GLYCOGENESIS

GLYCOGENESIS

The synthesis of glycogen from glucose is glycogenesis . Glycogenesis takes place in the cytosol and requires ATP and UTP, besides glucose.

1. Synthesis of UDP-glucose : The enzymes hexokinase (in muscle) and glucokinase (in liver) convert glucose to glucose 6-phosphate. Phosphoglucomutase catalyses the conversion of glucose 6-phosphate to glucose 1-phosphate. Uridine diphosphate glucose (UDPG) is synthesized from glucose 1-phosphate and UTP by UDP-glucose pyrophosphorylase.

2. Requirement of primer to initiate glycogenesis : A small fragment of pre-existing glycogen must act as a ‘primer’ to initiate glycogen synthesis. It is recently found that in the absence of glycogen primer, a specific protein—namely ‘glycogenin’—can accept glucose from UDPG. The hydroxyl group of the amino acid tyrosine of glycogenin is the site at which the initial glucose unit is attached. The enzyme glycogen initiator synthase transfers the first molecule of glucose to glycogenin. Then glycogenin itself takes up a few glucose residues to form a fragment of primer which serves as an acceptor for the rest of the glucose molecules.

3. Glycogen synthesis by glycogen synthase : Glycogen synthase is responsible for the formation of 1,4-glycosidic linkages. This enzyme transfers the glucose from UDP-glucose to the non-reducing end of glycogen to form - 1,4 linkages.

4. Formation of branches in glycogen : Glycogen synthase can catalyse the synthesis of a linear unbranched molecule with 1,4 - glycosidic linkages. Glycogen, however, is a branched tree-like structure. The formation of branches is brought about by the action of a branching enzyme, namely glucosyl -4-6 transferase. (amylo 1,4 1,6 trans- glucosidase). This enzyme transfers a small fragment of five to eight glucose residues from the non-reducing end of glycogen chain (by breaking -1,4 linkages) to another glucose residue where it is linked by -1,6 bond. This leads to the formation of a new non-reducing end, besides the existing one. Glycogen is further elongated and branched, respectively, by the enzymes glycogen synthase and glucosyl 4-6 transferase.

The overall reaction of the glycogen synthesis for the addition of each glucose residue is

(Glucose)n + Glucose + 2ATP (Glucose)n+1 + 2 ADP + Pi

Of the two ATP utilized, one is required for the phosphorylation of glucose while the other is needed for conversion of UDP to UTP.

   GLYCOGEN SYNTHESIS 

GLYCOGENESIS

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GENOMIC LIBRARY

A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. The organism's DNA is extracted from cells and digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. The vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.

STEPS:

1.     Isolating genomic DNA from the organism –

a) Isolation of DNA (purification) Eukaryotes: Prepare cell nuclei, remove proteins, lipids and other unwanted macromolecules by protease digestion and phase extraction.

b) Prokaryotes: Extracted DNA directly from cells.

2.     Cutting of DNA into suitable fragment size: Genomic DNA is then partially cleaved into thousands of fragments (of 5-100 kb) by restriction endonuclease to get inserts of desired size range, compatible with the cloning vector (such as plasmid, phage lambda, cosmid, bacteriophage P1,Bacterial Artificial Chromosome [BAC] or Yeast Artificial Chromosome [YAC]) used for library construction. Cleaving the entire genome of a cell with a specific restriction nuclease and cloning each fragment is sometimes called the “shotgun” approach to gene cloning. This technique can produce a very large number of DNA fragments. Digestion into small fragments can be achieved by physical shearing (Pipetting, mixing). Restriction enzyme digestion : Partial digestion is preferred to get a greater lengths of DNA fragments.

Selection of restriction enzymes: 1. Ends produced (sticky or blunt) & the cleaved ends of the to be vector 2. Whether the enzyme is inhibited by DNA modifications 3. Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.

3.     Incorporation of vector into suitable host: All the fragments are ligated into the cloning vector which is cleaved with the same restriction endonuclease and transformed into host organism which is commonly a population of Escherichia coli or yeast cells to produce a library with each cell containing one vector

molecule.

4.    Maintenance of clones: Each cloning vector contains a different fragment of the genome so that all DNA in the genome is represented among the clones in the library. All of the plasmids in a particular host cell harbour the same insert. As the plasmid multiplies, there will be 50-100 copies of the plasmid in each cell. Genomic DNA inserts can be sequenced by isolating individual recombinant cloning vectors from cells. By sequencing and analysing random clones from the library configs can be made and the whole genome sequence can be  recovered.

Application:

1. Determining the complete genome sequence of a given organism.

2. Serving as a source of genomic sequence for generation of transgenic animals through genetic engineering.

3. Study of the function of regulatory sequences in vitro.

4. Study of genetic mutations in cancer tissues.

CONSTRUCTION OF GENOMIC LIBRARY

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