20 Things You Should Know About Carbohydrates

- Carbohydrates are compounds containing carbon, hydrogen, and oxygen in the ratio (CH₂O)n

- Carbohydrate is a synonym for sugar

- Monosaccharides: simple sugars with multiple OH groups. Based on # of carbons, a monosaccharide is a triose, tetrose, pentose, or hexose. Disaccharide: 2 monosaccharides covalently linked. Oligosaccharides: a few monosaccharides covalently linked. Polysaccharides: polymer consisting of chains of monosaccharide or disaccharide units.

- A monosaccharide can be a aldose (having an aldehyde group at one end) or a ketose (having a keto group, usually at C2)

- Pentoses and hexoses can form rings as ketone or aldehyde reacts with OH group.

- Special type of bond called glycosidic bond joins two carbohydrate molecules: (R-OH + HO-R' ---> R-O-R' + H₂O)

- Condensation (dehydration synthesis) reactions join together smaller sugar molecules to form larger, more complex sugar molecules by forming a glycosidic bond between the smaller sugar molecules resulting in the release of one water molecule per glycosidic bond formed as a product.

- Hydrolysis reactions split complex sugar molecules by adding a water molecule to each glycosidic bond, causing the bond to break and form hydroxyl groups on both product molecules.

- alpha linkage is shaped like "A" or "V", beta linkage is shaped like " / " or " \ "

- Common disaccharides include: maltose [glucose + glucose with a(1→4) glycosidic bond], lactose [galactose + glucose with B(1→4) bond], sucrose [glucose + fructose with a(1→2) bond]

- Plants store glucose in polymer form as amylose or amylopectin, collectively called starch. Glucose storage in polymer form minimizes osmotic effects.

- Amylose is a glucose polymer with a(1→4) linkages. It adopts a helical structure.

- The end of a polysaccharide with an anomeric C1 not involved in a glycosidic bond is called the reducing end.

- Amylopectin is a glucose polymer with mainly a(1→4) linkages, but also has branches formed by a(1→6) linkages. Branches produce a compact structure and provide multiple chain ends at which enzymatic cleavage can occur.

- Glycogen, the glucose storage polymer in animals, is similar to amylopectin, but it has more a(1→6) branches. The highly branched structure allows the rapid release of glucose. The ability to rapidly mobilize glucose is more important in animals than in plants (ex. during strenuous physical activity).

- Cellulose, the material in plant cell walls, consists of long linear chains of glucose with B(1→4) linkages. In cellulose, every other glucose is flipped over due to beta linkages. This promotes intra-chain and inter-chain H-bonds and Van der Waals interactions that cause cellulose chains to be straight and rigid, and pack with a crystalline arrangement in bundles called microfibrils.

- Multisubunit Cellulose Synthase complexes in the plasma membrane produce very strong microfibrils consisting of 36 parallel, interacting cellulose chains. Cellulose gives strength and rigidity to plant cell walls, making them able to withstand high hydrostatic pressure gradients and prevent osmotic swelling.

- Oligosaccharides are sugars that are often covalently attached to proteins or membrane lipids. May be linear or branched chains.

- Lectins are glycoproteins (proteins that contain oligosaccharide chains covalently bonded to polypeptide side chains) that recognize and bind to specific oligosaccharides.

- Selectins are proteins in the plasma membrane with lectin-like domains that protrude on the outer surface of mammalian cells. They are involved in cell-cell recognition and binding.

Section 6.1 - Biotechnological Tools and Techniques + Section 6.2 - Genetic Engineering (text p.278-294)

These 2 sections in our textbook covered 4 important topics: restriction endonucleases, gel electrophoresis, plasmids, and transformation.

Restriction Endonucleases

• Restriction endonucleases (aka restriction enzymes) - enzymes that are able to cleave double-stranded DNA into fragments at specific sequences, known as recognition sites
• Recognition site - a specific sequence within double-stranded DNA, usually palindromic and consisting of 4 to 8 nucleotides, that a restriction enzyme recognizes and cleaves
• Restriction endonucleases produce DNA fragments with both sticky ends and blunt ends.
• Sticky ends - fragment ends of a DNA molecule with short single-stranded overhangs, resulting from cleavage by a restriction enzyme. These are more useful to molecular biologists, since they can be joined more easily to other sticky-end fragments produced by the same restriction endonuclease.
• Blunt ends - fragment end of a DNA molecule that are fully base paired
• Frequency of cuts of a restriction endonuclease depends on the length of their recognition sites. The more base pairs there are in the recognition site, the lower the frequency of cuts.
• Restriction enzymes are produced from bacteria, who use them to defend against the foreign DNA of viruses.

Gel Electrophoresis

• Gel electrophoresis - separation of charged molecules on the basis of size by sorting through a gel meshwork
• Each nucleotide has the same charge-to-mass ratio. The only difference between fragments of DNA of differing lengths is the number of nucleotides.
• Gel electrophoresis is like a molecular sieve. A shorter fragment will travel through the gel faster because it is able to navigate through the pores of the gel more easily. Longer fragments travel more slowly through the gel because they have a harder time moving through the pores of the gel.
• Solution containing DNA fragments are placed in a well in the gel. Gel consists of a buffer containing electrolytes and agarose, or polyacrylamide. Using direct current, a negative charge is placed at the end of the gel where the wells are, and a positive charge is placed at the opposite end of the gel. DNA will migrate down towards the positively charged electrode, with the shorter fragments migrating faster than the longer ones.
• After process is complete, gel is stained. Most commonly used stain is ethidium bromide, a molecule that fluoresces under UV light. Size of fragments can be determined using a molecular marker as a standard. Desired fragments can also be excised out of the gel for further study.
• Also applied to proteins, using polyacrylamide gels because they have smaller pores and proteins are generally smaller in size than nucleic acids.

Plasmids

• Plasmids are small circular pieces of DNA that can exit and enter bacterial cells. Using bacterial enzymes and ribosomes, DNA contained in plasmids can be replicated and expressed.
• Bacteria benefit from presence of plasmids. Plasmids carry genes for antibiotic resistance, resistance to toxic heavy metals, and the ability to break down certain chemicals. Relationship between bacteria and plasmids is endosymbiotic.
• Plasmids have a copy number - the number of copies of a particular plasmid found in a bacterial cell. More copies of a plasmid lead to more protein being synthesized.
• Artificial plasmids may be engineered to contain a multiple-cloning site - a region in the plasmid that has been engineered to contain recognition sites of a number of restriction endonucleases. Recognition sites are present only once, so only one cut can be made in the DNA.
• If foreign gene was excised using the same restriction enzyme, it will have the same complementary ends as the cut plasmid. When placed together, the sticky fragments will anneal. The foreign gene will permanently become part of the plasmid after the phosphodiester bonds are re-established with DNA ligase. Plasmid is now recombinant DNA. It can be introduced into bacterial cells, where it replicates to form many copies, thereby cloning the gene.

Transformation

• Transformation - introduction of foreign DNA, usually by a plasmid or virus, into a bacterial cell
• Plasmids can be used as vectors - vehicles by which DNA may be introduced into host cells
• Competent cell - a cell that readily takes up foreign DNA. Most bacteria are not naturally competent, but can be chemically induced to become so by being treated with a solution of calcium chloride at 0 degC, adding the plasmids, and then subjecting the solution to a quick heat shock treatment at 42 degC for 90 seconds, creating a draft that sweeps the plasmids into the bacterial cells.
• Selective plating is a method used to isolate cells with recombinant DNA. The plasmid vector also contains an antibiotic-resistance gene. The successfully transformed bacteria will be able to grow on media that contains the antibiotic. To check if the gene exists in the transformed bacteria, colonies are grown until enough plasmid DNA can be extracted. Plasmid DNA is subjected to restriction enzyme digestion to release cloned DNA fragment. DNA is put through gel electrophoresis. If expected pattern of bands appears on the gel, then the colony carries the recombinant DNA plasmid with the desired gene.
• New methods of transformation include electroporators and electrical "gene guns".