Blog Assignment 3: Nucleotide Sequence of Human Preproinsulin Complementary DNA

Abtract of the article: http://www.sciencemag.org/cgi/content/abstract/208/4439/57

Insulin Review Article Questions:

1. In what journal did this article appear? When? The article was published in Science (Volume 208) on April 4, 1980.
2. What is the primary purpose of this paper? The primary purpose of the paper is to present how cloned human preproinsulin cDNA and information from different sequences (mRNA, nucleotide and intervening) will help in isolating human insulin gene.
3. What is the structural difference between insulin and proinsulin? Insulin is made up of two polypeptide chains referred to as A chain and B chain. These chains are linked together by two disulfide bonds. Proinsulin is a prohormone precursor of insulin and C peptide is a part of its composition. Synthesis of proinsulin would produce insulin and C peptide.
4. What is complementary DNA (cDNA)? Complementary DNA (cDNA) is DNA in which the sequence of the nitrogenous bases (adenine, thymine, cytosine and guanine) on one strand of the double stranded structure chemically matches the sequence on the other strand.
5. What are "recombinant plasmids"? A plasmid is a circular DNA molecule that replicates independently of the chromosome. A recombinant plasmid is a DNA that has been artificially manipulated to combine genes from two different sources. The recombinant plasmid is one of the factors of industrial biotechnology whereby genetically engineered bacteria are inserted to produce industrially and medically important proteins such as enzymes, cytokines, growth hormones, antigens or live bacterial vaccines.
6. What does the article mean when it says "Escherichia coli x1776 was transformed with the recombinant plasmids"? A transformant is a cell that is genetically altered by taking up a plasmid from its environment through the cell wall and a new genetic material is inserted. These exogenous plasmids are produced outside this transformation process and are left unaltered so that it can be reused. In this case, Escherichia coli X1776 is the genetic material inserted in the recombinant plasmid to produce transformants to do further studies on the sequences that were obtained.
7. What is meant by the "polyA tail" or "polyadenylation" of a gene? Polyadenylation is the addition of a poly(A) tail to an RNA molecule. A poly (A) tail is a long stretch of (about ten to 200 or more) adenine nucleotides (adenine bases) added to the "tail" or 3' end of the pre-mRNA. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation.
8. What is meant by the statement that "insulin A and B chains are highly conserved"? The sequences of the chains of insulin of human is similar to other species. Sequence similarities serve as evidence for structural and functional conservation. They can be studied further, easily accessible and can be used when if experimentation is proven successful.
9. Which chain is most highly conserved? Although in most species, A chain consists of 21 amino acids and B chain has 30, in this review, the A chain contains most of the amino acids and shows the most highly conserved.
10. What do the researchers believe is the purpose of the C chain? The C Chain holds the insulin together (the A chain and the B chain) to maintain the three-dimensional form. It is located in the center of the two chains of insulin. When proinsulin is broken down into insulin and C peptide, the high amount of amino acid sequence in the C peptide doesn’t hinder its functionality. Further research states that newly diagnosed diabetes patients often get their C-peptide levels measured as a means of distinguishing type 1 diabetes and type 2 diabetes.
11. Why does it make sense that the C chain is more variable (less highly conserved) than the A chain and B chain? C peptide is sometimes not found in the homology with other species.
12. What do the researchers believe is the purpose of the pre-peptide (D chain)? The D chain is to serve as the signal for transfer of this protein into the endoplasmic reticulum.
13. How does the human preproinsulin gene differ from rat preproinsulin (rat I and rat II)? They differ in their untranslated region where human preproinsulin mRNA has 73 more nucleotides extending after the termination codon while the preproinsulin of both rat I and rat I mRNA 3' untranslated region are only 20 nucleotides.
14. What is the first codon in the coding region of the gene (at the start of the pre-peptide) and what is the first amino acid in the polypeptide? The first codon in the coding region is AUG and the first amino acid in the polypeptide is Methionine.