Question

1. Describe in detail how a protein-encoding gene in a eukaryote is transcribed as mRNA, and what events happen to the mRNA before it can be translated into a protein.

2.You want to investigate the effect of a probiotic on your gut microbiome- the population of bacteria living in your digestive tract. You collect faecal samples prior to and after consumption of the probiotic. Describe in detail how you would sequence the metagenome of the bacteria in these faecal samples and what bioinformatic analyses you would undertake.

3 Describe the principles behind and the applications of the following:

a) Northern blotting b) Site-directed mutagenesis c) DNase l footprinting d) Fusion protein vectors e) Sanger Sequencing of DNA

4a. Describe three differences between DNA replication in bacteria compared with eukaryotes.

4b. Explain the difference between DNA mismatch repair and nucleotide excision repair.

5.When phage infect a bacterial cell, they typically have a temporal program of gene expression, i.e., they express particular genes at particular times. What are two regulatory mechanisms phage use to control their gene expression?

5b. What is alternative splicing? Draw an example of two alternative spliced products

Answer 1

Answer:

1.The genes in DNA encode protein molecules, which are the workhorses of the cell, carrying out all the functions necessary for life. The enzymes which are necessary for all the metabolic reactions of the cell are synthesized by the genes which are present in DNA. During this process initially DNA is transcribed in to mRNA by the process of transcription. The resulting mRNA is a single stranded copy of the gene, which next must be translated into a protein molecule.

Before going to be translated into the protein the synthesized mRNA undergoes several structural modifications. Some of these modifications include 5’ capping, 3’polyadenylation and splicing.

2. The structure and function of the microbe suggested that diet may have a direct impact on the intestinal microbiota and human health status. Probiotics have been proposed preventive and therapeutic measures, in order to restore the healthy composition and function of the gut microbiome.

3. a). Principle of northern blot technique:

In Northern blot, RNA samples are separated on the basis of size by using gel electrophoresis. The isolated RNA fragments are transferred to a support membrane and then treated with a DNA probe.

Applications of Northern blot technique:

It includes, Gene expression studies, diagnosis of several studies and detection of viral microbes etc.

b) Principle of Site directed mutagenesis:

Site directed mutagenesis is one of the most important techniques in laboratory for introducing a mutation into a DNA sequence. The basic procedure requires the synthesis of short DNA primer.

Applications of Site directed mutagenesis:

Site directed mutagenesis is used as an investigative tool and also it has a lot of commercial applications.

c) Principle of DNase I foot printing:

This technique is used to identify specific site of DNA binding proteins. It not only finds the target protein that bins to specific DNA, but also identifies which sequence the target protein is bound. This technique can be used to study interactions between proteins and DNA molecules.

Applications of DNase I foot printing:

In vivo foot printing is a technique used to analyse the protein-DNA interactions that are occurring in a cell at a given time point. DNase I can be used as a cleavage agent if the cellular membrane has been permeabilized. However the most common cleavage agent used is UV irradiation because it penetrates the cell membrane without disrupting cell state and can thus capture interactions that are sensitive to cellular changes.

d) Principle of fusion protein vectors:

Fusion proteins are created by joining of two or more genes that originally coded for separate proteins. Recombinant fusion proteins are crated artificially by r-DNA technology for use in biological research.

Applications of fusion protein vectors:

Fusion proteins vectors are engineered with cleavage sites for proteases or chemical agents that enable the liberation of the two separate proteins.

e) Principle of Sanger sequencing of DNA:

This method is used for the determination of a sequence of nucleotide in a piece of DNA.

In this method, the desired DNA molecules are copied for several times for making fragments of different lengths.

Applications of Sanger sequencing of DNA:

It is a gold standard for sequencing technology in that it provides a high degree of accuracy. It has the long read of capability and flexibility to support a diverse range of applications in many research areas.

4. a). Three differences between DNA replication in bacteria compared with eukaryotes:

In prokaryotes, replication occurs at only one point of origin site at two opposing directions at the same side. But in case of eukaryotes it is originated at multiple points of origin. In eukaryotes replication occurs unidirectional with in the nucleus. Whereas, in prokaryotes it occurs as bi directional. By using two types of RNA polymerases prokaryotes undergo replication but in eukaryotes more than two types of RNA polymerases are used for replication.

b). Difference between DNA mismatch repair and nucleotide excision repair:

The errors if any found during the process of DNA synthesis are repaired by Mismatch repair mechanism. Nucleotide excision repair method is used to repair damaged bases within a string of nucleotides and replaced with DNA as directed by the undamaged template strand. In contrast to nucleotide excision repair, mismatch repair does not operate on bulky adducts or major distortions to the DNA helix.