Molecular Biology ;Answered

Q1. Give a stepwise overview, with diagrams, of the procedure used to clone a DNA sequence encoding one of the insulin subunits.  Clearly indicate the use of any enzymes and briefly state the function of each and the temperature at which the reaction would be performed.

Q2 Describe the major feature(s) of a plasmid vector that make it suitable for DNA cloning and expression.

Q3 Explain how E. coli cells that had transformed a vector could be ‘selected’ from untransformed E. coli.  Would further screening be required to distinguish E. coli containing vector DNA only from E. coli containing vector with inserted insulin A (or B) chain DNA?

Q4 For the insulin A and B chains to be expressed by the E. coli the DNA must be inserted downstream of a promoter in the vector DNA (in this instance the promoter for the b-galactosidase gene). Describe the structure of bacterial transcription promoters. How does the structure affect the ‘strength’ of the promoter?

Q5 For expression of protein of the correct sequence the inserted DNA should be cloned ‘in frame’. What does this mean and why is it important?

Q6 Compare and contrast the advantages and disadvantages of producing a protein for therapeutic use in humans in a bacterial system rather than a mammalian system.

Q7 Give another example of a human protein that has been cloned and is produced in bacteria?  Describe its use.

Q8 Explain one proposed hypothesis for the function of introns.

 

Give a stepwise overview, with diagrams, of the procedure used to clone a DNA sequence encoding one of the insulin subunits.  Clearly indicate the use of any enzymes and briefly state the function of each and the temperature at which the reaction would be performed.

 

 

 

The plasmid is cut across both strands by a restriction enzyme, leaving loose, sticky ends to which DNA can be attached. Special linking sequences are then added to the human cDNA so that it will fit precisely into the loose ends of the opened plasmid DNA ring. The plasmid containing the human gene, also called recombinant plasmid, is now ready to be inserted into another organism, such as a bacterial cell.

 

The recombinant plasmids and the bacterial cells are mixed up. Plasmids enter the bacteria in a process called transfection. With the recombinant DNA molecule successfully inserted into the bacterial host, another property of plasmids can be exploited – their capacity to replicate. Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen copies. When the bacteria divide, the plasmids are divided between the two daughter cells and the plasmids continue to reproduce. With cells dividing rapidly (every 20 minutes), a bacterium containing human cDNA (encoding for insulin, for example) will shortly produce many millions of similar cells (clones) containing the same human gene.

 

 

 

Describe the major feature(s) of a plasmid vector that make it suitable for DNA cloning and expression.

 

Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors. They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA. The complete DNA sequence of many plasmids is known; hence, the precise location of restriction enzyme cleavage sites for inserting the foreign DNA is available. Plasmids are smaller than the host chromosome and are therefore easily separated from the latter, and the desired DNA is readily removed by cutting the plasmid with the enzyme specific for the restriction site into which the original piece of DNA was inserted (2000).

 

 

 

Explain how E. coli cells that had transformed a vector could be ‘selected’ from untransformed E. coli.  Would further screening be required to distinguish E. coli containing vector DNA only from E. coli containing vector with inserted insulin A (or B) chain DNA?

 

  1. coli cells that are capable of forming colonies must either contain the vector without a chromosomal DNA insert or a vector with a chromosomal DNA insert. If the correct ratio of chromosomal DNA to vector DNA is used, rarely does a vector have two distinct pieces of chromosomal DNA inserted into it.

 

 

 

For the insulin A and B chains to be expressed by the E. coli the DNA must be inserted downstream of a promoter in the vector DNA (in this instance the promoter for the b-galactosidase gene). Describe the structure of bacterial transcription promoters. How does the structure affect the ‘strength’ of the promoter?

 

Bacterial transcription promoters are relatively simple. They are approximately 40 nucleotides (40 bp or four turns of the DNA double helix) in length, a region sufficiently small to be covered by an E. coli RNA holopolymerase molecule. In this consensus promoter region are two short, conserved sequence elements. Approximately 35 bp upstream of the transcription start site there is a consensus sequence of eight nucleotide pairs to which the RNAP binds to form the closed complex. More proximal to the transcription start site, about 10 nucleotides upstream, is a 6-nucleotide-pair AT-rich sequence. The TATA box is thought to ease the dissociation between the two DNA strands so that RNA polymerase bound to the promoter region can have access to the nucleotide sequence of its immediately downstream template strand. This is called the open complex(2000).

 

 

 

For expression of protein of the correct sequence the inserted DNA should be cloned ‘in frame’. What does this mean and why is it important?

 

The inserted DNA should be cloned ‘in frame’ means that the DNA sequence must be cloned in a way that the reading frame of the gene is maintained. Cloning vectors can incorporate many different features including transcription and translation signals. To clone into a vector and have the protein of interest be expressed from the regulated promoter, the DNA sequence must be cloned so that the reading frame of the gene is maintained.

 

 

 

Compare and contrast the advantages and disadvantages of producing a protein for therapeutic use in humans in a bacterial system rather than a mammalian system.

 

Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used. The resulting offspring from mammalian clones have to arise from sexual reproduction.

 

 

 

Give another example of a human protein that has been cloned and is produced in bacteria?  Describe its use.

 

Another example of a human protein that has been cloned and is produced in bacteria is the antibody. In 1976 it became possible to ‘immortalise’ single antibody-producing cells (from mice); such clones would be ideal for us, but very tantalisingly, this method has never worked well for human cells. In the present decade, however, the ability to clone the antibody genes has proved a promising alternative. If they are sampled from a suitable source, they can be cloned, identified and then mass-produced in bacteria, so that many single antibodies can be compared with each other (1998).

 

 

 

Explain one proposed hypothesis for the function of introns.

 

The coding regions of DNA, the transcripts of which ultimately appear in the cytoplasm as single mRNA molecules, are usually interrupted in the eukaryotic genome by large intervening sequences of noncoding DNA called introns. The function of the intervening sequences, or introns, is not clear. One proposed hypothesis is that they may serve to separate functional domains (exons) of coding information in a form that permits genetic rearrangement by recombination to occur more rapidly than if all coding regions for a given genetic function were contiguous. Such an enhanced rate of genetic rearrangement of functional domains might allow more rapid evolution of biologic function (2000).