PYQ – MZO-002: Genetics and Animal Biotechnology (Solved Q&A) | MZO-002 | MSCZOO | M.Sc.Zoology | IGNOU | December 2024
M.Sc. (Zoology) (MSCZOO)
Term-End Examination
December, 2024
MZO-002 : GENETICS AND ANIMAL BIOTECHNOLOGY
Time : 2 Hours| Maximum Marks : 50
Note: Attempt any five questions. All questions carry equal marks.
1. (a) Discuss the different categories of transcription factors. (5 Marks)
Transcription factors are proteins that help in controlling gene expression by regulating transcription. They do not make RNA themselves but guide RNA polymerase to start or stop the process. They are important in development, cell cycle, stress response and hormone signaling.
Transcription factors are mainly divided into two categories:
1. Based on Their Function
There are two types:
a) General Transcription Factors
These transcription factors are needed for the transcription of almost all protein-coding genes. They help in forming the transcription initiation complex near the promoter region. They guide the RNA polymerase II to attach at the correct place and begin transcription. These factors are not gene-specific.
Example: TFIID has a TBP part that recognizes the TATA box.
b) Specific Transcription Factors
These transcription factors control only certain genes. They work in special conditions such as during stress, hormone action, or development. They bind to enhancer or silencer sequences and either activate or block the transcription of target genes. They are gene-specific and time-specific.
Example: Estrogen receptor activates genes when estrogen is present.
2. Based on Their Structure and DNA-Binding Domain
There are four major types:
a) Helix-Turn-Helix
This type has two alpha helices connected by a short loop or turn. One of the helices fits into the major groove of DNA and helps in DNA binding.
b) Zinc Finger
These proteins have finger-like loops that are held together by zinc ions. These loops bind to DNA in a specific way and are very common in eukaryotic transcription factors.
c) Leucine Zipper
These proteins have a leucine amino acid at every 7th position which helps in forming a dimer. Two such proteins come together to hold the DNA tightly, like a zip and help in gene regulation.
d) Basic Helix-Loop-Helix
This type has two helices connected by a loop. They usually form dimers and bind to specific DNA sequences called E-boxes. These are common in developmental genes.
(b) Explain in brief about Lyon hypothesis. (5 Marks)
Lyon Hypothesis (Also called X-Chromosome Inactivation Hypothesis) was proposed by British geneticist Mary F. Lyon in 1961. According to this hypothesis, in female mammals, who have two X chromosomes, one of the X chromosomes becomes inactive during early embryonic development. This inactivation is random, meaning either the maternal or the paternal X can be inactivated. Once an X chromosome is inactivated in a cell, all the daughter cells formed from it retain the same X chromosome as inactive. This process is known as X-chromosome inactivation and it leads to genetic mosaicism in females.
The inactive X chromosome becomes highly condensed and appears as a dense structure called a Barr body, which is usually located near the nuclear membrane. This mechanism helps maintain dosage compensation, ensuring that females (XX) and males (XY) express equal levels of X-linked genes, because males have only one X chromosome.
The molecular control of this process involves a gene called XIST (X-inactive specific transcript), which is located on the X chromosome itself. The XIST gene produces a long non-coding RNA that coats the X chromosome from which it is transcribed and triggers its inactivation.
A well-known example of this hypothesis is the Calico cat, where females show patches of black and orange fur. This is because the fur color gene is located on the X chromosome and different X chromosomes are active in different skin cells, leading to patchy coat color.
2. Discuss in detail the steps for the construction of C-DNA library. (10 Marks)
A cDNA library is a collection of complementary DNA (cDNA) sequences synthesized from mRNA. It represents only the expressed genes of a cell or tissue at a particular time. cDNA libraries are used widely in molecular biology to study gene expression, isolate genes and produce eukaryotic proteins in prokaryotic systems.
There are five major steps in the construction of a cDNA library:
1. Isolation of mRNA
Total RNA is extracted from cells and mRNA is separated using oligo-dT cellulose columns. These columns bind specifically to the poly-A tails found at the 3' end of eukaryotic mRNA.
2. Synthesis of First Strand cDNA
The enzyme reverse transcriptase uses the mRNA template to synthesize the first cDNA strand. An oligo-dT primer binds to the poly-A tail and initiates synthesis from the 3' end of the mRNA.
3. Synthesis of Second Strand cDNA
After the first strand is made, the RNA strand is degraded by RNase H. Then DNA polymerase I synthesizes the second DNA strand using the RNA fragments as primers. DNA ligase helps in joining the DNA fragments to complete the double-stranded cDNA.
4. Addition of Linkers or Adapters
Short synthetic DNA sequences called linkers or adapters, containing restriction enzyme sites, are added to the ends of cDNA. This step helps in inserting the cDNA into suitable cloning vectors.
5. Cloning into Vectors and Library Formation
The modified cDNA is inserted into vectors like plasmids or phages and then introduced into competent E. coli cells through transformation. These transformed cells are stored as clones, forming a cDNA library that represents the actively expressed genes of the source organism.
3. Discuss the different applications of gene therapy. (10 Marks)
Gene therapy has many applications in modern medicine. It involves altering the genetic material of a person's cells to treat or prevent disease. There are two major types of gene therapy applications: somatic gene therapy and germline gene therapy. Somatic gene therapy affects only the patient and is not passed to offspring, while germline therapy affects future generations but is still not allowed in humans due to ethical concerns.
There are the following major applications of gene therapy, each based on different disease categories and treatment strategies:
1. Treatment of Genetic Disorders
This is the most widely used and studied application. Gene therapy helps in correcting monogenic disorders like Severe Combined Immunodeficiency (SCID), cystic fibrosis, thalassemia and Duchenne muscular dystrophy. For example, in SCID, a functional ADA gene is introduced into lymphocytes. Instead of simply managing symptoms, gene therapy targets the root genetic cause.
2. Cancer Treatment
Gene therapy is used to kill cancer cells, make them more sensitive to existing treatments or boost the immune system. For example, insertion of tumor suppressor genes like p53 can stop uncontrolled cell growth. In immunotherapy, genes encoding interleukins (like IL-2) are used to activate immune cells against tumors. It is also used to deliver suicide genes that make cancer cells respond to specific drugs.
3. Treatment of Viral Infections
Gene therapy can make immune cells resistant to viral infections. In HIV treatment, T-cells can be modified using CRISPR or zinc finger nucleases to block receptors like CCR5 that HIV uses to enter cells. Other approaches include inserting antiviral genes that prevent viral replication. Though in early stages, this method holds promise for diseases like HIV and hepatitis B.
4. Cardiovascular and Muscular Disorders
Gene therapy helps repair damage in heart and muscle tissues. In ischemic heart disease, genes like VEGF are delivered to promote blood vessel formation and improve blood flow. For muscular dystrophies, gene therapy tries to restore functional dystrophin protein, helping muscle maintenance.
5. Regenerative Medicine and Gene Editing
Gene therapy is also applied in tissue engineering and precision genome correction. Modified stem cells are used for repairing spinal cord injury, bone damage, or skin burns. Tools like CRISPR-Cas9 offer precise editing of faulty genes and are being explored for a range of diseases, though ethical and safety concerns remain.
4. What is an operon? With suitable diagrams, explain how is gene expression regulated in the trp operon by the amino acid tryptophan. (2 + 8 Marks)
An operon is a functional unit of DNA found in prokaryotes like E. coli. It consists of a group of related genes that are regulated together. These genes are controlled by a common promoter and transcribed into a single long mRNA strand, which is called polycistronic mRNA. This allows the cell to coordinate the production of several proteins from one mRNA.
The key parts of an operon include:
- Promoter: the site where RNA polymerase binds
- Operator: the regulatory region where a repressor protein may bind
- Structural genes: the actual genes that are transcribed
- Regulatory gene: may be located elsewhere, codes for repressor protein
This system helps bacteria conserve energy by expressing genes only when needed. Based on how they are regulated, operons can be of two types:
- Inducible operons, such as the lac operon, are usually off and can be turned on in response to specific signals
- Repressible operons, like the trp operon, are usually on and can be turned off when their product is in excess
This system of gene regulation is only found in prokaryotes, especially bacteria and is not seen in most eukaryotes.
Regulation of Gene Expression in the trp Operon by Tryptophan
Among several operons found in E. coli, the trp operon is a well-studied example which shows how gene expression is controlled based on the level of a specific amino acid, tryptophan.
The trp operon in E. coli controls the production of tryptophan (an essential amino acid). It consists of five structural genes: trpE, trpD, trpC, trpB and trpA, which together code for enzymes required for the biosynthesis of tryptophan. The operon also has a promoter, an operator and a regulatory gene (trpR) located elsewhere in the genome.
Tryptophan regulates the trp operon through two mechanisms: repression and attenuation. Both mechanisms act as negative feedback systems and work together to ensure that the trp operon functions only when tryptophan is needed, which helps prevent unnecessary energy use by the cell.
Repression Mechanism
When tryptophan levels in the cell are low, the repressor protein made by the trpR gene remains inactive and cannot bind to the operator. This allows RNA polymerase to bind to the promoter and transcribe the structural genes. As a result, enzymes for tryptophan synthesis are produced.
When tryptophan levels become high, some tryptophan molecules bind to the inactive repressor. This forms an active repressor-tryptophan complex, which binds to the operator region of the operon. This blocks RNA polymerase from initiating transcription. In this way, tryptophan acts as a corepressor, turning off its own synthesis when it is abundant.
Attenuation Mechanism
Attenuation is a second level of control in the trp operon. This occurs at a region called the leader sequence (trpL), which lies just before the structural genes.
When tryptophan is present in high amount, the ribosome translates the leader sequence quickly. This allows the mRNA to form a terminator hairpin loop structure, which signals RNA polymerase to stop transcription early, before the structural genes are copied.
But when tryptophan is low, the ribosome slows down or stops at the tryptophan codons in the leader sequence. This delay causes the mRNA to form a different shape called an anti-terminator loop. This structure allows RNA polymerase to continue transcribing the full operon. So, the enzymes for tryptophan synthesis are produced.
In this way, attenuation acts like a sensor that depends on how much tryptophan is available during translation. It fine-tunes the operon activity along with repression.
5. What are the steps of gene cloning? Describe the different DNA manipulation enzymes used in this technique. (4 + 6 Marks)
What are the steps of gene cloning?
Gene cloning is a molecular biology technique used to make exact copies of a specific gene or DNA segment. The main goal is to isolate a particular gene and make multiple identical copies of it. This technique is widely used in genetic research, biotechnology, medicine and agriculture. It helps in studying gene functions, producing proteins like insulin, making genetically modified organisms and more.
There are following major steps in gene cloning:
1. Isolation of DNA (Gene of Interest)
The first step involves extracting the desired gene from the source DNA. This is done using restriction enzymes that cut the DNA at specific sequences or by PCR (Polymerase Chain Reaction) to amplify the required gene.
2. Selection of a Suitable Cloning Vector
A vector is a carrier molecule, usually a plasmid, which is used to transport the gene into a host organism. A good vector contains an origin of replication, multiple cloning sites and a selectable marker like an antibiotic resistance gene.
3. Insertion of Gene into the Vector
Both the vector and the gene are treated with the same restriction enzyme to produce compatible ends. The gene is then ligated into the vector using DNA ligase, forming recombinant DNA.
4. Introduction into Host Cell (Transformation)
The recombinant plasmid is introduced into a bacterial host cell. This is usually done by chemical treatment or electroporation which makes the cell membrane permeable.
5. Selection of Recombinant Cells
Transformed cells are grown on a selective medium, usually containing an antibiotic. Only those cells that have taken up the recombinant DNA will survive and form colonies.
6. Cloning and Amplification
The selected bacterial colonies are allowed to grow and divide. With each division, the recombinant plasmid and the inserted gene are replicated, resulting in many identical copies of the gene.
DNA Manipulation Enzymes Used in Gene Cloning
DNA manipulation enzymes are used in gene cloning to cut, join, modify, or process DNA or RNA molecules. These enzymes allow scientists to carry out steps like restriction, ligation, amplification and cleaning of DNA.
The main types of DNA manipulation enzymes used in gene cloning are as follows:
1. Nucleases
- Nucleases are enzymes that cut nucleic acids. They are of two types: exonucleases remove nucleotides from the ends and endonucleases cut at internal sites. Restriction endonucleases are used in gene cloning to cut DNA at specific sequences.
2. S1 Nuclease
- S1 Nuclease cuts single-stranded DNA or RNA. It is used to remove loops or single-stranded regions from DNA to make it fully double-stranded.
3. Mung Bean Nuclease
- Mung Bean Nuclease is another single-strand-specific enzyme. It is used to clean up DNA overhangs and make blunt ends, which are helpful in blunt-end ligation during cloning.
4. Ribonuclease A
- Ribonuclease A (RNase A) breaks down RNA molecules present in DNA samples. It is used after DNA isolation to remove RNA contamination.
5. Ribonuclease H
- Ribonuclease H (RNase H) breaks the RNA strand in RNA-DNA hybrids. This is helpful when converting RNA into DNA during cDNA synthesis.
6. DNA Ligase
- DNA Ligase is used to join DNA fragments by making bonds between sugar and phosphate. It is needed to insert the gene of interest into the vector.
7. DNA Polymerases
- DNA Polymerases, such as Taq polymerase or Klenow fragment, are used for synthesizing new DNA strands or filling in overhangs. They are also important in PCR amplification.
8. DNA Modifying Enzymes
- DNA Modifying Enzymes include:
- Alkaline Phosphatase: Removes 5′ phosphate groups from DNA to prevent vector self-ligation.
- T4 Polynucleotide Kinase: Adds phosphate groups to the 5′ ends of DNA or RNA to aid in ligation.
- Terminal Deoxynucleotidyl Transferase (TdT): Adds nucleotides at 3′ ends without needing a template, useful in labeling or tailing DNA ends.
- DNA Methyltransferase: Adds methyl groups to specific DNA bases, protecting them from digestion by restriction enzymes and regulating expression.
6. (a) Explain the methodology of the production of recombinant growth hormone with the help of a suitable diagram. (5 Marks)
Recombinant human growth hormone (rhGH) is an artificially synthesized hormone used in the treatment of growth-related disorders. It is produced through genetic engineering by inserting the human growth hormone gene into a microbial host system like Escherichia coli (E. Coli) for large-scale expression and purification.
The following steps are involved in the production of recombinant growth hormone:
1. Identification and isolation of the gene
The gene coding for human growth hormone is identified from human DNA and isolated using restriction enzymes or PCR. This gene contains the required coding sequence to produce the hormone.
2. Insertion of the gene into a plasmid vector
The isolated gene is inserted into a plasmid. This plasmid acts as a vehicle and includes a bacterial promoter and antibiotic resistance gene. DNA ligase is used to join the gene and plasmid.
3. Transformation into host cells
The recombinant plasmid is introduced into competent E. coli cells. These cells take up the plasmid carrying the human gene.
4. Selection of transformed cells
The bacteria are grown on an antibiotic medium. Only those cells survive which have successfully taken up the plasmid due to the resistance gene.
5. Expression of the inserted gene
In selected bacteria, the promoter starts transcription and translation of the hGH gene, producing the hormone inside bacterial cells.
6. Extraction and purification of hormone
The hormone is extracted from bacterial cells. It is often misfolded, so it is refolded and purified using chromatographic techniques.
7. Final processing and quality testing
The purified hormone is tested for its biological activity and safety. Then it is formulated into a dosage form for therapeutic use.
(b) Describe the mechanism of gene known-down by RNA interference. (5 Marks)
RNA interference (RNAi) is a natural method used by cells to reduce or block the expression of specific genes. This happens by breaking down the messenger RNA (mRNA) before it can make a protein. Scientists use this process to study the role of genes and to control harmful gene expression in various organisms.
[Note:- Knock-down means reducing the expression of a gene, usually by interfering with its mRNA, so that the amount of protein it produces becomes lower but not completely absent.]
There are the following major steps involved in gene knockdown through RNAi:
1. Introduction of Double-stranded RNA (dsRNA):
The process begins when double-stranded RNA (dsRNA) enters the cytoplasm. It may come from outside the cell (injection or vector-based delivery) or be transcribed inside using expression systems.
2. Cleavage by Dicer Enzyme:
A special enzyme called Dicer recognizes the dsRNA and cuts it into small interfering RNA (siRNA) molecules. These siRNAs are around 21–23 nucleotides long and have two strands, one is the guide strand and the other is the passenger strand.
3. Formation of RISC Complex:
The siRNA then binds to a group of proteins called RISC (RNA-induced silencing complex). Inside this complex, the passenger strand is removed and the guide strand remains attached.
4. Target Recognition and mRNA Degradation:
The guide strand in RISC helps the complex bind to a complementary mRNA molecule in the cytoplasm. If the base pairing is nearly perfect, the Argonaute protein present in RISC cuts the mRNA.
5. Gene Silencing:
The targeted mRNA is degraded and cannot be translated into protein. Thus, the expression of the gene is reduced or silenced, achieving gene knockdown.
7. Write short notes on the following: (2.5 × 4 = 10 Marks)
(a) Karyotyping
Karyotyping is the process of arranging and analyzing the complete set of chromosomes in a cell. It is used to detect chromosomal abnormalities and to study chromosome number and structure. The process involves collecting cells (usually from blood or amniotic fluid), arresting them in metaphase using colchicine, staining them with a dye like Giemsa and photographing the chromosomes under a microscope. These chromosomes are then arranged in pairs based on size, centromere position and banding pattern. This arranged image is called a karyogram. Karyotyping is important in prenatal diagnosis, detection of genetic disorders like Down syndrome (trisomy 21), Turner syndrome (XO) and in cancer diagnosis to detect chromosomal translocations.
(b) Transposable elements
Transposable elements, also called transposons or jumping genes, are DNA sequences that can change their position within the genome. They were first discovered by Barbara McClintock in maize. There are two main types: Class I elements (retrotransposons) which move through an RNA intermediate using reverse transcriptase and Class II elements (DNA transposons) which move directly through a cut-and-paste mechanism using transposase enzyme. These elements can create mutations by inserting themselves into or near genes. While they can disrupt normal gene function, they also contribute to genome evolution, genetic diversity and regulation of gene expression.
(c) Histone modifications
Histone modifications are chemical changes made to the histone proteins around which DNA is wrapped in the chromatin. These modifications occur mainly on the N-terminal tails of histones and include acetylation, methylation, phosphorylation, ubiquitination, etc. They play a major role in the regulation of gene expression by altering chromatin structure. For example, acetylation of histone H3 and H4 (by histone acetyltransferases) leads to relaxed chromatin and active gene transcription, while deacetylation leads to condensed chromatin and gene silencing. Methylation can activate or repress genes depending on the site and type of histone. These modifications are part of epigenetic regulation and are reversible.
(d) Dominant epistasis
Dominant epistasis is a type of gene interaction where a dominant allele of one gene completely masks or suppresses the expression of another gene at a different locus. This happens even if the second gene also carries dominant alleles. It is different from Mendel's original principle because the phenotype is not only controlled by individual gene pairs but also by how genes interact.
In dominant epistasis, the phenotypic ratio in a dihybrid cross is usually modified from the Mendelian 9:3:3:1 to 12:3:1. This means 12 individuals will show the dominant epistatic trait, 3 will show the trait controlled by the hypostatic gene and 1 will show the recessive trait of both genes.
A clear example is found in summer squash (Cucurbita pepo). In this plant, the dominant allele W prevents pigment formation. So, plants with genotypes WW or Ww always appear white, regardless of the alleles present at the second gene, which can be Y or y. This second gene controls yellow or green pigment, but it can only show its effect when the first gene is ww. In that case, wwYY or wwYy gives yellow color and wwyy gives green color.
So, the dominant W allele is epistatic to both Y and y alleles.
Comments
Post a Comment