as opposed to the normal microbial cell. Disadvantages: 1. The same technology is also used in the development of toxins / microbial biomass that could be used in a bio-warfare which is even more dangerous than an atomic war fare. 2. In case of genetically modified plant containing the endotoxin of the Bacillus thuringiensis microbe, if at all there is a change in the genetic map of the weed, which will make the weed resistant to the toxin making it even difficult to remove the unwanted plants.

Outline the methods of direct and indirect selection of mutants.

Mutant strains of certain products are termed auxochromes. For example a microbe which is not capable of synthesizing the amino acid tryptophan is termed a tryptophan auxotroph. The microbe which is capable of synthesizing all the required nutrients by itself is known as prototroph or in common the wild type of the microbial strain without any mutation. These microbes could be selected through the usage of replica plating technique. In this replica plate technique the master plate contains the set of microbes (both auxotroph and prototroph) would be placed on minimal media in two plates separately one plate containing the amino acid tryptophan and the other one devoid of it. The auxotrophic strain of the microbe will not grow in the plate devoid of the amino acid and grows only in the plate containing it. Such techniques could be isolated for the selection of microbes possessing antibiotic sensitivity as well.

Why are mutation and recombination important in the process of natural selection and the evolution of organisms?

Mutation and recombination provide genetic diversity. Mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. Genetic recombination results in a reshuffling of the different alleles within a haplotype and none of the haplotypes will dominate the population. Environmental factors select for the survival of organisms through natural selection. Genetic diversity is necessary for the survival of some organisms through the process of natural selection. Organisms that are selected by nature further undergo genetic change, resulting in the evolution of species.

Describe two ways mutations can be repaired.

Mutation can occur through physical and chemical agents. In case of UV illuminated thymine dimer in a DNA, the alteration takes place with the help of visible light. Bacteria produce photolyases in the presence of light that acts on the dimer which separates and rectifies the damage. In other method mutation is corrected through excision repair mechanism. In this phenomenon, a wrong base pair that was added to the DNA during replication would be removed as a result of the endonucleases and the wrong nucleotide would be corrected. The wrong nucleotide added would be identified through the help of enzymes methylases. These methylase enzyme methylates the entire DNA leaving the wrong nucleotide without methylation that acts the identifying point for the restriction endonucleases that removes the wrong nucleotide. After the correct base pair is added the DNA would be ligated starting back the replication process. Not only during replication, nucleotide excision repair can be induced anywhere where mutation occurs.

How can a mutation be beneficial?

Mutation causes a permanent change in the genetic material of an organism. Although mutations causes many deleterious effects there are a few cases where in mutation could end up as a beneficiary effect. For example HIV virus can only attacks the T4 lymphocytes by joining through the GP120 protein to the lymphocyte. If the receptor molecule in the lymphocyte is mutated it causes a change in the protein component which ultimately fails to form an establishment with that of the lymphocytes. For example a specific 32 base pair deletion in human CCR5 gene confers HIV resistance or a delay in the infection process. Thus it could be ascertained that there are certain circumstances where in the mutated organism is more efficient and safe than the wild type organism.

Describe the effect of mutagens on the mutation rate.

Mutation is a factor which announces a change in the genetic blue print of an organism either naturally or through artificial induction with the help of mutagenic agents. Natural mutation occurs at a considerable slow rate when compared to the induced mutation. The frequency of mutation increases exponentially with the introduction of a mutagen (physical or chemical). For every one in every ten thousand the mutation rate is expressed in 10^-4 Spontaneous mutation takes place at the rate of one in every 10^9 generations. But with the introduction of a mutagen the frequency of mutation increases up to 10 to 1000 times more than the spontaneously induced mutation.

Classify mutations by type.

Mutation is a term used to denote a change in the genome of an organism. It could be natural or artificial. The chemical that brings about a change in the blue print of an organism is known as mutagen. These mutagens increase the chance of mutation in an organism. Mutagens are generally deleterious in their effect but in very rare circumstances mutation proves to be beneficial. Mutations are classified into different types which include: • The most common type of mutation is the base substitution mutation or point mutation. This may occur during DNA replication, where a wrong nucleotide is added to the growing DNA molecule and is not determined by the proof reading mechanism of the polymerase enzyme. • When a mutation takes place in the middle of the coding region of the DNA there would be a change in the amino acid that is added to the growing polypeptide chain. This change in DNA is also known as missense mutation. One deleterious effect of the missense mutation is the formation of sickle cell anemia. This results in the change of an adenosine molecule by a thymine molecule. The change in the nucleotide changes the amino acid to be added as valine instead of glutamic acid. When the entire polypeptide chain is formed due to the formation of tertiary and quarternary structure of the protein the RBC of human system takes a sickle shape which proves fatal. • The next type of mutation is the nonsense mutation. In this type of mutation, a change in the nucleotide of the DNA inserts a stop codon in the middle before the complete protein is formed. • Another type of mutation is the frame shift mutation which is caused as a result of insertion or deletion. As a result of either deletion or addition of a nucleotide the entire amino acid frame gets shifted (reading of tRNA on the mRNA during protein synthesis changes) and as a result the entire protein formed would be different as opposed to the desired product.

How do mutagens affect the mutation rate?

Mutation rate is probably a gene that will mutate when a cell divides. The mutation rate is stated as a power of 10. Mutation is a natural process that takes place very rarely (i.e., one in every 10,000 or 10^-4 or one in every 10^9 replicated base pairs). With the introduction of a mutagen the rate of mutation increases rapidly by up to 10 - 1000 times.

How can mutagens be repaired?

Mutations can be repaired through excision repair mechanism and through photolyases. Thymine dimers could be broken down through photolyases which cleaves the dimers formed due to UV mutation. In the case of an excision repair mechanism any base pair that is mismatched could be repaired. The base pair that was added wrongly would not be methylated by the enzyme methylase. As a result of the non-methylated nucleotide would be removed by the action of endonucleases.

How are selection and mutation used in biotechnology?

Natural selection is a process by which the microbial cell is capable of enhancing the survival ability against the odd of time. Artificial selection is a process by which microbe with specific property could be identified. In biotechnology, however, the artificial selection to select desirable microbe could be done. There are over 2000 different microbial strains that were found to produce antibiotics, were isolated from the soil. Mutation is a process which changes the genome of the microbe through mutagens. Though mutation causes deleterious effect, there are a few rare occasions which prove to be beneficial as well. Through site directed mutations, a specific change in a gene of an organism could be made. For example, a change in one amino acid in a protein makes the enzyme work in cold water too, then with the help of site directed mutagenesis the process could be accomplished.

Describe the use of plasmid and viral vectors.

Plasmids and viral genome could be used as vector for the genetic manipulation of an organism. Often, plasmids are used for the incorporation of a small fragment, when compared with a relatively larger fragment of gene that is used in viral vector. Plasmids vectors should have the following genes in their system in order to make sure that recombination has taken place. It should have a gene for any antibiotic resistance, origin of replication, gene for transfer of the plasmid, and a restriction site in the antibiotic resistance gene using an endonuclease that is used in the restriction of the donor. When the donor and plasmid is restricted using the endonuclease, there are three possible combinations that could arise as a result of ligation (the plasmid could re-circularize, the donor fragment might ligate, the donor and the plasmid could ligate removing the antibiotic resistivity of the organism against the antibiotic). In order to select the recombinant clone from the entire mass of cells, replica plating technique could help us in identifying the microbe that is incorporated with the recombinant plasmid. The microbe that has lost the capacity of growing in the plate containing the antibiotic and grows only in its absence would be selected as genetically recombinant strain. The viral genome, on the other hand, accepts a bigger donor gene to be incorporated in them than the plasmids. The inserted sequence would be incorporated in the viral head, which would be allowed to infect a bacterium in which recombination occurs and the donor gene gets integrated into the recipient cell. Gene therapy for specific ailments could be done using the viral vectors.

What types of genes do plasmids carry?

Plasmids are extra chromosomal DNA present in the bacteria. The three most sorted out plasmids which include are: • F (Fertility) plasmid, • R (Resistance) plasmid and • Col plasmid The F plasmid carries the genes necessary for the formation of sex pili, origin of replication for initiating the plasmid multiplications, for the transfer of plasmid to the other cell. The R plasmid codes for resistance of a microbe against a particular antibiotic (by synthesizing enzymes that can neutralize or denature the antibiotic that they code for). Col plasmid codes for toxin production and for bacterial attachment to the intestinal cells. Plasmids also code for proteins to enhance their pathogenicity of the microbes.

Describe the functions of plasmids and transposons.

Plasmids are extra chromosomal genetic materials that are capable of specifically imparting additional features to the microbes that they do not possess. The plasmids are of three types basically and are termed as: • Fertility (F) plasmid,• Resistance (R) plasmid and the • Col plasmid. The F plasmid is necessary for the transfer of plasmids from one cell to the other cell. It is because the F plasmid gives the genes necessary for the synthesis of the sex pili, origin of replication and the transfer gene. The R plasmid resistance is developed against specific antibiotic for the microbe whereas the col plasmid synthesis is specific toxin. Without the presence of the F plasmid there is no possibility of the R and col plasmid to be transferred to the other microbial cell. Transposons are short gene sequences that are also known as jumping genes. These are a part of the genetic material that moves from one part in a genome to another. The simple form of the transposon is the insertion sequences which harbours the gene that codes for the enzyme transposase (required for the endonuclease activity for the transfer of the part), and recognition sites that the transposase recognizes. At times these transposons could carry with them certain specific genes as well. A transposon could be about 700-40000 base pairs long. These transposons are capable of inactivating a particular gene by inserting themselves in between an active coding region.

How do plasmids differ from transposons?

Plasmids differ from transposons because plasmids are self-replicated outside the chromosome. Since, they have their own origin site. Transposons cannot self-replicate because they do not have their own origin site.

Outline the steps in PCR, and provide an example of its use.

Polymerase Chain Reaction: Polymerase Chain Reaction (PCR) is a process, through which multiple copies of a specific gene from a genome could be made within hours. For running a PCR, we need four types of nucleotides, polymerase (taq polymerase isolated from Thermus aquaticus), DNA template, and primers. The entire genome of an organism could not be used for making multiple copies in a PCR. Mechanism of PCR: The mechanism behind the working of a PCR is the double stranded DNA would be separated through increasing the temperature, after which the primers would be ligated to the gene of specific interest. The polymerase enzyme acts on the DNA and starts synthesizing the copies of the gene. Once a copy of the gene is formed the entire mixture would be heated again to remove the hydrogen bonds to form individual strands of DNA that would again act as a template for the primers to attach. Thus, in a PCR the copies of the gene formed increases exponentially. The polymerase specifically used in the case of a PCR is the taq polymerase, which is isolated from a thermophilic microbe, whose enzyme is capable of withstanding high temperatures for longer period of time. In a PCR, during every cycle the temperature has to be increased in order to break the hydrogen bonds, for the double strand to break, so that the single stranded DNA could act as the template. Uses of PCR: • The PCR products could be used in criminal cases in order to find out the suspect and to confirm it. • It could also be used in the identification of specific gene like that of the gene of the bacterium Mycobacterium tuberculosis.

Explain post-transcriptional regulation of gene expression.

Post transcriptional regulation does not take place in all the microbes except in eukaryotes. In eukaryotic system the organism has the presence of different organelles, all these organs have the same genetic blue prints. The gene product present in one organ differs from the other organ. The same gene could be used in the production of more number of gene products in a eukaryote through the post transcriptional modification. The main factor that involves with that of the post transcriptional modification is the micro RNA (miRNA) molecules. These miRNA are approximately around 22 amino acids in length and are capable of binding with the transcribed product (mRNA) through around 6 base pair long. When such a binding occurs the portion of the mRNA that binds would be restricted and the remaining part would be ligated to form a transcribed product. The process of cutting the non-coding regions of an mRNA from the coding region is termed as splicing and the enzymes involved are the spliceosomes. With the help of both splicesosomes and miRNA molecule the desired gene product would be formed at the end of translation.

For what is each of the following used in PCR: primer, DNA polymerase, 94°C?

Primer - Primer designed for a specific gene gets attached to its complementary strand on the DNA, so that the polymerase adds nucleotides form the 3' end, thereby enabling the growth of the DNA strand. DNA polymerase - It adds nucleotides from the 3' end of the primer and continues producing the copies of the gene. 94 0 C - For the complete separation of the double stranded DNA, the temperature of the content would be raised to 940C. Since the polymerase enzyme is from Thermus aquaticus, a thermophile, the enzyme would be active at such high temperature.

Why is a vector used in rDNA technology?

Prokaryotes though do not have a proper immune system, have a restriction modification system to prevent the bacterium from the attack of the viral infection. In the case of an infection, the restriction modification system methylates the cytosine residues in the host DNA and the restriction enzymes act only on DNA that is not methylated. The use of vector helps in two aspects. It carries the gene of interest, as well as, it prevents the gene from being denatured by the host restriction system. Also, since the genome of the microbe cannot be accessed as such for the incorporation of a specific gene, vector is used.

Describe protein synthesis, including transcription, RNA processing, and translation.

Protein synthesis initiates with the attachment of RNA polymerase enzyme to the promoter region of a particular gene. The RNA polymerase enzyme runs over the coding region of the DNA molecule and forms a copy of mRNA molecule (Thymine would be replaced by Uracil). Once the RNA polymerase reaches the stop codon it gets detached from the gene. This transcribed product (mRNA) would be translated to protein before the transcript is processed. Processing of mRNA takes place in a eukaryotic system and not in a prokaryote. In eukaryotes the mRNA synthesized would be transversed by both the coding (exon) and non-coding regions (intron). Through the process of splicing the introns would be removed from the premature mRNA transcript and the exons get ligated. The ligated and processed transcript would be transferred through the nuclear membrane to the cytoplasm. The ribosomes (small unit and a large unit) in the cytoplasm form a complex with mRNA forming the protein factory. The mRNA molecule would be read by the tRNA molecule that brings the amino acid as coded by the triplet code of the mRNA. Once the stop codon is reached the protein synthesized would be cut off and the complex would be dismantled. The mRNA molecule could be used by another ribosome complex for synthesizing the same protein again.

Define RNAi.

RNA interference is abbreviated as RNAi, which holds a promising feature for the suppression of tumor cells. The RNA molecules of short lengths could be used in the silencing of genes, or for a differential expression of products formed from the same gene as in the case of eukaryote. These short RNA interacts with the mRNA and the double stranded forms get digested. Thus, the introns present in the gene of eukaryote would be removed. It is only because of these interfering RNA, the different organs exhibit different products, though they have the same genetic blue print.

Define the following terms: random shotgun sequencing, bioinformatics, and proteomics.

Random short gun sequencing is a concept of sequencing a microbe at random spots in short sequences in a genome. All these sequences would be integrated with the aid of computers and the missed spots would be further sequenced. The use of short gun sequencing is employed in the human genome project. Bioinformatics, on the other hand, makes use of the sequences developed as a result of the short gun sequencing and analyze them for a probable gene of interest and their effect. It is a science of understanding the function of genes through computer assisted analysis. The DNA sequences are stored in web-based databases that are called as gene bank. Proteomics is another branch of science, which deals with the expressed product formed as a result of the gene. It gives a list of all the proteins that could be expressed from a gene.

Explain how DNA technology can be used to treat disease and to prevent disease.

Recombinant DNA technology could be very well used for both treatment as well as for the prevention of diseases. The genes encoding for the antigens of the pathogenic strains could be incorporated into a normal microbial strains that expresses the same gene producing the antigen. When this microbial strain was introduced into a human system, the immune cells can generate antibodies against them. Such a type of vaccine is termed as the sub-unit vaccine. For treatment purposes, the technique, gene therapy can be employed. The defective gene could be bumped off and be replaced by the normal unmutated DNA molecule, thus removing the mutation effect caused. Gene therapy has been studied for suppressing cancer. The gene p53 carried by the adeno virus, which acts as the vector, can be made use to be incorporated into the cancerous cells. It is only the mutation in both the strands of p53 that makes the cells grow without any control towards the cancer. If the p53 gene is incorporated, then the multiplication of the cancerous cells could very well be under control.

List at least five applications of DNA technology.

Recombinant DNA technology has many credits to its usage. The following are some of its credentials. 1. Recombinant DNA technology is used for the synthesis of products that were not a part of their system. For ex: Human insulin. 2. It could be used for the synthesis of subunit vaccines and through the incorporation of plasmids the disease could be prevented by expressing the genes in non-pathogens, or commensals. 3. Gene silencing could be used for switching of a particular gene of interest. For example: Tumor gene. 4. It could be used in the identification of diseases that an offspring could attain from its parents through southern blotting techniques. 5. Using DNA finger printing, a crime suspect could very well be confirmed.

Describe an rDNA experiment in two or three sentences. Use the following terms: intron, exon, DNA, mRNA, cDNA, RNA polymerase, reverse transcriptase.

Recombinant DNA technology includes the following steps: • Isolation of desired gene. • Isolation of desired vector. • Insertion of gene into vector (rDNA). • Transformation of rDNA into host. • Selection of cells containing rDNA. • Cloning or Expression of gene to desired product. In a eukaryotic cell, RNA polymerase enzyme transcribes DNA; RNA processing removes the introns, leaving the exons in the mRNA. cDNA can be made from the mRNA by reverse transcriptase.

How are recombinant clones identified?

Recombinant clones are identified based on the blue white colonies when the foreign DNA is incorporated in a plasmid that has β-Galactosidase gene and Ampicillin resistance gene. On restriction, the β-Galactosidase gene gets broken down and the foreign DNA would be incorporated. Due to this, the β-Galactosidase enzyme activity would be lost by the microbe. Another probability that might arise as a result of the ligation is that, the plasmid might re- circularize again having the β-Galactosidase gene secreting the product. Thus, after transformation of the plasmid when the microbial cell is plated on media containing x-gal and ampicillin, there would be the presence of white and blue colored colonies. The formation of blue colored colonies is due to the presence of β-Galactosidase enzyme. X-gal acts as a substrate for the β-Galactosidase enzyme, which splits it into galactose and a blue colored indigo compound. The microbial colony that remains white is color is due to its inability to produce β-Galactosidase enzyme. It could be ascertained from this that the white colonies where those that have the foreign DNA in them.

What is the value of restriction enzymes in rDNA technology?

Restriction enzymes acts at a specific point in a DNA of a microbe producing blunt and sticky ends. The formation of the blunt and sticky ends depends upon the endonuclease used. For recombinant DNA technology, the donor and the vector DNA would be restricted using the same restriction endonuclease, so that the flanked ends formed as a result get ligated easily. It is highly important that the selection of restriction enzyme plays a vital part in the formation of a recombinant DNA molecule.

Define restriction enzymes, and outline how they are used to make rDNA.

Restriction enzymes are those that act on the genetic material of the organism, primarily built to provide immunity to a prokaryote from the viral infections. This is done by methylating the host DNA during infection, so that the viral genome entering would be broken down neglecting the effect of infection. These restrictions are also used in natural genetic recombinations and in DNA repair mechanisms, during replication and during any possible mutations. The specific characteristic of the restriction enzyme is that, these enzymes are highly specific and attack a specific area in the genome of a microbe, producing sticky or blunt ends. The sticky ends are highly important in rDNA technology and the use of enzymes for producing sticky ends is highly appreciated. The DNA to be incorporated into the vector (plasmid), and the vector, both the components should be cut using the same restriction endonuclease, thus producing the same sticky ends. When these two are allowed to join using the ligase enzyme, there are three possible combinations available. They are the plasmid could rejoin, the donor could rejoin, and the plasmid and the donor could be rejoined. The recombinant vector could be identified by the presence of certain markers like the antibiotic resistant gene. The restriction enzyme should be selected in such a manner that its activity should occur at the gene incorporating level, giving resistance for the microbe, against a particular antibiotic. If a recombinant appears, then it loses its property of antibiotic resistance and the microbial colony can be identified by replica plating technique.

Why do RFLPs result in DNA fingerprint?

Restriction fragment length polymorphism (RFLP) is a part of the DNA finger printing technique. When it is subjected to digestion by the restriction enzyme, it gives rise to specific number of DNA fragments. When a specific genome is restricted using different restriction enzymes, hundreds of fragments could be developed based on the size of the genome under study. These fragments are then run on an agarose gel, giving specific bands based on their molecular weight. The number of bands formed on the gel and the distance it has traveled are specific and vary for each person. Therefore, this resembles and is equally significant just like a finger print is for a person as RFLP is also of such a high specificity.

Compare selection and mutation.

Selection is both a natural, and an artificial phenomenon. The theory of survival of the fittest by microbes, based on its inherited nature, to survive when the environment changes is the best example of a natural selection theory. In the case of artificial selection, the inherited nature of a specific gene can be used in selecting a microbe. For example, the microbe capable of producing the enzyme laccase can be isolated by plating them in a medium containing guaiacol, which gets oxidized to a brownish red color, indicating the production of laccase. Mutation is a term used to denote any change in the genome of a microbe which can be incorporated either naturally, or artificially. Mutation in general are deleterious owing to the level of mutation happened, but under certain rare circumstances mutation can also be beneficial.

How are shotgun sequencing, bioinformatics, and proteomics related to genome projects?

Shot gun sequencing is a concept of sequencing the genome of the microbe at random spots in short sequences in a genome. All these sequences would be integrated with the aid of computers and the missed spots would be further sequenced. The use of shot gun sequencing is employed in the human genome project. Bioinformatics, on the other hand, makes use of the sequences developed as a result of the shot gun sequencing and analyze them for a probable gene of interest and their effect. Proteomics is another branch which deals with the expressed product formed as a result of the gene. It gives a list of all the proteins that could be expressed from a gene. In a human genome project, since there were more than 25,000 different types of genes to be studied, it is a mammoth task to make sure that the sequences that were done through shot gun technique are well organized. This is the main part, as only when the sequence done is of the right order, the details of the gene as well as their gene product would be understood. Even with the aid of such sophistication, it took about 14 years to finish the human genome project, as the genome was only sequenced. It is now understood from the genome project that more than 98% of the gene codes for the miRNA and only about 2% of the gene of the entire genome codes for a characteristic product. All these details were understood only with the usage of such high end support techniques.

What is southern blotting?

Southern blotting is a technique of finding a specified gene or a part of DNA in a genome. In southern blotting technique, the genome to be tested is restricted with the help restriction enzymes. The restricted fragments are separated on a gel. The separated fragments of DNA are then loaded on to a nitro cellulose fiber. The nitrocellulose fiber is then incubated with a target probe (which was radio labelled previously) that will attach to its complementary strand present in the restricted fragments. When the incubated nitrocellulose fiber is read through an x-ray sheet or subjected to autoradiography, the presence of bands on the x-ray denotes the presence of a specific target band of DNA in the test genome. Southern blotting is used to analyze the genetic disorders that a child can develop based on the paternal and maternal chromosomes.

Diagram DNA fingerprinting, and provide an example of its use.

The DNA finger printing technique is used to identify a specific gene of interest during the following conditions: • An outbreak of a disease.• In rape cases. • In paternity and maternity disputes. • In diagnosis and cure of inherited diseases. • In personal identification. • In criminal cases and forensics.

The DNA probe, 3ʹ-GGCTTA, will hybridize with which of the following? a. 5′-CCGUUA b. 5′-CCGAAT c. 5′-GGCTTA d. 3′-CCGAAT e. 3′-GGCAAU

The DNA probe being 3′-GGCTTA binds to 5'-CCGAAT since they are complementary to each other while being anti-parallel. The probe cannot bind to 3'-CCGAAT since it is not anti-parallel even though they are complementary base pairs.

What is the role of cAMP in regulating gene expression?

The cAMP represents the level of burnt down energy as it is synthesized from the hydrolysis of ATP energy molecule. cAMP level is inversely proportional to the level of glucose present within the cell. Once the cAMP level is low, it shows the microbe in having enough nutrients, but when the level of cAMP increases it shows that the microbe is devoid of energy and it starts looking for other polysaccharides for energy acquirement through hydrolysis.

What criteria must a vector meet?

The vector to be involved in the development of a genetically modified microbe, it should have the following properties: • The vector should be self-replicative. • It should have a marker (ex: Antibiotic resistance gene). • It should be able to carry a larger DNA fragment. • It should be able to be digested by the same restriction enzyme as the donor is being cut, in order to make sure that the sticky ends are ligated once recombination occurs.

Explain pre-transcriptional regulation of gene expression in bacteria.

Transcription is a process through which a part of DNA, a gene is copied to an mRNA molecule which in turn would be translated to form the desired protein. Transcription is an important factor that determines the cell protein and enzymatic content. The synthesis of protein and the entire transcription and translation process is an energy consuming process. It is highly important that the microbes present in a supplement depleted environment has to make use of the available nutrients and its reserve source for thriving in the environment. Hence there are a few systems that are prevailing in the microbial system that keeps the genes on only at required times and keeps it off at other times. Repression and induction are two most prevailing systems that regulate the gene products in a particular cell. Repression is a phenomenon by which the gene would be activated only if a particular required nutrient is absent in the medium. But if the same compound is present in the medium the gene expression stops. For example in the tryptophan operon, in the presence of tryptophan, the gene shuts down and in its absence the microbe starts synthesizing the amino acid. In its presence though the amino acid (which acts as the corepressor) binds to the repressible protein and binds itself to the operator thereby preventing transcription. The default status of a repressible gene is switch-on state. Induction is a phenomenon in which the gene would be in a default switch-off state, with the gene being stopped for transcription by a repressor molecule. If an inducer is added to the medium it binds to the repressor molecule removing it from the operator thereby enabling transcription and hence the gene product. Lactose operon acts using this principle.

Which sequence is the best target for damage by UV radiation: AGGCAA, CTTTGA, or GUAAAU? Why aren't all bacteria killed when they are exposed to sunlight?

UV radiation damages DNA by forming thymine dimers between adjacent thymine residues of DNA. Endospores and pigments offer protection against UV radiation. Additionally, repair mechanisms can remove and replace thymine polymers. Hence CTTTGA can be targeted for thymine dimers by UV radiation.

List the four properties of vectors.

Vector is a carrier molecule that should be able to incorporate the genetically modified DNA fragment into the microbial system. Vectors should have some basic properties, such as the following: • The vector should be self-replicative. • It should have a marker (ex: Antibiotic resistance gene). • It should be able to carry a larger DNA fragment. • It should be able to be digested by the same restriction enzyme as the donor is being cut, in order to make sure that the sticky ends are ligated, once recombination happens.

In one sentense, describe how a vector and clones are used.

Vector is the carrier molecule that carries the specific gene of interest into the appropriate microbial cell and clone is the group of cells containing the same gene of interest. Thus, both vector and clone are described in a single sentence.

Identify the roles of a clone and a vector in making recombinant DNA.

Vector is the component of a carrier vehicle, which takes the specific gene of interest that was engineered outside the cell to inside the cell, expressing the product of interest. Plasmids and viral genome can be used as a vector that can be engineered by inserting specific gene of interest through the usage of restriction endonucleases and then introducing the same inside, through transformation or viral infection. The size of the new gene to be introduced also prefers a suitable vector. For example, the viral carrier can accommodate a greater gene size than the plasmid. When this plasmid is introduced into a microbial cell and is allowed to multiply, the mass of cells containing the specified gene of interest in its system is termed as clone.

Compare conjugation between the following pairs: F+ ✕ F-, Hfr ✕ F-.

When conjugation occurs between a F+ and a F- strain, the fertility plasmid moves from the microbe that possess it to the microbe that lacks the F plasmid (Copy of the F plasmid moves to the recipient making both the microbes involved in conjugation as F+ strains). In the case of high frequency of recombination (Hfr) strain, the fertility plasmid of the microbe integrates with that of the chromosome of the microbe forming a high frequency recombinant microbial strain. In such a case when conjugation occurs the entire genomic material along with the integrated chromosome and plasmid traverses to the adjacent microbial cell that lacks the fertility plasmid.

Match the following choices to the statements in questions through. A gene that hybridizes with mRNA. a. antisense b. clone c. library d. Southern blot e. vector

a Any gene or RNA that hybridizes to mRNA is called antisense gene or RNA since mRNA is considered to be sense strand and the strand complementary to sense strand is called antisense strand. This antisense strand inhibits the activity of mRNA (protein synthesis) since it is not allowing mRNA to be present as single stranded fragment for translation to occur.

Which of the following is not a method of horizontal gene transfer? a. Binary fission b. Conjugation c. Integration of a transposon d. Transduction e. Transformation

a Binary fission is not a gene transfer method when compared to other tenchinies. It is a method of asexual reproduction.

Compare and contrast the following terms: a. cDNA and gene b. RFLP and gene c. DNA probe and gene d. DNA polymerase and DNA ligase e. rDNA and cDNA f. genome and proteome

a) Both are DNA. cDNA (complementary DNA) is the portion of mRNA (messenger RNA) that is transcribed reversely with the help of reverse transcriptase (RNA dependent DNA polymerase) to form DNA. This DNA is different from normal eukaryotic DNA by lacking intervening sequences called introns. They may not be genes every time. Gene is the unit of transcription. The portion of DNA that transcribes and codes for RNA or protein is called gene. Eukaryotic gene contains introns. Upon transcription, introns are removed. b) Both are DNA. Restriction fragment is the small double stranded segment of (about base pairs) DNA that serve as recognition site for restriction endonucleases to hydrolyze DNA. Restriction fragments are not genes. They exhibit palindromic symmetry (cyclic symmetry). Gene is the unit of transcription. The portion of DNA that transcribes and codes for RNA or protein is called gene. It does not require specific symmetry. c) Both are DNA. DNA probe is a short single-stranded DNA tagged with radiolabeled or a fluorescent dye. This is used to identify specific DNA fragments during hybridization techniques (such as Southern hybridization...) Gene is the unit of transcription. THe portion of DNA that transcribes and codes for RNA or protein is called genes. d) Both are enzymes that act on common substrate, DNA. DNA polymerase synthesizes DNA using DNA template and dNTPs in direction. It extends the primer from by adding each complementary dNTP at a time to the parent strand. DNA ligase enzyme binds of one nucleotide and of adjacent nucleotide in a DNA by a phosphodiester bond consuming one ATP of energy. e) Both are DNA rDNA (recombinant DNA) is the combination of two DNA from two different sources into a single entity (e.g. DNA resulting after conjugation, transduction, transformation and genetic engineering). cDNA (complementary DNA) is the portion of mRNA (messenger RNA) that is reverse transcribed with the help of reverse transcriptase (RNA dependent DNA polymerase) to form DNA. f) Genome is the complete set of genetic information of an organism. It is encoded mostly as DNA or as RNA in some viruses. Genome is the hereditary information of an organism. Proteome is the complete set of proteins expressed by the genome (genetic material) of an organism. An organism's genome is one complete copy of its genetic information. The proteins encoded by this genetic material comprise the proteome

The following is a code for a strand of DNA. DNA 3' A T A T _ _ _ T T T _ _ _ _ _ _ _ _ _ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 mRNA CGU UGA tRN UGG Amino Acid Met _________ a. Using the genetic code provided in Figure fill in the blanks to complete the segment of DNA shown. b. Fill in the blanks to complete the sequence of amino acids coded for by this strand of DNA. c. Write the code for the complementary strand of DNA completed in part (a). d. What would be the effect if C were substituted for T at base 10? e. What would be the effect if A were substituted for G at base 11? f. What would be the effect if G were substituted for T at base 14? g. What would be the effect if C were inserted between bases 9 and 10? h. How would UV radiation affect this strand of DNA? i. Identify a nonsense sequence in this strand of DNA.

a. A T A T T A C T T T G C A T G G A C T b. RNA synthesis starts with metionine, the genetic code formed from the DNA will be AUG AAA CGU ACC UGA. These codes for the following amino acid sequence Methionine-lysine-arginine- threonine-end. c. T A T A A T G A A A C G T A C C T G A. d. If T is replaced by C, the genetic code on mRNA would be AAG which again codes for lysine. Hence there would be no change. e. If G is replaced by A, the genetic code on mRNA would be UGU which codes for cysteine instead of arginine. f. If T is replaced by G, the genetic code on mRNA would be CCC which codes for proline instead of threonine. g. If C is inserted between 9 and 10 bases it leads to frameshift mutation. The genetic code would now be AUG AAC ACG UAC CUG A. h. UV radiation may cause thymine dimers at thymine residues between 4 & 5, 8 & 9 or 9 & 10. i. Nonsense sequence strand indicates stop codon. Here it is ACT.

Identify when (before transcription, after transcription but before translation, after translation) each of the following regulatory mechanisms functions. a. ATP combines with an enzyme, altering its shape. b. A short RNA is synthesized that is complementary to mRNA. c. Methylation of DNA occurs. d. An inducer combines with a repressor.

a. ATP combines with an enzyme and thereby alters the shape after translation. b. A short RNA is synthesized that is complementary to the mRNA after translation. This short stretch of RNA is called as the primer. c. Methylation of the DNA bases occurs before transcription. This is done so as to prevent the digestion of the DNA by its own restriction endonucleases. d. An inducer combines with a repressor before transcription. When an inducer, a molecule that initiates the gene expression, is present, then it can interact with the repressor protein and detach it from the operator. Then the RNA polymerase can transcribe the message thereby expressing the gene.

You are provided with cultures with the following characteristics: Culture 1: F+, genotype A+ B+ C+ Culture 2: F-, genotype A- B- C- a. Indicate the possible genotypes of a recombinant cell resulting from the conjugation of cultures 1 and 2. b. Indicate the possible genotypes of a recombinant cell resulting from conjugation of the two cultures after the F+ has become an Hfr cell.

a. In Conjugation Culture 1 remains the same while culture 2 will convert to but will have its original genotype. Hence culture 1 is F+, genotype A+ B+ C+and culture 2 is F-, genotype A- B- C-. b. The two cultures may have different combinations of A+ B+ C+ and A- B- C- such as A- B- C+ and A+ B+ C- , and A- B+ C+ and A+ B- C-, A+ B- C+ and A- B+ C-. If F plasmid is transferred to the recipient cell may become F+ or else is left as F-.

Differentiate the following terms. Which one is "hit and miss"— that is, does not add a specific gene to a cell? a. protoplast fusion b. gene gun c. microinjection d. electroporation

a. Protoplast fusion is the process of fusion of two protoplasts (wall less cells) to give rise to a different genotype. Here the addition of specific gene to the cell is not guaranteed. Hence protoplast fusion is "miss". b. In gene gun technique, tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls. c. Microinjection introduces DNA directly into an animal cell by glass micropipette of smaller diameter than cell. d. Electroporation uses electrical current to form microscopic pores in the membrane of cells of entry of DNA. Hence, Gene gun, microinjection, and electroporation ensure specific gene entry into the cell. Hence all these techniques are "hit" for specific gene entry.

Use the following metabolic pathway to answer the questions that follow it. Subtrate A → Enzyme A → Intermediate B → Enzyme B → End-product C a. If enzyme a is inducible and is not being synthesized at present, a (1) __________ protein must be bound tightly to the (2) __________ site. When the inducer is present, it will bind to the (3) __________ so that (4) __________ can occur. b. If enzyme a is repressible, end-product C, called a (1) __________, causes the (2) __________ to bind to the (3) __________. What causes derepression?

a. Repression is a response to decrease the rate of synthesis enzymes. Repressor protein binds tightly to the promoter site. It has the ability to block RNA polymerase to repress the genes. When the inducer is present, it will bind to the repressor so that transcription can occur. b. Corepressor causes the repressor protein to bind to the operator which causes derepression.

Match the following choices to the statements in questions through A population of cells carrying a desired plasmid a. antisense b. clone c. library d. Southern blot e. vector.

b A population of cells or organisms carrying same DNA are said to be clones. The cells are called clones.

The following enzymes are used to make cDNA. What is the second enzyme used to make cDNA? a. reverse transcriptase b. ribozyme c. RNA polymerase d. DNA polymerase

b Normally cDNA is formed by the following steps: 1. DNA (introns + exons) ↓ Transcription in presence of ↓ RNA polymerase 2. mRNA (only exons gets transcribed) ↓ Reverse transcription in presence of ↓ Reverse transcriptase 3. cDNA (only exons) Instead of following the above technique, ribozymes (RNA enzymes) that are present in the intron region of RNA are responsible for self splicing leading to formation of cDNA.

Which of the following is the fourth basic step to genetically modify a cell? a. transformation b. ligation c. plasmid cleavage d. restriction-enzyme digestion of gene e. isolation of gene

b Genetic engineering steps include the following: 1. Isolation of gene. 2. Restriction-enzyme digestion of gene. 3. Plasmid cleavage. 4. Ligation (of digested gene and plasmid). 5. Transformation.The fourth basic step is addition of Ligation of digested gene and plasmid.

Match the following choices to the statements in questions through. a. antisense b. clone c. library d. Southern blot e. vectorPieces of human DNA stored in yeast cells.

c Human DNA is vast compared to yeast vectors. Hence the human DNA is fragmented and each fragment is stored in each yeast vector. This ensures the representation and storage of complete human genome. This process is termed gene library of human genome.

If you put a gene in a virus, the next step in genetic modification would be a. insertion of a plasmid. b. transformation. c. transduction d. PCR. e. Southern blotting.

c • The uptake of DNA from surrounding medium is termed transformation. • The transfer of DNA from one bacterium to other with the aid of conjugation tube is called conjugation. • The transfer of DNA from one host to other through a virus is termed transduction. Since modified virus is used in genetic modification of host, it is called transduction.

Why isn't cDNA synthetic?

cDNA is synthesized from an mRNA molecule that belongs to an eukaryote. Since the mRNA molecule is an expressed product or a blue print of a particular gene, by using reverse transcriptase and DNA polymerase, the mRNA would be converted to a double stranded DNA molecule. Since it is only a matter selecting a particular gene, it is not said to be a synthetic one as opposed to a completely built human growth hormone gene. Also due to the size of the gene, it is highly difficult to produce a synthetic DNA molecule. cDNA is the simplest way of finding the sequence of a particular gene of interest that could be amplified by approaching different methodologies like mutation.

Two offspring cells are most likely to inherit which one of the following from the parent cell? a. a change in nucleotide in mRNA b. a change in nucleotide in tRNA c. a change in nucleotide in rRNA d. a change in nucleotide in DNA e. a change in a protein

d DNA is the genetic material which is inherited. Hence, changes in the DNA nucleotide may be inherited.

You have a small gene that you want replicated by PCR. You add radioactively labeled nucleotides to the PCR thermal cycler. After three replication cycles, what percentage of the DNA single strands are radioactively labeled? a. 0% b. 12.5% c. 50% d. 87.5% e. 100%

d In three cycles of PCR, totally 2^3 DNA fragments will be formed 2^3 = 8 fragments of which one of the DNA fragments is non-labeled parent strand. Hence, the number of radio-labeled DNA after 3 cycles of PCR = 8-1 =7 Percentaage of radio-labebed DNA = 7/8 x100 = 87.5%

Bacteria can acquire antibiotic resistance by all of the following except a. Mutation b. Insertion of transposons c. Conjugation d. snRNPs e. Transformation

d. snRNPs Antibiotic resistance occurs due to mutation or acquiring the resistance from transposon insertions or gene transfer mechanisms. Whereas, small nuclear ribonucleoproteins (snRNPs) have no role in antibiotic resistance.

Match the following choices to the statements in questions through. Self-replicating DNA for transmitting a gene from one organism to another. a. antisense b. clone c. library d. Southern blot e. vector

e A vector is the DNA that is used for transmitting gene from one organism to other. One of the prime property of vector is that, it should be self-replicating i.e. it should have its own origin of replicating (ori) site.

How does miRNA stop protein synthesis?

miRNA are short in length having about 22 nucleotides. These miRNA bind to the mRNA and are transcribed forming a double stranded molecule which is identified and would be enzymatically destroyed. This is done to make sure that specific genes are operated at specific junctures even though there are no switch off and switch on mechanisms for certain genes which keep on transcribing. They are highly important in higher order animals where each organ is required the services of specific gene products like heart, ear etc. A gene product that is highly important for heart functioning will not be activated in ear and even if it is transcribed the miRNA attaches to them and denatures them with the help of endo and exo nucleases.