Question

1.You have discovered a novel toxin-encoding gene in a bacterial pathogen. An adjacent gene encodes a putative transcriptional regulator protein. You decide to identify the promoter for the toxin gene and investigate whether the transcriptional regulator protein controls expression from this promoter. Describe your experimental approach to investigate these questions

2a.Describe how the lac repressor protein is involved in controlling expression of the lac operon.

2b.Explain the difference between homologous recombination and site-specific recombination.

3a.Most genome sequencing projects have used an approach known as "shotgun genome sequencing”. Briefly describe how shotgun sequencing works.

3b.DNA transposons and retrotransposons are both types of mobile elements. Describe these two mobile elements and explain how they differ from each other.

4a.For the following restriction enzymes: A) Xmal C|CCGGG (B) Smal CCC|GGG (C) Sdal CCTGCA|GG (D) Pstl CTGCA|G

(i) What is the length and nature (5’ or 3’) of the overhang generated by each restriction enzyme? (ii) Which pair of these enzymes generate sticky ends that are compatible with each other?

4b.The successful construction and transplantation into a cell of a synthetic Mycoplasma genome was an important milestone in synthetic biology. Briefly describe the approach used in this project

5a.What was different between the genome sequencing approaches used by the public human genome project and the private human genome project led by Craig Venter?

5b.What is systems biology? Give two examples of experimental approaches that have been used in systems biology research.

5c.Describe the principles behind and the applications of the following:

a) Reverse transcriptase-PCR b) Cloning DNA into a plasmid vector c) SDS-PAGE d) Restriction mapping e) Sanger Sequencing of DNA

Answer 1

When nothing is known about a particular promoter, people usual select 2-3kb upstream the Open Reading Frame (ORF) and assume that has a region that contains the promoter. There are some softwares that allow you to predict specific motifs in the DNA that might be binding regions for transcription factor. Usually by deleting these specific regions people can assess what are the regulators of a particular gene. Often people express the puitative promoter region with consecutive deletions in order to narrow down the region that is necessary to trigger transcription. Besides this, 5'RACE or 5' Rapid amplification of cDNA ends- a method combined with high throughput sequencing can be used to map the transcription start site of a single gene.

Once the region is identified, the sequence of that region can easily be deduced. After sequencing, the promoter region of the gene of interest can easily be PCR amlified and inserted before a GFP or Green Fluoroscent Protein sequence into a plasmid followed by transformed into a host a bacterium. In order to determine whether the promoter interacts with the regulatory protein, the bacterium is cotransfected with another plasmid encoding only that protein. If there is any interaction, then that can be observed and assessed by the level of expression of GFP. If the protein represses the expression by binding to the promoter and blocking the transcription, the GFP expression will be low. The reverse will happen if the protein upregulates the promoter activity. But in this case, one should check if there is plasmid incompatibility issues between the two plamids used.

Another approach can be based on EMSA or Electrophoretic mobility shift assay. As we have both the protein and the DNA, we can show by EMSA whether the protein binds to the primer or not. The control lane (DNA probe or promoter without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. However, if the protein is capable of binding to the fragment, the lane with a protein that binds present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to protein which is 'shifted' up on the gel (since it has moved more slowly).

2a) Lac operon is an inducible operon. In the absence of lactose, the lac repressor, lacI, halts production of the enzymes encoded by the lac operon. The lac repressor is always expressed unless a co-inducer binds to it. In other words, it is transcribed only in the presence of small molecule co-inducer like allolactose and isopropylthiogalactoside. Lac repressor (LacI) operates by a helix-turn-helix motif in its DNA-binding domain, binding base-specifically to the major groove of the operator region of the lac operon, with base contacts also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. This DNA binding causes the specific affinity of RNA polymerase for the promoter sequence to increase sufficiently that it cannot escape the promoter region and enter elongation phase, and so prevents transcription of the mRNA coding for the Lac proteins.Binding of the inducer, either allolactose or IPTG, to the repressor can alter the shape of the represor in such a manner so that it can not bind to the DNA and thus RNA polymerase then becomes free to transcribe the operon.

2b) Homologous recombination occurs between DNA with extensive sequence homology anywhere within the homology. Site-specific recombination occurs between DNA with no extensive homology (although very short regions may be critical) only at special sites.

Homologous recombination requires some post-recombination DNA repair synthesis and subsequent DNA ligation must occur. Sie-specific recombination does not require any post recombination nick sealing or gap joining.

Homologus recombination may involve the removal of a strand of DNA (damaged strand or mutated strand) but as the strand exchange during site-specific recombination occurs by precise break/join events and does not involve any DNA loss or DNA resynthesis.

3a) Shotgun sequencing is a method used for sequencing long DNA strands. In shotgun sequencing, DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method (discovered by Sanger) to obtain reads. Multiple overlapping reads for the target DNA are obtained (called 'contig') by performing several rounds of this fragmentation and sequencing. Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence.

1. Genomic DNA is sheared or restricted to yield random fragments of the required size.
2. The fragments are cloned into a universal vector.

[Note that it is important to sequence both strands because certain areas on one strand may be difficult to sequence accurately (for example, because of local secondary structure formation). The complementary strand, however, may sequence well. Using primers from opposite ends will give you sequence for both strands. ]

3. Sequencing reactions are performed with a universal primer on a random selection of the clones in the shotgun library.

4. A software can find regions of overlap (shown as hatch marks above) and properly align them into the complete original sequence.

3b. Transposons are segments of DNA that can move around to different positions in the genome of a single cell. In the process, they may cause mutations, increase (or decrease) the amount of DNA in the genome of the cell, and if the cell is the precursor of a gamete, in the genomes of any descendants. These mobile segments of DNA are sometimes called "jumping genes". Retrotransposons are first transcribed into RNA, then converted back into identical DNA sequences using reverse transcription, and these sequences are then inserted into the genome at target sites.

Transposon	Retrotransposon
Transposons on the chromosome divides and “jumps” to any random position in the chromosome and may get excised from the original locus thus creating mutation (‘cut and paste’ mechanism)	Retrotransposons move by keeping a copy of it in the original locus. This is called ‘copy and paste’ mechanism but the copy is made of RNA, not DNA. The RNA copies are then transcribed back into DNA — using a reverse transcriptase — and these are inserted into new locations in the genome.
Transposon copy number is low and if integration of a transposon into a new site has created a mutation, excision of this transposon will reverse the effect of mutation.	Retrotransposons maintain a high copy number since they are not excised from the donor site unlike transposons.

4a.i)

ii) Sda1 and Pst1 generate sticky ends that are compatible with each other (evident from the cutting pattern).