Chem 330 test 2

Osmotic Pressure

pressure that must be applied to prevent osmotic movement across a selectively permeable membrane

Saponifiable Lipids

(Complex Lipids) Hydrolyzed to yield fatty acids

Higher Temperatures for membranes

(Disordered State): Rapid rotation about C-C single bonds of fatty acid residues prevents tight packing of hydrocarbon chains (high membrane fluidity)

Lower Temperatures for Membranes

(Ordered State): Lower degree of thermal motion allows tighter packing of fatty acid side chains - at sufficiently low temperatures the bilayer becomes a gel-like solid

Non-Saponifiable Lipids

(Simple lipids) Do not give fatty acids upon hydrolysis

Conformations of Fatty Acids

-Saturated -Unsaturated -Essential

Glucose storage polysaccharides

-Starch -Glycogen

Major Classes of Nonsaponfiable Lipids

-Terpenes and terpenoids -Steroids -Eicosanoids

Major Classes of Saponifiable Lipids

-Waxes -Triacylglycerols (Triglycerides) -Glycerophospholipids (Phosphoglycerides) -Sphingolipids

Central Dogma of Molecular Biology

-m-RNA -translation via t-RNA --All these processes rely upon template-directed synthesis in which one strand of DNA or RNA is used as a template (i.e., instructions) for the synthesis of a new molecule (DNA, RNA, or protein) via the base- pairing mechanism

Cholesterol also affects the fluidity of membranes by broadening the transition temperature range

1. Cholesterol intercalates fatty acid side chains near the surface of the membrane, decreasing the overall mobility of lipids within the bilayer. By providing more order between fatty acid side chains near the surface of the membrane, this helps to prevent the membrane from becoming too fluid at higher temperatures. 2. Cholesterol also acts as spacer between phospholipid head groups, resulting in more disorder of fatty acid side chains in the interior portion of the bilayer, which helps to preserve membrane fluidity at lower temperatures.

PCR Cycle

1. Denaturation: Double-stranded DNA is heated to about 95 oC to denature the DNA. 2. Annealing: The temperature is lowered to allow the DNA primers to associate with (anneal to) the complementary strand of DNA. 3. Elongation: DNA polymerase elongates each of the primer strands of DNA.

Determination of Disaccharide Structure - Lactose:

1. Hydrolysis of lactose gives equal amounts of D-glucose and D-galactose. -Lactose is a disaccharide of D-glucose and D-galactose. 2. Lactose is a reducing sugar and undergoes mutarotation. -One monosaccharide unit is a glycoside, but the other is not. The next step in the determination of the structure of lactose is to determine which monosaccharide (glucose or galactose) is the glycoside. 3. Oxidation of lactose with bromine-water followed by acid hydrolysis gives D-galactose and D-gluconic acid -Bromine-water oxidizes aldoses to aldonic acids, but it does not oxidize glycosides. Since the glucose unit of lactose is oxidized to gluconic acid, it must be the subunit that exists in the hemiacetal form. Lactose is an O-glycoside of D- galactose with glucose as the alcohol portion of the glycoside. -The next step is to determine whether galactose is in the pyranose (left) or furanose (right) form 4. Complete methylation of lactose followed by hydrolysis gives the methylated sugar derivatives shown below. Galactose must be in the pyranose form since the hydroxyl group at position 5 of galactose is not methylated in the reaction products - if galactose was in the furanose form, 2,3,5,6-tetra-O-methyl-D-galactose would be formed. This experiment also gives some information concerning the glycosidic bond to the glucose subunit. The observation that hydroxyl groups at positions 4 and 5 of the glucose unit are not methylated via the reaction sequence leads to two possible structures for lactose: a. The glucose unit in the O-methylglycoside is in the pyranose form and the glycosidic bond is attached to C-4 of the glucose unit (below left). b. The glucose unit in the O-methylglycoside is in the furanose form and the glycosidic bond is attached to C-5 of the glucose unit (below left). 5. Methylation of lactobionic acid (product step 3 (above)) followed by hydrolysis gives the methylated sugar derivatives shown below. This experiment shows that the glycosidic bond to the glucose unit is to the hydroxyl group at C-4 - the linkage between galactose and glucose in lactose is a (1,4)-glycosidic bond 6. Determination of the stereochemistry of glycosidic bonds is most easily determined by enzymatic hydrolysis - some enzymes are very specific towards hydrolysis of β-linkages, others are very specific towards hydrolysis of α-linkages For lactose, hydrolysis is catalyzed by β-galactosidases, but not by α-galactosidases. The linkage between galactose and glucose in lactose is a β (1,4)-glycosidic bond

Experiment: Fluorescence Recover after Photobleaching (FRAP)

1. Phospholipids tagged with fluorescent molecules are incorporated into a lipid bilayer. 2. A short pulse of intense laser light used to destroy the fluorescent tags in a small region of the bilayer. 3. Redevelopment of fluorescence in the photobleached region is monitored FRAP and other studies have determined that diffusion rates for lipids in bilayers can be very large. Similar experiments have shown that membrane proteins are also mobile within the lipid bilayer. It is also important to note that while lateral motion of membrane lipids can occur at a very rapid rate, transverse motion (lipid molecules 'flipping' from one side of the bilayer to the other) occurs only very slowly (t1/2 ~ days) in the absence of specific proteins (flippases) that catalyze the process.

Basic steps of DNA replication

1. RNApol binds to DNA and synthesizes a short section of RNA (primer). 2. DNApol binds to the DNA-RNA complex and extends the growing polynucleotide chain with deoxynucleotides. 3. The RNA primer is excised and replaced with DNA 4. Under natural conditions, double-stranded parent DNA does not dissociate into two strands of single- stranded DNA prior to replication as implied in the above figures. The two strands of daughter DNA are synthesized simultaneously with the strands of parent DNA being separated as new double-stranded DNA is formed. Since DNApol can only synthesize DNA in the 5' to 3' direction, only one strand of daughter DNA can be synthesized continuously in this direction (leading strand). The other strand (lagging strand) must be synthesized discontinuously

The basic structure of amylopectin can be determined using the results of the above data.

1. Since the major product is 2,3,6-tri-O-methylglucose, most of the glucose units are connected by (1→4) glycosidic bonds. Finding maltose as the major disaccharide from partial hydrolysis indicates these glycosidic bonds have the α configuration. 2. Since 2,3,4,6-tetra-O-methylglucose is formed from glucose units at the non-reducing ends of polymer chains (above), finding 4-5% of this compound after methylation and hydrolysis indicates that the average length of linear chains in amylopectin is 20-25 glucose units. 3. The 2,3-di-O-methyl glucose isolated after methylation and hydrolysis are those that act as branch points in the amylopectin molecule (i.e., are bound to three other glucose molecules). From the methylation pattern, the branches occur via formation of glycosidic bonds to carbon 6 of these glucose units; other studies show that these glycosidic bonds have the α configuration. Overall, amylopectin contains chains of 20-25 glucose units connected by α(1→4) glycosidic bonds. These chains are interconnected by α(1→6) glycosidic bonds

Stearic Acid

18:0

Oleic Acid

18:1n-9

DNA Sugar

2'-D-Deoxyribose

Oligosaccharides

A few" (typically 3 to 10) monosaccharide units linked by glycosidic bonds. Some oligosaccharides are naturally found in the "free" form, but very often are covalently bound to protein (glycoprotein) or lipid (glycolipid) molecules (bioconjugates).

Fluorescent Dyes

A number of dyes have been developed that will fluoresce strongly only when intercalated with DNA (i.e., specifically detect double stranded DNA).

Gangliosides

Acidic Glycosphingolipids Similar in structure to above, but contain more complex carbohydrate head groups, which include, in mammals, N-acetylneuraminic acid

Purines

Adenine and Guanine

DNA Bases

Adenine, Thymine, Guanine, Cytosine

RNA Bases

Adenine, Uracil, Cytosine, Guanine

Detection of DNA

After DNA is separated by electrophoresis, some method must be employed to visualize the DNA molecules.

Micelles vs. Lipid Bilayers

Amphipathic molecules with one hydrocarbon tail group tend to form micelles - with a single hydrocarbon tail, both the head groups (surface of micelle) and the tail groups (interior of micelle) can pack closely together Amphipathic molecules with two hydrocarbon tail groups (e.g., phospholipids) tend to form bilayer vesicles such as liposomes. Since amphipathic molecules with two tail groups are much wider at their nonpolar ends, they cannot form a micelle structure where both the head groups and tail groups are tightly packed. For example, tight packing of head groups would leave an empty cavity (unstable) or a pocket of water (also unstable) at the core of a micelle. Formation of bilayer vesicles allows tight packing of both head groups and tail groups with both head group surfaces in contact with aqueous solution. Amphipathic substances with a single tail group, such as soaps and detergents, disrupt lipid bilayer structures and can lead to their degradation at sufficient concentration

Conformations of RNA

As with peptide chains in proteins, certain types of RNA fold into specific 3D structures. 1. Single-stranded RNA forms a right-handed helix with stacking interactions between bases (figure 8-22 in text). 2. Complementary sequences of bases within a single strand of RNA can base pair to form regions of double-helical structure

Gel Electrophoresis

As with proteins, gel electrophoresis is an important method for separating nucleic acids by molecular weight, but there are a few differences. One major difference is that for the separation of proteins by size, SDS (sodium docecyl sulfate) must be added to give the different peptides approximately the same mass-to-charge ratio. However, with DNA this is not necessary because DNA's of different size have naturally about the same mass-to charge ratio. In a buffer of about pH 7, every nucleotide residue contains one negatively-charged phosphate group (two negative charges per base pair for double-stranded DNA). Because the two purines and the two pyrimidines have similar molecular weights, and, for double-stranded DNA, the number of purines equals the number of pyrimidines, DNA molecules of different sizes have approximately the same mass-to-charge ratio. Therefore, with DNA no additional treatment is required to give separation on the basis of size (with smaller DNA's migrating at faster rates). The two most common materials used to prepare gels for the separation of DNA are agarose and polyacrylamide. Agarose is a linear polysaccharide isolated from certain species of seaweed. Agarose gels do not have a uniform pore size. The variability in pore size for agarose gels allows for separation of DNA's over a wide range of size (50-20,000 bp), but resolution of molecules of similar size is relatively poor. Polyacrylamide is a synthetic polymer prepared from acrylamide with bisacrylamide to give a gel with reasonably uniform pore. Using polyacrylamide gels it is possible to separate small DNA's (5-500 bp) with a level of resolution that allows for separation of DNA fragments that differ in size by a single nucleotide.

Peripheral Membrane Proteins:

Associated with the face of a membrane via noncovalent interactions with lipid head groups (ionic attraction and/or hydrogen bonding). These proteins can usually be separated from the membrane by mild extraction with agents that disrupt hydrogen bonding (various salts, EDTA, urea). In most cases these proteins are localized to a specific face of the membrane (i.e., some are found only on the inner side of the membrane while others are only on the outer face)

Ribonucleic Acid - RNA: Linear Polymer of Ribonucleotides

Backbone of RNA contains alternating ribose and phosphate groups - phosphate groups link the 3' and 5' positions of ribose subunits via phosphodiester bonds - strands of RNA are directional (5'-end and 3'-end). Naturally-occurring RNA is usually single-stranded; various classes of RNA molecules range in size from about 20 to several thousand nucleotides. Most types of RNA contain primarily adenosine, guanosine, cytidine and uridine residues (exception: transfer-RNA's (tRNA) contain a fairly high percentage of other nucleotides)

Sphingolipids

Based on sphingosine or related long-chain amino alcohol; second most abundant class of lipids in plant and animal membranes. Note that the C=C double bond in sphingosine has the trans configuration. Ceramide: Sphingosine with a fatty acid residue attached via an amide bond at position 2; the fatty acid group is usually large (18-26 carbons) and is either unsaturated or mono unsaturated -Different classes of sphingolipids differ in the structure of their head groups. Sphingolipids are amphipathic molecules with charged or polar head group and two nonpolar tails -Sphingomyelin -Neutral Glycosphingolipids -Gangliosides

Deoxyribonucleosides and deoxyribonucleotides

Basic structures are analogous to those of ribonucleosides and ribonucleotides with the exception that the sugar unit is 2'-deoxyribose

Lipid Rafts

Because sphingolipids contain a larger proportion of unsaturated and monounsaturated hydrocarbon tail groups than glycerophopholipids, cholesterol tends to associate more tightly with sphingolipids. This "clumping" of sphingolipids and cholesterol results in the formation of microdomains with lower membrane fluidity than other regions of the membrane. The average length of the hydrocarbon tail groups in sphingolipids also tends to be longer than for glycerophospholipids so the membrane is also somewhat thicker in these regions. These lipid rafts can then "float" through areas of the membranes with larger proportions of glycerophospholipids. Lipid rafts usually do not appear to be permanent structures, but rather are continuously being formed and degraded. Some proteins appear to be preferentially localized to lipid rafts, especially those anchored to the membrane by two saturated fatty acid groups

RNA Polymerase (RNAP)

Catalyzes synthesis of an RNA chain from a DNA template in analogous fashion to DNApol (e.g., adds nucleotides to 3' end of growing strand), but unlike DNApol, it can initiate synthesis of RNA from single-stranded DNA

Plasmalogens

Class of glycerophospholipids in which the hydrocarbon group at position 1 attached via an enol ether linkage (usually trans) rather than by an ester bond (R1 and R2 are both long hydrocarbon chains) - ethanolamine (shown below) and choline are the most common head groups. In humans, these compounds are found abundantly in the central nervous system.

Amylose

Complete hydrolysis of amylose gives only D-glucose. Upon partial hydrolysis, the only disaccharide isolated is maltose. Amylose is a linear polymer, with typical chain lengths 300-3000 glucose units, in which all glucose units are linked by α(1→4) glycosidic bonds. Amylose chains form helical structures (see structures at end of section).

Glycosphingolipids

Components of cell membranes

Steroids

Compounds containing the characteristic four-ring system shown below.

Neutral Glycosphingolipids

Contain an uncharged carbohydrate, attached via a β-glycosidic bond, as head group. Cerebrosides - Contain a single monosaccharide unit (usually galactose or glucose) as a head group. Other neutral glycosphingolipids contain head groups of up to four monosaccharide units in linear arrangement

Renaturation

Conversion of single-stranded DNA to double helical DNA. If two strands of DNA are not completely separated (incomplete denaturation), the native double helix structure usually reforms rapidly when the sample is cooled. -When denatured DNA is rapidly cooled to temperatures far below Tm, the native double helical structure is generally not formed. Small sections of DNA with complementary sequences associate via base pairing, but at low temperatures are locked into non-native conformations. When denatured DNA is cooled slowly to about 25oC below Tm, native double helical DNA forms; any improperly matched segments of DNA can dissociate and re-associate until base pairing is maximized in the native structure.

RNA Sugar

D-Ribose

Watson-Crick Double Helix Structure of DNA (B-DNA):

DNA Molecule contains two strands of DNA running in opposite (antiparallel) directions. One strand runs in 5' to 3' direction Opposing strand runs in 3' to 5' direction -The diameter of the double helix is about 20 A. The sugar phosphate backbone of DNA strands forms the outside edges of the helix - electrostatic repulsion between negatively charged phosphate groups is minimized, and phosphate groups can hydrogen bond with water. Base pairs occupy the core of the helix and are situated nearly perpendicular to the axis of the helix. -The pitch (rise per turn) of B-DNA is 36 A and each turn of the helix contains about 10.5 base pairs - separation of base pairs is around 3.4 A (base pairs are closely stacked).

Overview of functions of nucleic acids

DNA encodes the information required for the development and functioning of all known organisms (and many viruses), and in conjunction with RNA, provides for the synthesis of proteins molecules that mediate cellular functions.

Reaction of DNApol

DNApol catalyzes formation of a phosphodiester bonds between the α-phosphate group of deoxynucleoside 5'-triphosphate and the 3' hydroxyl group of a growing nucleotide chain. a. The new strand of DNA is only synthesized in the 5' to 3' direction - DNApol "reads" the template strand in the 3' to 5' direction. b. Incorporation of the correct nucleotide on the growing strand of DNA is determined by Watson-Crick base pairing. c. DNApol cannot initiate formation of double-stranded DNA from single-stranded DNA alone - it can only extend a growing polynucleotide chain attached to the DNA template.

DNA Fingerprinting

Different individuals of the same species (except identical twins) have slightly different DNA sequences. In humans it is estimated that any two person's genomes will differ by one base pair per 1250 base pairs - well over 10^6 different base pairs in the entire genome. The change of one base pair from one individual to another may create or eliminate a given restriction site. If so, the restriction enzyme specific for that sequence will produce different sized DNA fragments from the two individuals. Example: Two individuals have two restriction sites at the same position of a given chromosome, but B has a third restriction site between the other two. Differences in the pattern of sizes of DNA fragments produced by that restriction enzyme are easily determined by gel electrophoresis (right).

Hyaluronic Acid

Disaccharide: D-Glucuronic acid with β(1→3) glycosidic linkage to N-acetyl-D-glucosamine. Disaccharide units linked by β(1→4) glycosidic bonds. Typically contains 250-25,000 disaccharide units - every other monosaccharide units contain a group that is negatively-charged at neutral pH.

Maltose and cellobiose

Disaccharides of two glucose units linked by (1→4) glycosidic bonds; the only difference in structure of these two compounds is the stereochemistry of glycosidic bond -Maltose and cellobiose are both reducing sugars - each has one glucose unit in the hemiacetal form.

Cycle 1 PCR

Each individual strand of the original DNA ("full length" DNA (FL-DNA)) is used as a template for the synthesis of a complementary strand of DNA. These strands of DNA ("random length" DNA (RL-DNA)) are shorter than the original strands as the primers do not anneal to the 3'-ends of FL-DNA, but are longer than the desired section of DNA.

Cycle 2 PCR

Each strand of double-stranded DNA from cycle 1, containing one strand of FL-DNA and one strand of RL-DNA, undergoes the same denaturation, annealing and elongation steps of cycle 1. As shown above for one of the double-stranded products of cycle 1, the FL-DNA strand gives another FL/RL double strand (same as in cycle 1). However, the new strand of DNA formed using the RL strand from cycle 1 as the template is limited in length by the positions of the primers in both cycles 1 and 2 - gives formation of a DNA strand of the desired length ('unit-length" DNA (UL-DNA)). The UL-DNA strand complementary to the one shown in the above diagram will be formed from the other FL/RL double strand from cycle 1.

Waxes

Esters of fatty acids and large, nonpolar alcohols Beeswax: Mixture of waxes with mp > 60 oC; used to construct honeycomb Waxes often serve protective functions in plants and animals Plants: Wax coating on leaves inhibits evaporation of water Birds and Mammals: Waxes on feathers/fur acts as water repellent

Integral Membrane Proteins

Estimated that on the order of 20% of the proteins expressed by a cell fall into this category. Large portions of the protein are embedded in the hydrophobic core of the membrane, in many cases spanning the entire bilayer. Due to the very strong association between the protein and membrane, drastic disruption of membrane structure (e.g., with detergents or organic solvents) is required to separate these proteins from the lipid bilayer, and many are inactivated when removed from the membrane. The folding pattern of integral membrane proteins is quite different from cytosolic proteins in which hydrophobic groups are largely localized to the interior of the protein in the native state. Incorporation of the protein into the hydrophobic interior of the membrane requires large numbers of nonpolar amino acid side chains at the membrane surface. It is also found that the aromatic amino acids tyrosine and tryptophan occur in rather high proportion at the surface of the membrane. The orientation of transmembrane proteins is directional

Cycles 3, 4, 5... PCR

FL and RL strands will produce RL and UL strands as shown above, but once formed, the UL strands will only form additional UL strands (i.e, the primers will now bind at the 3' ends of these strands).

Triacylglycerols (Triglycerides)

Fatty Acid Triesters of Glycerol The three fatty acids residues may be identical or different. Triacylglycerols can be hydrolyzed with aqueous acid or aqueous base* to yield glycerol and three equivalents of fatty acid (ester hydrolysis). Biochemically, degradation of triacylglycerols is accomplished by lipases - enzymes that catalyze the hydrolysis of ester linkages. *Soap: Sodium salts of fatty acids made by boiling animal fat with lye (NaOH) - saponification. Triacylglycerols are major components of fats and oils. In the strict sense, fats are solid or semisolid at room temperature while oils are liquid at room temperature (some fats from plants are commonly referred to as "vegetable oils" even though they are solid at room temperature). The average molecular weight of the fatty acid residues makes some contribution to the melting temperature of triacylglycerols, but the major factor is the ratio of saturated to unsaturated fatty acid groups in the substance; as with the fatty acids, melting temperature decreases with the number of cis-double bonds. Typical animal fats contain 40-50% saturated fatty acid residues with fairly small amounts of the polyunsaturated variety. Many vegetable oils contain only 10-15% saturated compounds, but up to 70% polyunsaturated fatty acids

Functions of Triacylglycerols

Fatty acids can be oxidized for production of ATP, and a major function of triacylglycerols is storage of fatty acids as energy reserves (below), but they can also serve other specialized purposes. For example, the bodies of many marine mammals are covered in the fat-rich substance blubber. The blubber layer provides insulation from cold environments (poor thermal conductor) and helps to modulate the buoyancy of the animal (less dense than water). Triacylglycerols - Highly efficient compound for storage of biochemical energy reserves. 1. The average oxidation state of carbon in fatty acids is lower than for carbohydrates and proteins - more energy is available upon oxidation. 2. In animals, glucose is stored as glycogen, a large polymer of glucose monomers. Due to the large number of hydroxyl groups in glucose, glycogen is very hydrophilic and binds about two- times its mass in water under physiological conditions. On the other hand, triacylglycerides are nonpolar (hydrophobic) and thus exclude water (anhydrous). Overall, triacyglycerols can provide about six-times more metabolic energy per gram than hydrated glycogen

Structure of Biological Membranes

Fluid Mosaic Model A lipid bilayer composed primarily of phospholipids (phosphoglycerides and sphingomyelins) provides the framework for the membrane. The two layers of phospholipids are arranged in a tail-to-tail fashion so that the head groups (charged or polar) are in contact with the aqueous environment. The interior of the membrane is hydrophobic. The plasma membrane in animals also contains a high proportion of cholesterol. In addition to lipids, biological membranes contain large amounts of protein, which varies in amount depending on the function of the membrane. Inner Mitochondrial Membrane: 75-80% Protein Myelin Membrane (Nerve Cells): Up to 75% Lipid

Recognition Sequences - Palindromes

For most restriction enzymes recognition sequences are short segments of DNA (often 4 to 6 base pairs) in which the two complementary strands of DNA have the same sequence of bases in the 3'-to-5' direction. Some restriction enzymes (e.g., EcoRV) cleave at the center of the recognition sequence to yield "blunt-end" DNA fragments. Others (e.g., EcoRI) cleave at specific off-center sites, which give DNA fragments with "sticky ends".

Glycogen

Glucose storage polymer in animals, fungi and bacteria. Bonding is analogous to that of amylopectin, but with a more highly branched structure; chain lengths of 10-12 glucose and branch points about every 6 glucose units. In animals, glycogen particles can contain >10^5 glucose surrounding a protein (glycogenin) core

Glycerophospholipids amphipathic molecules

Glycerophospholipids are amphipathic molecules - molecules with regions of opposite behavior with respect to their solubility properties in water. Head Group: Charged or Zwitterionic at pH 7 - can form strong electrostatic and/or hydrogen bonding interactions with water molecules (hydrophilic). Fatty Acid Groups: The long hydrocarbon chains of the two fatty acid moieties cannot form strong intermolecular interactions with water molecules (hydrophobic)

Hydrocarbon Tails of Lipid Bilayer

Glycerophospholipids usually contain one saturated and one unsaturated fatty acid group (mono- or poly-unsaturated) whereas sphingomyelins usually contain a saturated or monounsaturated fatty acid group along with the saturated hydrocarbon chain of shpingosine. As a result, outer layer of the membrane (higher percentage of sphingomyelin) contains a larger proportion of tail groups with unsaturated chains which provide less fluidity to that layer

Lipids

Group of structurally-diverse, water-insoluble biomolecules; can be extracted from cells using organic solvents of low polarity (e.g., diethyl ether or chloroform (CHCl3)). Lipids are traditionally divided into two major classes based on their hydrolysis products

Determination of Gylcerophospholipids Structure

Hydrolysis under acidic conditions cleaves all carboxylate and phosphate ester bonds (A-D), while hydrolysis in aqueous base cleaves only the carboxylate ester bonds (A and B; see problem set). Phospholipases, enzymes that catalyze hydrolysis of ester linkages in glycerophospholipids, can be used to cleave specific bonds -phospholipases A1, A2, C, and D catalyze the hydrolysis at the specific sites as shown below; phospholipase B cleaves both fatty ester bonds (A1 and A2)

Radioactivity

In some techniques (e.g., sequencing of DNA), the DNA sample is prepared with the incorporation of a radioactive isotope (most often 32 P in the phosphate group) which can be detected via radioactive emission (autoradiogram).

Cellulose

Like amylose, cellulose is a linear polymer of D-glucose units linked by (1→4) glycosidic bonds. The difference in their structure is in the stereochemical configuration of the glycosidic bonds - in amylose the glucose units are linked by α(1→4) glycosidic bonds, while in cellulose the glycosidic bonds are β(1→4) linkages (see structure of the disaccharide cellobiose). The number of glucose units in cellulose molecules varies from several hundred to thousands depending on the source. The difference in the glycosidic bonds in amylose and cellulose results in different shapes and properties of the molecules. The α(1→4) glycosidic bonds of amylose allow the molecule to adopt a helical structure, which is soluble in water. Cellulose molecules form extended chains which associate with one another through hydrogen bonding to give a strong, fibrous material which is insoluble in water. Cellulose microfibrils are an important component of the cell walls of plants.

Fatty Acids

Long-chain carboxylic acids; more than 100 have been isolated and identified from natural sources; several common fatty acids are listed below. In the above system for describing the structure of fatty acids, the first number gives the number of carbons in the molecule and the second the number of C=C double bonds; the superscripted numbers refer to the position(s) of the C=C bond(s) (the carboxylate carbon is position 1) - all C=C bonds have the cis-configuration unless otherwise noted.

Fluidity of Membranes

Maintaining the proper fluidity of membranes is essential to proper functioning of cells and subcellular components - the fluidity of lipid bilayers varies with temperature

Aggrecan

Major proteoglycan component of cartilage The central region of aggrecan (the core protein in the figure shown below) is an extended chain that covalently binds about 100 chondroitin sulfate chains with up to 1000 disaccharide units each and about 30 chains of keratan sulfate with up to 250 disaccharide units each. Overall, carbohydrate groups account for nearly 90% of the total mass of the proteoglycan. Aggrecans associate non-covalently strands of hyaluronic acid via link proteins at regularly spaced intervals (200-300 Å). Large hyaluronic acid molecules can bind with up to about 100 aggrecan molecules to form what is often called a "bottle brush" type of structure.

Restriction Endonucleases

Many methods involved in the characterization of DNA require large DNA molecules to be cleaved into smaller pieces. There are a number of chemical reagents that will cleave DNA, but these are not particularly useful as their action is non-specific (i.e., cleave at random positions). However, cleavage at well-defined positions can be accomplished using restriction endonucleases. Restriction Endonucleases (Restriction Enzymes): Bacterial enzymes that cleave double- stranded DNA (both strands) at an internal position of the polynucleotide chain as a mechanism for protection against from viral infections. These enzymes can recognize certain specific sequences of base pairs in viral DNA, which initiates cleavage of that strand of DNA. Bacterial DNA is protected from cleavage by methylation of specific nucleotide bases within the recognition sequence (i.e., bacteria mark their own DNA to distinguish it from foreign DNA). Some restriction enzymes cleave DNA at sites remote from the recognition sequence (in some cases more than 1000 bp's distance), but others cleave at the recognition sequence - the remaining discussion focuses on these enzymes.

Polysaccharides

Many monosaccharide units (tens to thousands) linked by glycosidic bonds

Melting points of Fatty Acids

Melting points of cis-monounsaturated fatty acids are typically more than 50oC lower than saturated fatty acid with the same number of carbons; cis-conformation of the C=C bond impedes tight packing of hydrocarbon chains in the solid state. The melting points of fatty acids decrease with number of cis-C=C bonds

Starch

Mixture of polysaccharides in plants containing a water-soluble component (amylose) and a water-insoluble compound (amylopectin).

Glycerophospholipids (Phosphoglycerides):

Most abundant type of lipid in cell membranes. -As with triacylglycerols, glycerol and fatty acids are components of glycerophospholipids, but rather than having three fatty acid residues, glycerophospholipids bear only two fatty acid groups. -The third hydroxyl group of glycerol forms a phosphate ester bond - this group may be a phosphate monoester or diester (see below). The acidity of the phosphate group is sufficient that it will be ~100% ionized (with a net negative charge) at pH 7. -Fatty Acid Groups: Position 1 (R2): Usually a saturated fatty acid residue Position 2 (R1): Usually an unsaturated acid residue (mono- or poly-unsaturated) -Head Groups: Different classes of glycerophospholipids are distinguished by the group attached to the phosphate moiety of the compound - a few examples are given below (at pH 7).

Amylopectin

Much larger than amylose - can contain over 10^5 glucose units. Partial hydrolysis of amylopectin gives maltose as the major disaccharide product, indicating that most glucose units are linked by α(1→4) glycosidic bonds, but, unlike amylose, it is not a linear polymer. Complete methylation of amylopectin followed by hydrolysis gives three methylated glucose derivatives in the amounts given below If amylopectin was simply a large linear polymer, ~100% tri-O-methylglucose would be obtained. In a linear chain, the only glucose unit that could form 2,3,4,6-tetra-O-methylglucose would be the one at the non-reducing end of the chain (bonded to only one other glucose via its anomeric carbon). For example, a linear chain of 10,000 glucose units would yield one molecule of tetra-O-methylglucose (0.01%) and 9,999 molecules of tri-O-methylglucose.

Membrane Proteins

On average, about 50-60% of the mass of the membrane - proteins may be either tightly or loosely associated with the lipid bilayer. --Peripheral Membrane Proteins --Integral Membrane Proteins --Lipid-Anchored Membrane Proteins

Chondroitin 4-Sulfate

Other GAGs usually contain 50-1000 disaccharide units per chain. D-Glucuronic acid with β(1→3) glycosidic linkage to N-acetyl-D-galatosamine 4-sulfate. Disaccharide units linked by β(1→4) glycosidic bonds.

Bacterial Cell Walls - Peptidoglycans

Polysaccharide chain consists of alternating N-acetylglucosamine and N- acetylmuramic acid units. Each muramic acid unit is attached to a short chain of amino acids (peptide) via an amide bond. Cross-linking of the peptide chains forms a large sack-like molecule that encompasses the entire cell. Bacterial cell walls are a good target for organisms to protect themselves against bacterial infections.

Saturated Fatty Acids

Prefer extended conformation (all C-C bonds in the anti-conformation)

Lysozyme

Protective enzyme found in a number of extracellular secretions in animals (e.g., tears, saliva, mucus, milk) and in egg whites - catalyzes hydrolysis of β(1→4) glycosidic bonds between NAM and NAG units of bacterial cell walls

Lipid-Anchored Membrane Proteins

Proteins covalently bonded to one or more hydrocarbon chain that can be embedded in the nonpolar region of the membrane. In some cases, the protein is attached to the head group of a phospholipid. In others, a fatty acid group or isoprenoid chain (terpene) is attached to a group on the protein. Fatty Acids: Myristic Acid (14:0) - Usually linked via amide bond to amino group of an N-terminal glycine. Palmitic Acid (16:0) - Usually linked via thioester bond to a thiol group of cysteine. Isoprenoid Chains - Linked via sulfide bond to cysteine

Proteoglycans

Proteins covalently bound to at least one GAG chain. In some cases, proteoglycans associate with other GAGs and/or proteins to form complex structures.

Nitrogenous Bases

Purines and Pyrimidines

Type O

R=H

Type A

R=N-Acetyl-D-galactosamine

Ribozymes (RNA Enzymes)

RNA molecules that catalyze chemical reactions, most often those involved in RNA metabolism and protein synthesis (below right).

Plasma Membrane

Separates the contents of the cell from the external environment while allowing for selective passage of specific compounds in and out of the cell. Eukaryotic cells contain numerous membrane-enclosed subcellular structures that perform specialized functions - for example, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and chloroplasts (some structures (e.g., mitochondria) contain multiple membranes)

Differences in Structure of DNA and RNA

Since DNA contains the coded instructions for all process carried out by a cell, it is important that the cell stringently preserve its structure. On the other hand, the various types of RNA are "turned over" (synthesized, used, and decomposed) on a regular basis.

Explanation of Sanger Dideoxy Method

Since the concentration of the dideoxynucleotides is much lower than the deoxynucleotides, most of the DNA strand formed will contain only deoxynucleotides. Occasionally, however, a dideoxynucleotide nucleotide will be randomly added to the growing strand of DNA. Since the dideoxynucleotide does not have a hydroxyl group at the 3'-position, synthesis of that strand of DNA is terminated at that point. Consider the sample to which ddATP was added - in addition to forming a full double-strand of DNA, the following two partially double-stranded DNA's will also be formed (A* at the 3'-end of each of these strands is the dideoxynucleotide ddATP) Since smaller pieces of DNA run faster on PAGE, the DNA fragment on the left (5'-A-T) will appear near the bottom of the autoradiogram while the fragment on the right appears near the top. Likewise, the sample to which ddGTP was added will give two partially double-stranded DNA's. Overall, reading the pattern of bands starting from the bottom of the gel gives the sequence of bases of the strand of DNA formed during the sequencing reaction (5'-to-3' direction). The sequence of the original piece of single-stranded DNA can be determined using rules of base pairing. A modification of this technique is used in automated DNA sequencers. Rather than using radioactivity for detection, the dideoxynucleotides are labeled with fluorescent dyes - each different base is tagged with a dye that fluoresces at a different wavelength (different color). This allows all four of the dideoxynucleotides to be reacted simultaneously with the analyte DNA (i.e., rather than running four separate samples). At the end of the reaction the DNA sample is separated by capillary gel electrophoresis with a fluorescence detector that identifies the base at the 3'-end of each differently-sized DNA by its color. Automated systems can sequence segments of DNA of over 1000 nucleotides at a rate of ~19,000 bases per hour.

Deoxyribonucleic Acid - DNA: Linear Polymer of Deoxyribonucleotides

Single-stranded DNA has the same basic bonding pattern as RNA - alternating sugar and phosphate groups with phosphate groups linking the 3' and 5' positions of sugar subunits via phosphodiester bonds.

Plasma Membrane of Human Erythrocyte

Spingomyelin: Inner Layer < 5% Outer Layer ~40% Phosphatidylcholine: Inner Layer ~15% Outer Layer ~40% Phosphatidylethanolamine: Inner Layer ~50% Outer Layer ~13% Phosphatidylserine: Inner Layer ~30% Outer Layer < 5%

Why store glucose as a polymeric molecule?

Storage of glucose in polymeric form prevents osmotic pressure damage to cells. Osmotic pressure ( ) is a colligative property of solutions - depends on the number of particles dissolved (II= MRT, were M = molarity). Combining hundreds to thousands of individual glucose units into a single molecule decreases the osmotic pressure several orders of magnitude

Function of Glucose Storage Polysaccharides

Storage of glucose in polymeric form prevents osmotic pressure damage to cells. Osmotic pressure () is a colligative property of solutions - depends on the number of particles dissolved ( = MRT, were M = molarity). Combining hundreds to thousands of individual glucos units into a single molecule decreases the osmotic pressure several orders of magnitude

Transcription

Synthesis of a strand of messenger RNA (mRNA) complimentary to a portion of one strand of a DNA molecule

Replication

Synthesis of an exact copy of a DNA molecule

Translation

Synthesis of protein from the instructions encoded in mRNA - involves interactions between mRNA, ribosomal RNA (rRNA; component of ribosomes) and transfer RNA (tRNA)

Polymerase Chain Reaction (PCR)

Technique used to produce multiple copies of a specific segment of DNA. Synthesize two DNA primers, one complementary to the 3'-end of each DNA strand flanking the DNA section interest. The double-stranded DNA is mixed with the DNA primers, the four deoxyribonucleoside triphosphates, and a heat-stable DNA polymerase* in a thermocycler (increases and decreases the temperature at pre-set time/temperature intervals). *Most proteins are irreversibly denatured at the temperatures used in PCR, but certain thermophilic bacteria (which thrive at high temperatures) produce DNA polymerases that are stable at 95oC.

Terpenes and Terpenoids

Terpenes are hydrocarbons that are biochemically-synthesized from two or more units of the five-carbon precursor isopentenyl diphosphate (isoprene units); the number of carbon atoms in terpenes is always a multiple of 5. Terpenoids are also formed from isopentenyl diphosphate (with number of carbon atoms equal to a multiple of 5), but also contain oxygen. Terpenes and terpenoids have a wide variety of structures as shown below in three examples of monoterpene/monoterpenoid (10 carbon) compounds.

Monounsaturated Fatty Acids

The C=C is most often between carbons 9 and 10

Sanger Dideoxy Method

The Sanger method is used to sequence relatively short pieces of single-stranded DNA (less than 1000 bases). This requires cleavage of larger DNA molecules (e.g., using restriction enzymes) followed by denaturation and separation of single-stranded DNA

Lipid Bilayer

The framework of the membrane - composed primarily of phospholipids (phosphoglycerides and sphingomyelins), but often contains significant amounts of other sphingolipids, especially in specialized membranes. Cholesterol is also a major component of the plasma membranes in animals (≥ 20% of lipid). In most biological membranes the inner and outer layers of lipid bilayer have different lipid compositions The asymmetric distribution of lipids in the two layers of bilayer can impart differences in the physical properties of the inner and outer layers of lipid bilayer as described below for the erythrocyte plasma membrane

Head Groups of Lipid Bilayer

The head group of phosphatidylserine is anionic while head groups with choline and ethanolamine (includes most sphingomyelins) are zwitterionic at pH 7; the inner layer has a much higher proportion of negatively-charged groups at the membrane surface

Biological membranes are Dynamic Structures (Fluid Mosaic Model)

The lipid bilayer of membranes is not a rigid structure - weak interactions between hydrocarbon tails allow components of membranes, both lipid molecules and proteins, to diffuse in the lateral direction of the bilayer. The lipid bilayer behaves as a two-dimensional fluid, which is essential to proper functioning of cells and subcellular components.

Transition Temperature ("Melting" Temperature)

The midpoint of the temperature range over which a lipid bilayer changes from its ordered state (gel - low temp) to its disordered state (fluid - high temp) The transition temperature of a membrane is largely dependent on the fatty acid composition of the bilayer lipids. A major determinant of transition temperature is the percentage of unsaturated fatty acid residues in the lipid bilayer. Since the cis-conformation of C=C bonds in unsaturated fatty acid residues impedes tight packing of hydrocarbon side chains, larger amounts of unsaturated groups decrease the transition temperature.

Permeability of Biological Membranes

The nonpolar interior of the lipid bilayer restricts the types of molecules that can pass through the membrane. Nonpolar Compounds: Can diffuse through the lipid bilayer - sizes of molecules range from small gases (O2 and CO2) to certain steroid hormones (mw > 300 D). Polar and Ionic Compounds: Impermeable to the lipid bilayer - transport across the membrane relies upon membrane proteins. Although in very few cases (e.g., the outer mitochondrial membrane) proteins form large non- selective pores through which molecules smaller than the pore size can freely diffuse, membrane transport proteins typically have a high selectivity towards the substance transported. As an example, the potassium ion (K+) channel protein allows diffusion of K+ at a rate about 10,000 times faster than for Na+

Modifications to Fluid Mosaic Model

The original fluid mosaic model supposes a random distribution of membrane components, but recent studies show that some regions of the membrane have higher degree of order than others. -Lipid Rafts

Charge of Gycerophospholipids

The overall charge of the lipid depends on the group at this position (the head group). For example, the head group of phosphatidylcholine contains equal numbers of positive and negative charges, so is electrically neutral (zwiterionic) at pH 7, while phosphatidylserine bears a net negative charge at this pH

Chitin

The structure of chitin, a polysaccharide component of the exoskeletons of insects and crustaceans, is analogous to cellulose (i.e., only β(1→4) glycosidic bonds), but rather than being composed of glucose, it is synthesized from the glucose derivative N-acetylglucosamine (2-(acetylamino)-2-deoxy-D-glucose). The amide groups provide for stronger hydrogen bonding between polysaccharide chains than in the cellulose

Disaccharide

Two monosaccharides linked via a glycosidic bond (acetal functional group)

Characteristics of Common Fatty Acids in Mammals

Unbranched compounds with an even number of carbon atoms (16-22 most common). In most cases, the only functional group besides the carboxylate group is the C=C double bond, which is almost always in the cis configuration.

Glycoaminoglycans (GAG's)

Unbranched polysaccharides usually composed of alternating uronic acid and hexosamine residues; in several types specific hydroxyl groups are sulfonated. GAGs are major constituents of ground substance, the gel-like matrix between cells. The viscosity and elasticity of solutions of GAGs also makes them excellent biological lubricants (synovial fluid) and shock absorbers (vitreous humor).

Ribose vs. Deoxyribose

Use of 2'deoxyribose in DNA increases its stability in aqueous solution. Although phosphodiesters are usually unreactive towards base-promoted hydrolysis, hydrolysis of RNA (but not DNA) is a notable exception due to intramolecular catalysis of the reaction. Rather than attacking phosphate directly, hydroxide ion deprotonates the 2'-hydroxyl group of the ribose, which acts much more efficiently as a nucleophile in cleaving the polynucleotide chain. The 2',3'-cyclic diphosphate (center, can be isolated from the reaction mixture) undergoes subsequent hydrolysis to give a mixture of phosphorylated products. Since DNA lacks a 2'-hydroxyl group, it cannot undergo hydrolysis by this mechanism. Not only does this mechanism account for the greater stability of DNA, but also provides a basis for the selectivity of nucleases (enzymes that cleave polynucleotides) - enzymes operating by this mechanism will cleave RNA, but not DNA.

Replication of DNA in E. coli

a. Leading strand is synthesized continuously in 5' to 3' direction by DNApol3. b. Lagging strand is synthesized in 5' to 3' direction by DNApol3 in relatively short segments of about 1000 bp called Okazaki fragments - synthesis of each of these fragments requires a separate RNA primer c. DNApol1: In addition to having DNA polymerase activity, DNApol1 can act as a 5'-to-3' exonuclease - it can cleave both individual ribonucleotides and deoxyribonucleotides from the 5' end of a polynucleotide (DNA or RNA). Using the 3'-end of an Okazaki fragment as its primer, DNApol1 extends that Okazaki fragment in the 5' to 3' direction, removing the RNA primer from the preceding Okazaki fragment in the process. However, DNApol1 is not able to link the growing 3' end of the growing Okazaki fragment with the 5' end of the lagging strand - this leaves a nick in the lagging strand. d. The enzyme DNA ligase closes the nick by catalyzing formation of a phosphodiester bond between the 3' end of the Okazaki fragment and the 5' end of the lagging strand.

Pyrimidines

cytosine, thymine, uracil

In the (x→y) or (x,y) designation of the glycosidic bond

the first number refers to the anomeric carbon atom of the monosaccharide group that is the glycoside (acetal). The second number refers to the number of the carbon atom of the monosaccharide to which the glycosidic bond is attached. The letter (β or α) preceding the numbers indicates the stereochemistry of the glycosidic bond.

Maltose and cellobiose are only two of the elevn known disaccharides of glucose

the other nine have different glycosidic linkages as shown below (3 stereoisomers for the (1,1) linkage and 2 stereoisomers for each of the others)

Denaturation of DNA

the separation of the two strands of the double helix into individual (random coil) -High temps

Maltose

α(1,4) glycosidic bond

Cellobiose

β(1,4) glycosidic bond

DNA Strands

Double stranded

Watson-Crick Base Pairing

-Adenosine in one strand pairs with a thymidine in the opposite strand - Two Hydrogen Bonds -Guanosine in one strand pairs with a cytidine residue in the opposite strand - Three Hydrogen Bonds

Denaturation and Renaturation of DNA

All DNA bases absorb fairly strongly in UV (maxima about 260 nm), but their absorption in the random coil is more intense than in double helix (hyperchromic shift), so monitoring the change in absorbance is a convenient method to observe the helix-to-coil transition. Melting Temperature (Tm) of DNA: Temperature at which 50% of double helical DNA is converted to random coil. As discussed above, the stability of a sample of DNA will be influenced by its composition. In particular, G-C pairs are not only stabilized by a higher degree of hydrogen bonding (three hydrogen bonds for a G-C pair vs. two for A-T pairs), but also interact more strongly with adjacent base pairs in base stacking. Consequently, the Tm of DNA increases with the percentage of G-C base pairs in its structure (above right), and measurement of the Tm of a sample of DNA can be used to obtain a reasonable estimate of its composition.

Eicosanoids

Class of lipids derived from arachidonic acid.

Biological Membranes

Complexes of Lipids and Proteins

Ribonucleosides

Contain a ribose sugar unit in the furanose form with a heterocyclic base (purine or pyrimidine) bound at the 1' position via an N-glycosidic bond. Locants with primes (') refer to the positions of the sugar.

Liver Cells

Contain up to 10% glycogen by mass -Semipermeable membrane containing 2% solution of glucose in water -If placed in water would need to increase by a 2.7 fold to reach II= 1 atm -if all glucose is in the form of glycogen, II=0 atm

transfer-RNA (tRNA)

Deliver amino acids to ribosomes in the order required for synthesis of a specific peptide (below left)

Restriction Map

Diagram of a DNA molecule showing recognition sites (cleavage sites) of different restriction enzymes.

Sucrose

Disaccharide of glucose and fructose linked by a (α1→β2) glycosidic bond (or (α1,β2) glycosidic bond). The glycosidic bond in sucrose involves the anomeric carbon of both monosaccharide units - because the glycosidic linkage between glucose and fructose involves the anomeric carbon of each monosaccharide unit, sucrose is a non- reducing sugar (i.e., both sugar units are glycosides - neither can form an open chain structure).

Replication of DNA - Basic Principles

During replication, each strand of the original DNA (parent DNA) serves as a template for the synthesis of a new complementary strand. Replication of DNA is semi-conservative; each daughter DNA gets one strand of original DNA and a copy of the complementary strand.

Summary - Cycles of PCR

Each FL strand gives one complementary RL strand. Each RL strand gives one complementary UL strand. Each UL strand gives one complementary UL strand. After ten cycles, about 99% of the DNA in the sample is of the desired length as dictated by the DNA primers added at the start of the procedure. In each subsequent cycle, the amount of UL-DNA is roughly doubled, so that by the end of 16 cycles more than 100,000 copies of the desired section of DNA have been synthesized, increasing to more than one million copies at the end of 19 cycles.

Unsaturated Fatty Acids

Each cis-C=C double bond produces a ~30o bend in carbon chain

DNA Polymerase

Enzyme that synthesizes a new strand of DNA using another DNA strand as a template. Cells usually have more than one type of DNA polymerase (abbreviated DNApol) - all catalyze synthesis of DNA but have other specific functions.

β-Lactam Antibiotics (e.g., penicillins (below), cephalosporins)

Inhibitors of the bacterial enzymes that cross-link peptidoglycan chains. Many bacteria with resistance to β-lactam antibiotics express enzymes (β-lactamases) that catalyze hydrolysis of the amide bond in these compounds

Ribonucleotides

Phosphate esters of ribonucleosides existing largely as dianions at pH 7. In naturally occurring ribonucleotides, the phosphate group is usually located at the 5'-position (below middle), but in some cases is found at the 2' or 3' (below right) position.

Sphingomyelin

Phosphodiesters of ceramide with choline (below) as the most common head group constituent - head group is zwitterionic at pH 7

Type B

R=D-galactose

RNA Strands

Single-stranded

Basic Procedure of Sanger Dideoxy Method

The DNA strand (single-stranded) to be sequenced is mixed with a short DNA primer complementary to its 3'-end and the four 2'-deoxyribonucleotide-5'-triphosphates (dATP, dGTP, dCTP, and dTTP) - a small amount of one of these nucleotides (usually dATP) is radioactively labeled at the -phosphate group with 32P. This solution is divided into four tubes. A small amount (e.g., much smaller than the amounts of dATP, dGTP, etc.) of a different 2',3'-deoxyribonucleotide 5'-triphosphate (ddNTP; below) is added to each of the four tubes. dCTP, and dTTP) - a small amount of one of these nucleotides (usually dATP) is radioactively labeled at the-phosphate group with 32P. This solution is divided into four tubes. A small amount (e.g., much smaller than the amounts of dATP, dGTP, etc.) of a different 2',3'- deoxyribonucleotide 5'-triphosphate (ddNTP; below) is added to each of the four tubes. DNA polymerase is added to each tube to give formation of double-stranded DNA using the sample DNA as a template. When the reaction is complete, samples of DNA from the four tubes are analyzed by polyacrylamide gel electrophoresis (PAGE), with the four samples being run in side-by-side lanes on the same gel. Example: Diagram of the autoradiogram obtained by sequencing of (3') A-T-C-G-G-C-T-A-G (5') (not including primer segment)

Stability of B-DNA

The double helix is maintained by the sum of a large number of individually weak interactions. Hydrogen bonding between the two strands via base pairing provides some stabilization, but there are other forces involved. Stacking of the base pairs in close contact maximizes the strength of van der Waals attractions in the interior of the helix, with stacking of adjacent G-C pairs giving the strongest attraction. These interactions are augmented by the hydrophobic effect - since water is excluded from the core of the helix, stacking of the bases requires less organization of water molecules than if the bases were to be exposed to aqueous solution. -Because the sugar-phosphate backbone contains one negatively charged group per nucleotide, DNA is also stabilized by the presence of cations (eg Mg2+). -The stabilization of DNA via a large number of individually weak interactions is important for several reasons, including providing the conformation flexibility to allow DNA to be packed within a cell. As well, the ability of DNA to be dissociated into separate strands is required for its biological function

Polyunsaturated Fatty Acids

The first C=C is often between carbons 9 and 10 and have one methylene group (CH2) between the C=C bonds (nonconjugated C=C bonds

Fidelity of DNA replication

The inherent mistake rate of DNA polymerases is incorporation of one incorrect base per 10^4-10^5 nucleotides added to a growing strand of DNA. Proofreading & Editing: Many DNA polymerases also contain 3'-to-5' exonuclease activity which is activated by non-Watson-Crick base pairing as a "proof-reading" mechanism - if such a mismatch is detected, the polymerase "backs up" to delete several nucleotides from the growing chain, then continues synthesis of DNA synthesis in the 5'-to-3' direction. This mechanism improves the mistake rate by a factor of ~100. Other enzyme systems detect and correct errors occurring during replication, as well as replacing sections of DNA damaged by exposure to chemical reagents (mutagens) and exposure to UV light. Overall, the fidelity of replication is on the order of one incorrectly incorporated nucleotide per 10^8-10^10 bases.

Uracil vs. Thymine

The use of thymine, rather than uracil, as a base in DNA appears to be a consequence of the chemistry of the third common pyrimidine in in nucleic acids, cytosine -deamination of cytosine via nucleophilic attack of water gives uracil. In a typical mammalian cell, deamination of cytidine residues in DNA occurs at a rate of about 100 times per day, converting G-C pairs in double-stranded DNA into G-U pairs. However, since DNA does not normally contain U, when the enzymes responsible for maintaining the integrity of DNA identify this mismatched base pair, the uridine residue will be cleaved from the chain and replace by cytidine. On the other hand, if DNA used U and C, rather than T and C, as pyrimidine bases, the DNA repair enzymes could not correctly identify which residue of the mismatched pair should be replaced - that is, replace U with C to give a GC pair, or replace G with A to give an AU pair.

All monosaccharides are reducing sugars:

disaccharides may be reducing or non-reducing sugars depending upon the arrangement of their glycosidic bonds