Biochemistry Exam #3

2D Gel Electrophoresis

- 2D gel electrophoresis allows the separation and display of up to 1,000 different proteins on a single gel. Mass spectrometry can then be used to partially sequence individual protein spots and assign each to a gene. The appearance and nonappearance (or disappearance) of particular protein spots in samples from different tissues, from similar tissues at different stages of development or from tissues treated in ways that simulate a variety of biological conditions can help define cellular function. - Because many proteins are displayed at once in these gels, the technique is also applicable to systems biology. For example, a bacterial pathogen may evolve so as to become resistant to one or more antibiotics. The pattern of protein expression in that bacterial strain is likely to change, and multiple proteins may be affected.

DNA Library

- A DNA library is a collection of DNA clones, gathered together as a source of DNA for sequencing, gene discovery or gene function studies. The library can take a variety of forms depending on the source of the DNA.

Cellular Function

- A description of the network of interactions engaged in by a protein at the cellular level. Interactions with other proteins in the cell can help define the kinds of metabolic processes in which the protein participates.

Selectins

- A family of plasma membrane lectins that mediate cell-cell recognition and adhesion in a wide range of cellular processes. One such process is the movement of immune cells (neutrophils) through the capillary wall, from blood to tissues, at sites of infection of inflammation. - At an infection site, P-selectin on the surface of capillary endothelial cells interacts with a specific oligosaccharide of the glycoproteins of circulating neutrophils. This interaction slows the neutrophils as they adhere to and roll along the endothelial lining of the capillaries. - A second interaction between integrin molecules in the neutrophil plasma membrane and an adhesion protein on the endothelial cell surface now stops the neutrophil and allows it to move through the capillary wall into the infected tissues to initiate the immune attack. • Two other selectins participate in this "lymphocyte homing": E-selectin on the endothelial cell and L-selectin on the neutrophil bind their cognate oligosaccharides on the neutrophil and endothelial cell respectively. - Human selectins mediate the inflammatory responses in rheumatoid arthritis, asthma, psoriasis, MS, and the rejection of transplanted organs, and thus there is great interest in developing drugs that inhibit selectin-mediated cell adhesion.

Cellulose

- A fibrous, tough, water-insoluble substance found in the cell walls of plants. - Like amylose, the cellulose molecule is a linear unbranched homopolysaccharide, consisting of 10,000-15,000 D-glucose units. However, in cellulose, the glucose residues have the beta configuration. - The glucose residues in cellulose are linked by beta 1-4 glycosidic bonds. - Glycogen and starch ingested in the diet are hydrolyzed by α-amylases and glycosidases, enzymes in saliva and the intestine that break α 1-4 glycosidic bonds between glucose units. - Most animals cannot use cellulose as a fuel source because they lack an enzyme to hydrolyze the beta 1-4 linkages.

Hyaluronan

- A glycosaminoglycan that contains alternating residues of D-glucuronic acid and N-acetylglucosamine. - With up to 50,000 repeats of the basic disaccharide unit, hyaluronan has a molecular weight of several million. It forms clear, highly viscous solutions that serve as lubricants in the synovial fluid of joints and gives the vitreous humor of the vertebrate eye its jelly like consistency. - Also a component of the ECM of cartilage and tendons, to which it contributes tensile strength and elasticity as a result of its strong interactions with other components of the matrix.

cDNA Library

- A library that includes only those genes that are expressed - transcribed into RNA - in a given organism or even in certain cells or tissues. Such a library focuses on those portions of a genome relevant to the function of a tissue or cell type.

Chitin

- A linear homopolysaccharide composed of N-acetylglucosamine residues in beta 1-4 linkage. - The only chemical different from cellulose is the replacement of the hydroxyl group at C2 with an acetylated amino group. - Chitin forms extended fibers similar to those of cellulose, and like cellulose cannot be digested by vertebrates. - Chitin is the principal component of the hard exoskeletons of arthropods.

Nucleoside

- A nucleotide without a phosphate group.

Unusual Structures of DNA - Palindrome - Hairpin - Cruciform - Mirror Repeat

- A rather common type of DNA sequence is a palindrome. A palindrome is spelled identically read either forward or backward. The term is applied to regions of DNA with inverted repeats of base sequence having twofold symmetry over two strands of DNA. In the DNA of living cells, sites recognized by many sequence-specific DNA-binding proteins are arranged as palindromes. - Such sequences are self-complementary within each strand and therefore have the potential to form hairpin or cruciform structures. - When the inverted repeat occurs within each individual strand of the DNA, the sequence is called a mirror repeat. Mirror repeats do not have complementary sequences within the same strand and cannot form hairpin or cruciform structures.

Lectins - Protection from Degradation - Erythrocytes

- A similar mechanism is apparently responsible for removing old erythrocytes from the mammalian bloodstream. Newly synthesized erythrocytes have several membrane glycoproteins with oligosaccharide chains that end in Neu5Ac. When the sialic acid residues are removed by withdrawing a sample of blood from experimental animals, treating it with sialidase in vitro and reintroducing it into the circulation, the treated erythrocytes disappear from the bloodstream within a few hours; erythrocytes with intact oligosaccharides continue to circulate for days.

Green Fluorescent Protein

- A target gene fused with a gene for green fluorescent protein (GFP) generates a fusion protein that is highly fluorescent - it lights up. - GFP has a β-barrel structure, with a flurophore in the center of the barrel. The flurophore is derived from a rearrangement and oxidation of several amino acid residues in an autocatalytic reaction that requires only molecular oxygen. Thus the protein is readily cloned in an active form in almost any cell. Just a few molecules of this protein can be observed microscopically, allowing the study of its location and movements in a cell. - Careful protein engineering has generated mutant forms of GFP with a range of different colors and other properties and related proteins have recently been isolated form other species. - Immunofluorescence requires cell fixation (death of the cell).

Most Stable 3D Structure for α 1-4 Linked Chains - Starch - Glycogen

- A tightly coiled helix, stabilized by interchain hydrogen bonds.

Nucleotides as Enzyme Cofactors

- A variety of enzyme cofactors serving a wide range of chemical functions include adenosine as part of their structure. They are unrelated structurally except for the presence of adenosine. - In none of these cofactors does the adenosine portion participate directly in the primary function, but removal of adenosine generally results in a drastic reduction of cofactor activities. • For example, removal of the adenine nucleotide from acetoacetyl CoA, the coenzyme A derivative of acetoacetate, reduces its reactivity as a substrate for β-ketoacyl-CoA transferase (an enzyme of lipid metabolism) by a factor of 10^6. - The requirement for adenosine must involve the binding energy between enzyme and substrate (or cofactor) that is used both in catalysis and in stabilizing the initial enzyme-substrate complex. • In the case of β-ketoacyl-CoA transferase, the nucleotide moiety of coenzyme A seems to be a binding handle that helps to pull the substrate into the active site.

Sequence Tagging Site

- A well-characterized library may contain thousands of long contigs, all assigned to and ordered on particular chromosomes to form a detailed physical map. The known sequences within the library (each called a sequence-tagged site or STS) can provide landmarks for genomic sequencing projects.

Cloning Vector & Linkers

- After the target DNA fragment is isolated, DNA ligase can be used to join it to a similarly digested cloning vector - a vector digested by the same restriction endonuclease. - DNA ligase catalyzes the formation of new phosphodiester bonds in a reaction that uses ATP or a similar cofactor. The base pairing of complementary sticky ends greatly facilitates the ligation reaction. Blunt ends can also be ligated, albeit less efficiently. - Researchers can create new DNA sequences by inserting synthetic DNA fragments (linkers) between the ends that are being ligated. - Inserted DNA fragments with multiple recognition sequences for restriction endonucleases are called polylinkers.

Alkylating Agents

- Alkylating agents can alter certain bases of DNA. For example, the highly reactive chemical dimethylsulfate can methylate a guanine to yield O^6 - methylguanine, which cannot base pair with cytosine. • Many similar reactions are brought about by alkylating agents normally present in cells, such as S adenosylmethionine.

Monosaccharide Chirality

- All the monosaccharides except dihydroxyacetone (ketotriose) contain one or more asymmetric (chiral) carbon atoms and thus occur in optically active isomeric forms. - In general, a molecule with n chiral centers can have 2^n stereoisomers.

Nucleic Acid Polarity

- All the phosphodiester linkages in DNA and RNA have the same orientation along the chain giving each linear nucleic acid strand a specific polarity and distinct 5' and 3' ends. By definition, the 5' end lacks a nucleotide at the 5' position and the 3' end lacks a nucleotide at the 3' position. - Other groups (most often one or more phosphates) may be present on one or both ends. The 5' to 3' orientation of a strand of nucleic acid refers to the ends of the strand, not the orientation of the individual phosphodiester bonds linking its constituent nucleotides. - The sequence of a single strand of nucleic acid is always written with the 5' end at the left and the 3' end at the right in the 5'-3' direction.

Minor Bases

- Although nucleotides bearing the major purines and pyrimidines are most common, both DNA and RNA also contain some minor bases. In DNA the most common of these are methylated forms of the major bases; in some viral DNAs, certain bases may be hydroxymethylated or glucosylated. - Altered or unusual bases in DNA molecules often have roles in regulating or protecting the genetic information. - Minor bases of many types are also found in RNAs, especially in tRNAs.

Genomic Library

- Among the largest types of DNA library is a genomic library, produced when the complete genome of a particular organism is cleaved into thousands of fragments and all the fragments are cloned by insertion into a cloning vector.

Hyaluronidase

- An enzyme secreted by some pathogenic bacteria that can hydrolyze the glycosidic linkages of hyaluronan, rendering tissues more susceptible to bacterial invasion. - In many species, a similar enzyme in sperm hydrolyzes an outer glycosaminoglycan coat around the ovum, allowing sperm penetration.

Epitope Tag

- An epitope tag is a short protein sequence that is bound tightly by a well-characterized monoclonal antibody. The tagged protein can be specifically precipitated from a crude protein extract by interaction with the antibody. - If any other proteins bind to the tagged protein, those will precipitate as well, providing information about protein-protein interactions in a cell.

Depurination

- Another important reaction in the deoxyribonucleotides is the hydrolysis of the N-β-glycosyl bond between the base and the pentose, to create a DNA lesion called an AP (apurinic, apyrimidinic) site or abasic site - depurination. - This occurs at a higher rate for purines than for pyrimidines. As many as one in 10^5 (10,000 per mammalian cells) are lost from DNA every 24 hours under typical cellular conditions. - Depurination of ribonucleotides and RNA is much slower and generally is not considered physiologically significant. - In the test tube, loss of purines can be accelerated by dilute acid. Incubation of DNA at pH 3 causes selective removal of the purine bases, resulting in a derivative called apurinic acid.

Dextrans

- Bacterial and yeast polysaccharides made up of α 1-6 linked poly-D-glucose. - All have α 1-3 branches and some also have α 1-2 or α 1-4 branches. - Dental plaque, formed by bacteria growing on the surface of the teeth, is rich in dextrans. - Synthetic dextrans are used in several commercial products that serve in the fractionation of proteins by size-exclusion chromatography. The dextrans in these products are chemically cross-linked to form insoluble materials of various porosites, admitting macromolecules of various sites.

Glycosaminoglycans

- Basement membrane is a specialized ECM that underlies epithelial cells; it consists of specialized collagens, laminin and heteropolysaccharides. These heteropolysaccharides, the glycosaminoglycans are a family of linear polymers composed of repeating disaccharide units. - One of the two monosaccharides is always either N-acetylglucosamine or N-acetylgalactosamine; the other is in most cases a uronic acid. - Some glycosaminoglycans contain esterified sulfate groups. The combination of sulfate groups and the carboxylate groups of the uronic acid residues give glycosaminoglycans a very high density of negative charge. To minimize the repulsive forces among neighboring charged groups, these molecules assume an extended conformation in solution, forming a rodlike helix in which the negatively charged carboxylate groups occur on alternate sides of the helix. The extended rod form also provides maximum separation between the negatively charged sulfate groups. - The specific pattern of sulfated and nonsulfated sugar residues in glycosaminoglycans provide for specific recognition by a variety of protein ligands that bind electrostatically to these molecules. - The sulfated glycoasminoglycans are attached to extracellular proteins to form proteoglycans.

Glycogen Reducing Ends

- Because each branch in glycogen ends with a nonreducing sugar unit, a glycogen molecule with n branches has n+1 nonreducing ends, but only one reducing end. - When glycogen is used as energy source, glucose units are removed one at a time from the nonreducing ends. - Degradative enzymes that act only at nonreducing ends can work simultaneously on the many branches, speeding the conversion of the polymer to monosaccharides.

Glycans

- Carbohydrate polymers that serve as structural and protective elements in the cell walls of bacteria and plants and in the connective tissues of animals. - Others lubricate skeletal joints and participate in recognition and adhesion between cells. - Glycans differ from each other in the identity of their recurring monosaccharide units, in the length of their chains, in the types of bonds linking the units and in the degree of branching.

Glycoproteins

- Carbohydrate-protein conjugates in which the glycans are smaller, branches and more structurally diverse than the glycosaminoglycans of proteoglycans. - The carbohydrate is attached at its anomeric carbon through a glycosidic link to the OH of a Ser of Thr residue (O-linked) or through an N-glycosyl link to the amide nitrogen of an Asn residue (N-linked). - Some glycoproteins have a single oligosaccharide chain, but many have more than one; the carbohydrate may constitute from 1-70% or more of the glycoprotein by mass. - About half of all proteins are glycosylated and about 1% of all mammalian genes encode enzymes involved in the synthesis and attachment of these oligosaccharide chains. Sequences for the attachment of O-linked chains tend to be rich in Gly, Val and Pro residues. In contrast, the attachment of N-linked chains depends on the consensus sequence N-[P]-[ST]. - Not all potential sites are used.

Nucleotides as Regulatory Molecules

- Cells respond to their environment by taking cues from hormones or other external chemical signals. The interaction of these extracellular chemical signals (first messengers) with receptors on the cell surface often leads to the production of second messengers inside the cell, which in turn leads to adaptive changes within the cell interior. - Often, the second messenger is a nucleotide. One of the most common is adenosine 3',5' - cyclic monophosphate (cyclic AMP, cAMP) formed from ATP in a reaction catalyzed by adenylyl cyclase, an enzyme associated with the inner face of the plasma membrane. - Cyclic AMP serves regulatory functions in virtually every cell outside the plant kingdom. - Guanosine 3',5' - cyclic monophosphate (cGMP) occurs in many cells and also has regulatory functions.

Agar

- Certain marine red algae have cell walls that contain agar, a mixture of sulfated heteropolysaccharides made up of D-galactose and an L-galactose derivative ether-linked between C3 and C6. - Agar is a complex mixture of polysaccharides all with the same backbone structure, but substituted to varying degrees with sulfate and pyruvate.

Methylation of DNA Bases Overview

- Certain nucleotide bases in DNA molecules are enzymatically methylated. Adenine and cytosine are methylated more often than guanine and thymine. - Methylation is generally confined to certain sequences of regions of a DNA molecule. - All known DNA methylases use S-adenosylmethionine as a methyl group donor.

Fusion Protein

- Changes can also be introduced that involve more than one base pair. Large parts of a gene can be deleted by cutting out a segment with restriction endonucleases and ligating the remaining portions to form a smaller gene. - Parts of two different genes can be ligated to create new combinations. The product of such a fused gene is called a fusion protein.

Plasmids - Transformation - Electroporation

- Circular DNA molecules that replicate separately from the host chromosome. - Naturally occurring bacterial plasmids range in size from 5,000-400,000 bp. They can be introduced into bacterial cells by a process called transformation. The cells and plasmid DNA are incubated together at 0°C in a calcium chloride solution, then subjected to a shock by rapidly shifting the temperature to 37 to 43°C. Some of the cells treated in this way take up the plasmid DNA. - Some species of bacteria are naturally competent for DNA uptake and do not require the calcium chloride treatment. - In an alternative method, cells incubated with the plasmid DNA are subjected to a high-voltage pulse. This approach called electroporation, transiently renders the bacterial membrane permeable to large molecules. - Transformation of typical bacterial cell walls with purified DNA (never a very efficient process) becomes less successful as plasmid size increases and it is difficult to clone DNA segments longer than about 15,000 bp when plasmids are used as the vector.

Haworth Perspective Formulas

- Commonly used to show the stereochemistry of ring forms of monosaccharides. - The six-membered pyranose ring is not planar, but tends to assume either of two chair conformations. - Two conformations of a molecule are interconvertible without the breakage of covalent bonds, whereas two configurations can be interconverted only by breaking a covalent bond. - To interconvert alpha and beta configurations, the bond involving the ring oxygen atom would have to be broken, but interconversion of the two chair forms does not require bond breakage.

Glycoconjugates

- Complex carbohydrate polymers covalently attached to proteins or lipids act as signals that determine the intracellular location of metabolic fate of glycans.

Synteny

- Conserved gene order called synteny, provides additional evidence for an orthologous relationship between genes at identical locations within the related segments.

Monosaccharides

- Consist of a single polyhydroxy aldehyde or ketone unit. - Monosaccharides of four or more carbons tend to have cyclic structures. - Colorless, crystalline solids that are freely soluble in water but insoluble in nonpolar solvents.

Oligosaccharides

- Consist of short chains of monosaccharide units, or residues, joined by glycosidic bonds. - The most abundant are the disaccharides with two monosaccharide units. - In cells, most oligosaccharides consisting of three or more units do not occur as free entities, but are joined to nonsugar molecules (lipids of proteins) in glycoconjugates.

Starch

- Contains two types of glucose polymer: amylose and amylopectin. - Amylose consists of long, unbranched chains of D-glucose residues connected by α 1-4 linkages. Such chains vary in weight from a few thousand to more than a million. - Amylopectin also has a high molecular weight but unlike amylose is highly branched. The glycosidic linkages joining successive glucose residues in amylopectin chains are α 1,4; the branch points (occurring every 24-30 residues) are α 1-6 linkages.

Dermatan sulfate

- Contributes to the pliability of skin and is also present in blood vessels and heart valves. - Many of the glucuronate residues present in chondroitin sulfate are replaced by their 5-epimer, L-iduronate.

Chondroitin sulfate

- Contributes to the tensile strength of cartilage, tendons, ligaments and walls of the aorta.

Chemical Agents that Cause DNA Damage

- DNA also may be damaged by reactive chemicals introduced into the environment as products of industrial activity. - Such products may not be injurious per se but may be metabolized by cells into forms that are. - There are two prominent classes of such agents: • Deaminating agents, particularly nitrous acid (HNO2) or compounds that can be metabolized to nitrous acid or nitrites. • Alkylating agents.

Structural Variation of DNA

- DNA is a very flexible molecule. Considerable rotation is possible around several types of bonds in the sugar-phosphate backbone and thermal fluctuation can produce bending, stretching and unpairing (melting) of the strands. - Structural variation in DNA reflects three things: 1. The different possible conformations of the deoxyribose. 2. Rotation about the contiguous bonds that make up the phosphodeoxyribose backbone. 3. Free rotation about the C-1'-N-glycosyl bond. - Because of steric constraints, purines in purine nucleotides are restricted to two stable conformations with respect to deoxyribose, called syn and anti. Pyrimidines are generally restricted to the anti conformation because of steric interference between the sugar and the carbonyl oxygen at C-2 of the pyrimidine.

Disaccharides & Glycosidic Bonds

- Disaccharides consist of two monosaccharides joined covalently by an O-glycosidic bonds, which is formed when a hydroxyl group of one sugar reacts with the anomeric carbon of the other. - This reaction represents the formation of an acetal from a hemiacetal and an alcohol, and the resulting compound is called glycoside. - Glycosidic bonds are readily hydrolyzed by acid but resist cleavage by base.

Heparin Sulfate Domain Structure

- Domain structure is not random; some domains (typically 3-8 disaccharide units long) differ from neighboring domains in sequence and in ability to bind to specific proteins. - Highly sulfated domains (called NS domains) alternate with domains having unmodified GlcNAc and GlcA residues (N-acetylated or NA domains). - The exact pattern of sulfation in the NS domain depends on the particular proteoglycan; given the number of possible modifications of the GlcNAc-IdoA dimer, at least 32 different disaccharide units are possible. - Furthermore, the same core protein can display different heparan sulfate structures when synthesized in different cell types.

Stability of RNA vs. DNA Duplexes

- Duplexes of two RNA strands or one RNA strand and one DNA strand (RNA-DNA hybrids) can also be denatured. Notably, RNA duplexes are more stable than DNA duplexes. • At neutral pH, denaturation of a double-helical RNA often requires temperatures 20°C or more higher than those required for denaturation of a DNA molecule with a comparable sequence. - The stability of a RNA-DNA hybrid is generally intermediate between that of RNA and that of DNA.

Methylation of DNA Bases - E. Coli

- E. coli has two prominent methylation systems. One serves as part of a defense mechanism that helps the cell to distinguish its DNA from foreign DNA by marking its own DNA with methyl groups and destroying foreign DNA without the methyl groups - restriction-modification system. - The other system methylates adenosine residues within the sequence (5')GATC(3') to N^6-methyladenosine. This is mediated by the Dam (DNA adenine methylation) methylase, a component of a system that repaired mismatched base pairs formed occasionally during DNA replication.

Most Stable 3D Structure for Cellulose

- Each chair is turned 180° relative to its neighbors, yielding a straight extended chain. - All OH groups are available for hydrogen bonds with neighboring chains. With several chains lying side by side, a stabilizing network of interchain and intrachain hydrogen bonds produces straight, stable supramolecular fibers of great tensile strength. - Water content is low because extensive interchain hydrogen bonding between cellulose molecules satisfies their capacity for hydrogen-bond formation.

Proteoglycan Aggregates

- Enormous supramolecular assemblies of many core proteins all bound to single molecule of hyaluronan. - Aggrecan core protein (Mr ~ 250,000) has multiple chains of chondroitin sulfate and keratin sulfate, joined to Ser residues in the core protein through trisaccharide linkers, to give aggrecan monomer of Mr ~ 2x10^6. When a hundred or more of these decorated core proteins bind a single, extended molecule of hyaluronate, the resulting proteoglycan aggregate and its associated water of hydration occupy a volume about equal to that of a bacterial cell. - Aggrecan interacts strongly with collagen in the ECM of cartilage, contributing to the development, tensile strength and resiliency of this connective tissue.

Two Enzymes Involved in DNA Cloning

- First, restriction endonucleases recognize and cleave DNA at specific sequences (recognition sequences or restriction sites) to generate a set of smaller fragments. - Second, the DNA fragment to be cloned is joined to a suitable cloning vector by using DNA ligases to link the DNA molecules together. - The recombinant vector is then introduced into a host cell, which amplifies the fragment in the course of many generations of cell division.

Tetraplex

- Four DNA strands can also pair to form a tetraplex, but this occurs readily for DNA sequences with a very high proportion of guanosine residues. The guanosine tetraplex, or G tetraplex, is quite stable over a wide range of conditions. The orientation of strands in the tetraplex can vary between parallel and antiparallel.

Properties of Nucleotide Bases and Their Affect on the 3D Structure of Nucleic Acids

- Free pyrimidines and purines are weakly basic compounds and thus are called bases. - The purines and pyrimidines common in DNA and RNA are aromatic molecules, a property with important consequences for the structure, electron distribution and light absorption of nucleic acids. - Electron delocalization among atoms in the ring gives most of the bonds partial double-bond character. One result is that pyrimidines are planar molecules and purines are very nearly planar, with a slight pucker. - Free pyrimidine and purine bases may exist in two or more tautomeric forms depending on the pH. - All nucleotide bases absorb UV light, and nucleic acids are characterized by a strong absorption at wavelengths near 260nm.

Expression Vectors

- Frequently, it is the product of the cloned gene, rather than the gene itself, that is of primary interest. - Most eukaryotic genes lack the DNA sequence elements - such as promoters - required for their expression in E. coli cells, so bacterial regulatory sequences for transcription and translation must be inserted at appropriate positions relative to the eukaryotic gene in the vector DNA. - In some cases cloned genes are so efficiently expressed that their protein product represents 10% or more of the cellular protein; they are said to be overexpressed. At these concentrations some foreign proteins can kill an E. coli cell, so gene expression must be limited to the few hours before the planned harvest of the cells. - Cloning vectors with the transcription and translation signals needed for the regulated expression of a cloned gene are often called expression vectors. - The rate of expression of the cloned gene is controlled by replacing the gene's own promoter and regulatory sequences with more efficient and convenient versions supplied by the vector. Generally, a well-characterized promoter and its regulatory elements are positioned near several unique restriction sites for cloning, so that genes inserted at the restriction sites will be expressed from the regulated promoter element. - Some of these vectors incorporate other features such as bacterial ribosome binding site to enhance translation of the mRNA derived from the gene, or a transcription termination sequence.

Paralogs

- Genes similarly related to each other within a single species.

Orthologs

- Genes that occur in different species but possess a clear sequence and functional relationship to each other. - If the function of a gene has been characterized for one species, this information can be used to at least tentatively assign gene function to the ortholog found in the second species. The correlation is easiest to make when comparing genomes from relatively closely related species, although many clearly orthologous genes have been identified in species as distant as humans and bacteria. Sometimes even the order of genes on a chromosome is conserved over large segments of the genomes of closely related species.

mRNA

- Given that the DNA of eukaryotes is largely confined to the nucleus whereas protein synthesis occurs on ribosomes in the cytoplasm, some molecule other than DNA must carry the genetic message from the nucleus to the cytoplasm. - RNA was considered the logical candidate: RNA is found in both the nucleus and the cytoplasm, and an increase in protein synthesis is accompanied by an increase in the amount of cytoplasmic RNA and an increase in its rate of turnover. These and other observations led several researchers to suggest that RNA carries genetic information from DNA to the protein biosynthetic machinery of the ribosome. - The process of forming mRNA on a DNA template is transcription. - In bacteria and archaea, a single mRNA molecule may code for one or several polypeptide chains. If it carries the code for only one polypeptide, the mRNA is monocistronic; if it codes for two or more different polypeptides, it is polycistronic. - In eukaryotes, most mRNAs are monocistronic. Cistron refers to a gene.

Steric Factors: Glycogen, Starch and Cellulose

- Glycogen, starch and cellulose are composed of pyranoside subunits as are the oligosaccharides of glycoproteins and glycolipids. - Such molecules can be represented as a series of rigid pyranose rings connected by an oxygen atom bringing two carbon atoms (the glycosidic bond). - There is, in principle, free rotation about both C-O bonds linking the residues, but as in polypeptides, rotation about each bond is limited by steric hindrance by substituents. - The 3D structures of these molecules can be described in terms of the dihedral angles φ and ψ about the glycosidic bond. - The bulkiness of the pyranoses ring and its substituents, and electronic effects at the anomeric carbon place constraints on the angles φ and ψ; thus certain conformations are much more stable than others.

RNA Hairpin Loops

- Hairpin loops form between nearby self-complementary sequences. - The potential for base-paired helical structures in many RNAs is extensive, and the resulting hairpins are the most common type of secondary structure in RNA. Specific short base sequences (such as UUCG) are often found at the ends of RNA hairpins and are known to form particularly tight and stable loops. Such sequences may act as starting points for the folding of an RNA molecule into its precise 3D structure. - Other contributions are made by hydrogen bonds that are not part of standard Watson-Crick base pairs. • For example, the 2'-hydroxyl group of ribose can hydrogen-bond with other groups.

Keratin sulfate

- Has no uronic acid and a variable sulfate content. - Present in the cornea, cartilage, bone and a variety of horny structures formed of dead cells.

Glycoproteins - Overview

- Have one or several oligosaccharides of varying complexity joined covalently to a protein. They are usually found on the outer face of the plasma membrane (as part of the glycocalyx), in the ECM and in the blood. Inside cells they are found in specific organelles such as Golgi complexes, secretory granules and lysosomes. - The oligosaccharide portions of glycoproteins are very heterogenous and like glycosaminoglycans, they are rich in information, forming highly specific sites for recognition and high-affinity binding by carbohydrate proteins called lectins. - Some cytosolic and nuclear proteins can be glycosylated as well.

Denaturation

- Heat and extremes of pH denature double-helical DNA. Disruption of the hydrogen bonds between paired bases and of base stacking causes unwinding of the double helix to form two single strands, completely separate from each other along the entire length or part of the length (partial denaturation) of the molecule. No covalent bonds in the DNA are broken.

Heparan Sulfate & Heparin

- Heparan sulfate is produced by all animal cells and contains variable arrangements of sulfated and non sulfated sugars. The sulfated segments of the chain allow it to interact with a large number of proteins, including growth factors and ECM components, as well as various enzymes and factors present in plasma. - Heparin is a fractionated form of heparan sulfate, derived mostly from mast cells. It is a therapeutic agent used to inhibit coagulation through its capacity to bind the protease inhibitor antithrombin. - Heparin binding causes antithrombin to bind and inhibit thrombin, a protease essential to blood clotting. The interaction is highly electrostatic; heparin has the highest negative charge density of any known biological macromolecule.

Homopolysaccharides vs. Heteropolysaccharides

- Homopolysaccharides contain only a single monomeric species. - Heteropolysaccharides contain two or more different kinds.

PCR

- If we know the sequence of at least the flanking parts of a DNA segment to be cloned, we can hugely amplify the number of copies of that DNA segment, using the polymerase chain reaction (PCR). The amplified DNA can be cloned directly or used in a variety of analytical procedures.

pBR322 Plasmid

- Important pBR322 features include: 1. An origin of replication, ori, a sequence where replication is initiated by cellular enzymes. This sequence is required to propagate the plasmid and maintain it at a level of 10-20 copies per cell. 2. Two genes that confer resistance to different antibiotics (tetR, ampR) allowing the identification of cells that contain the intact plasmid or a recombinant version of the plasmid. 3. Several unique recognition sequences that are targets for different restriction endonucleases, providing sites where the plasmid can later be cut to insert foreign DNA. 4. Small size, which facilitates entry of the plasmid into cells and the biochemical manipulation of the DNA.

Most Stable 3D Structure for Amylose

- In amylose (with no branches) this structure is regular enough to allow crystallization and thus determination of the structure by X-ray diffraction. - The average plane of each residue along the amylose chain forms a 60° angle with the average plane of the preceding residue, so the helical structure has six residues per turn. - For amylose, the core of the helix is precisely the right dimensions to accommodate iodine as complex ions, giving an intensely blue complex.

Cyclic Structures of Monosaccharides

- In aqueous solution, aldotetroses and all monosaccharides with five or more carbon atoms in the backbone occur predominantly as cyclic structures in which the carbonyl group has formed a covalent bond with the oxygen of a hydroxyl group along the chain. - The formation of these ring structures is the result of a general reaction between alcohols and aldehydes and ketones to form derivatives called hemiacetals (aldehydes) or hemiketals (ketones), which contain an additional asymmetric carbon atoms and thus can exist in two stereoisomeric forms.

Methylation of DNA Bases - Eukaryotic

- In eukaryotic cells, about 5% of cytidine residues in DNA are methylated to 5-methylcytidine. Methylation is most common at CpG sequences, producing methyl-CpG symmetrically on both strands of the DNA. The extent of methylation of CpG sequences varies by molecular region in large eukaryotic DNA molecules.

Role of Sialic Acid in Lectin Recognition

- In humans, a family of 11 lectins that bind to oligosaccharide chains ending in sialic acid residues plays some important biological roles. - All of these lectins bind sialic acids at beta sandwich domains like those found in immunoglobulins and the proteins are therefore called siglecs 1-11 or sometimes sialoadhesins. - The interaction of a siglec with sialic scid (Neu5Ac) involves each of the ring substituents unique to Neu5Ac: the acetyl group at C5 undergoes both hydrogen-bond and van der Waals interaction with the protein; the carboxyl group makes a salt bridge with a conserved Arg residue; and the hydroxyls of the glycerol moiety hydrogen bond with the protein. - Siglecs regulate activities in the immune and nervous systems and in blood cell development.

Specificity and Affinity of Lectins

- In their carbohydrate-binding sites, lectins have a subtle molecular complementarity that allows interaction only with their correct carbohydrate cognates. The result is a high specificity in these interactions. - The affinity between an oligosaccharide and each carbohydrate binding domain (CBD) of a lectin is sometimes modest, but the effective affinity is in many cases greatly increased by lectin multivalency, in which a single lectin molecule has multiple CBDs. In a cluster of oligosaccharides - as is commonly found on a membrane surface, for example - each oligosaccharide can engage one of the lectin's CBDs, strengthening the interaction. When cells express multiple receptors, the avidity of the interaction can be very high, enabling highly cooperative events such as cell attachment and rolling. - In addition to very specific interactions, there are more general interactions that contribute to the binding of many carbohydrates to their lectins. For example, many sugars have a more polar and a less polar side; the more polar side hydrogen-bonds with the lectin, while the less polar undergoes hydrophobic interactions with nonpolar amino acid residues. The sum of all these interactions produces high-affinity binding and high specificity of lectins for their carbohydrates.

Sugar Code - why is an oligosaccharide so much more informative than a protein?

- Increased complexity and diversity in the oligosaccharides of glycoproteins and glycolipids. • Structural diversity of monosaccharides (18 pentoses and hexoses) • Pyranose and furanose form • α and β anomerism • Variety of glycosidic bond linkage: 1→2, 1→3, 1→4, 1→6, 2→3, 2→6 • Ability of oligosaccharide chain to branch out • Derivatives (aminotion, sulfation, etc) - In reality, only a subset of possible combinations is found, given the restrictions imposed by the biosynthetic enzymes and availability of precursors. Nevertheless, the enormously rich structural information in glycans does not merely but far surpasses that of nucleic acids in the density of information contained in a molecule of modest size. - Each of the oligosaccharides presents a unique 3D face readable by the proteins that interact with it.

Aggregates & Fibronectin Proteins

- Interwoven with these enormous extracellular proteoglycans are fibrous matrix proteins such as collagen, elastin and fibronectin, forming a cross-linked meshwork that gives the whole ECM strength and resilience. - Some of these proteins are multiadhesive, a single protein having binding sites or several different matrix molecules. - Interactions between cellular and extracellular molecules serve not merely to anchor cells to the extracellular matrix but also to provide paths that direct the migration of cells in developing tissue and to convey information in both directions across the plasma membrane.

DNA Cloning

- Involves separating a specific gene or DNA segment from a larger chromosome, attaching it to a small molecule of carrier DNA, and then replicating this modified DNA thousands or millions of times through both an increase in host cell number and the creation of multiple copies of the cloned DNA in each cell. The result is selective amplification of a particular gene or DNA segment.

Anomers

- Isomeric forms of monosaccharides that differ only in their configuration about the hemiacetal carbon atoms. - The hemiacetal (or carbonyl) atom is called the anomeric carbon. - The designation alpha indicates that the hydroxyl group at the anomeric center is, in a Fischer projection, on the same side as the hydroxyl attached at the farthest chiral center, whereas beta indicates that these hydroxyl groups are on opposite sides. - If in the cyclic Fischer projection the exocyclic oxygen at the anomeric centre is cis to the exocyclic oxygen attached to the anomeric reference atom (C5) (in the OH group) the anomer is alpha. If the two oxygens are trans (on different sides) the anomer is beta.

Watson-Crick Model for Structure of DNA

- It consists of two helical DNA chains wound around the same axis to form a right-handed double helix. The hydrophilic backbones of alternating deoxyribose and phosphate groups are on the outside of the double helix, facing the surrounding water. - The furanose ring of each deoxyribose is in the C-2' endo conformation. - The purine and pyrimidine bases of both strands are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and perpendicular to the long axis. The offset pairing of the two strands created a major grove and minor groove on the surface of the duplex. - Each nucleotide base of one strand is paired in the same plane with base of the other strand. Watson and Crick found that the hydrogen-bonded base pairs, G with C and A with T, are those that fit best within the structure. - Three hydrogen bonds can form between G and C, but only two can form between A and T. Other pairings of bases tend to destabilize the double-helical structure. - The two antiparallel polynucleotide chains of double-helical DNA are not identical in either base sequence or composition. Instead they are complementary to each other. - To account for the periodicities observed in the x-ray diffraction patterns of DNA fibers, Watson and Crick manipulated molecular models to arrive at a structure in which the vertically stacked bases inside the double helix would be 3.4 Å apart; the secondary repeat distance of about 34 Å was accounted for by the presence of 10 base pairs in each complete turn of the double helix. - In aqueous solution, the structure differs slightly from that in fibers, having 10.5 base pairs per helical turn.

Why is glucose not stored in its monomeric form?

- It has been calculated that hepatocytes store glycogen equivalent to a glucose concentration of 0.4M. The actual concentration of glycogen, which is insoluble and contributes little to the osmolarity of the cytosol is about 0.01 micromoles. - If the cytosol contained 0.4M glucose, the osmolarity would be threateningly elevated, leading to osmotic entry of water that might rupture the cell. - Furthermore, with an intracellular glucose concentration of 0.4M and an external concentration of about 5mM, the free energy change for glucose uptake into cells against this very high concentration gradient would be prohibitively large. - In addition, storing glucose as a polymer means an ordered structure (energy on demand) and low solubility means it stays stationary.

Specificity and Affinity of Lectins Example - Mannose 6-phosphate

- Lectins also act intracellularly. An oligosaccharide containing mannose 6-phosphate marks newly synthesized proteins in the Golgi complex for transfer to the lysosome. - A common structural feature on the surface of these glycoproteins, the signal patch, is recognized by an enzyme that phosphorylates (in a two-step process) a mannose residue at the terminus of an oligosaccharide chain. - The resulting mannose 6-phosphate residue is then recognized by the cation-dependent mannose 6-phosphate receptor, a membrane associated lectin with its mannose phosphate binding site on the lumenal side of the Golgi complex. When a section of the Golgi complex containing this receptor buds off to form a transport vesicle, proteins containing mannose phosphate residues are dragged into the forming bud by interaction of their mannose phosphates with the receptor; the vesicle then moves to and fuses with a lysosome, depositing its cargo therein. - Many, perhaps all, of the degredative enzymes (hydrolases) of the lysosome are targeted and delivered by this mechanism. - Some of the mannose 6-phosphate receptors can capture enzymes containing the mannose 6-phosphate residue and direct them to the lysosome. This process is the basis for "enzyme replacement therapy" to correct lysosomal storage disorders in humans. - The structure of the mannose 6-phosphate receptor/lectin reveals details of its interaction with mannose 6-phosphate that explain the specificity of the binding and the role for a divalent cation in the lectin-sugar interaction. - His105 is hydrogen-bonded to one of the oxygen atoms of the phosphate. When the protein tagged with mannose 6-phosphate reaches the lysosome (which has a lower internal pH than the Golgi complex), the receptor loses its affinity for mannose 6-phosphate. Protonation of His105 may be responsible for this change in binding.

Proteoglycans - Overview

- Macromolecules of the cell surface or ECM in which one or more sulfated glycosaminoglycan chains are joined covalently to a membrane protein or a secreted protein. - The glycosaminoglycan chain can bind to extracellular proteins through electrostatic interactions with the negatively charged groups on the polysaccharide. - Major components of ECMs.

DNA Microarrays

- Major refinements of the technology underlying DNA libraries, PCR and hybridization have come together in the development of DNA microarrays (sometimes called DNA chips), which allow the rapid and simultaneous screening of many thousands of genes. - DNA segments from known genes, a few dozen to hundreds of nucleotides long are amplified by PCR and placed on a solid surface using robotic devices that accurately deposit nanoliter quantities of DNA solution. Up to a million such spots are deposited in a predesigned array on a surface area of just a few square centimeters. - UV light cross-links the DNA to the glass slides. - An alternative strategy is to synthesize DNA directly on the solid surface, using photolithography. - Once the chip is constructed, it can be probed with mRNAs or cDNAs from a particular cell type or cell culture to identify the genes being expressed in those cells. - A microarray can answer such questions as which genes are expressed at a given stage in the development of an organism.

Proteoglycans

- Mammalian cells can produce over 40 types of proteoglycans, which act as tissue organizers. They influence various cellular activities such as growth factor activation and adhesion. - The basic proteoglycan units consists of a core protein with covalently attached glycosaminoglycan(s). The point of attachment is a Ser residue to which the glycosaminoglycan is joined through a tetrasaccharide bridge. - The Ser residue is generally in the sequence Ser-Gly-X-Gly (where X is any amino acid residue), although not every protein with this sequence has an attached glycosaminoglycan. - Many proteoglycans are secreted into the ECM, but some are integral membrane proteins. For example, the sheet-like ECM (basal lamina) that separates organized groups of cells from other groups contains a family of core proteins (Mr 20,000-40,000), each with several covalently attached heparan sulfate chains.

Gangliosides

- Membrane lipids of eukaryotic cells in which the polar head group and part of the lipid that forms the outer surface of the membrane is a complex oligosaccharide containing a sialic acid and other monosaccharide residues. - Some of the oligosaccharide moieties of gangliosides, such as those that determine human blood groups are identical with those found in certain glycoproteins, which therefore also contribute to blood group type. - Like the oligosaccharide moieties of glycoproteins, those of membrane lipids are generally, perhaps always, found on the outer face of the plasma membrane.

Glycolipids - Overview

- Membrane sphingolipids in which the hydrophilic head groups are oligosaccharides. - The oligosaccharides act as specific recognition sites by lectins. - The brain and neurons are rich in glycolipids, which help in nerve conduction and myelin formation. - Also play a role in signal transduction in cells.

Types of RNA

- Messenger RNA is only one of several classes of cellular RNA. - Transfer RNAs are adapter molecules in protein synthesis; covalently linked to an amino acid at one end, they pair with the mRNA in such a way that amino acids are joined to a growing polypeptide in the correct sequence. - Ribosomal RNAs are components of ribosomes. - There is also a wide variety of special-function RNAs including some (called ribozymes) that have enzymatic activity.

Microarrays in Evolution

- Microarrays are also used to examine changes in DNA, such as genetic changes induced by natural selection or simple variation in a population. - If a bacterial population has a new phenotype that signals the presence of one or more mutations, the mutations can often be identified quickly using microarrays. - A microarray containing the wild-type sequence information is probed with DNA from the mutant cells. Hybridization will be reduced wherever the mutant cell DNA and the wild-type DNA do not match, producing a signal at the relevant spots.

Reducing Sugars

- Monosaccharides can be oxidized by relatively mild oxidizing agents such as cupric (Cu2+) ion. The carbonyl is oxidized to a carboxyl group. - Glucose and other sugars capable of reducing cupric ion are called reducing sugars. They form enediols, which are converted to aldonic acids and then to a complex mixture of 2-, 3-, 4- and 6- carbon acids. - The reducing end is the anomeric position.

Nitrous Acid

- Nitrous acid, formed from organic precursors such as nitrosamines and from nitrite and nitrate salts, is a potent accelerator of the deamination of bases. Bisulfite has similar effects.

Pentoses in RNA & DNA

- Nucleic acids have two kinds of pentoses. The recurring deoxyribonucleotide units of DNA contain 2'-deoxy-D-ribose, and the ribonucleotide units of RNA contain D-ribose. - In nucleotides, both types of pentoses are in their β-furanose (closed five-membered ring) form. The pentose ring is not planar but occurs in one of a variety of conformations generally described as "puckered." - Although DNA and RNA seem to have two distinctions - different pentoses and the presence of uracil in RNA and thymine in DNA - it is the pentoses that define the identity of a nucleic acid. If the nucleic acid contains 2'-deoxy-D-ribose, it is DNA by definition even though it may contain some uracil.

Structure of a Nucleotide

- Nucleotides have three characteristic components: 1. A nitrogenous base. 2. A pentose. 3. A phosphate. - The base of a nucleotide is joined covalently (at N-1 of pyrimidines and N-9 of purines) in an N-β-glycosyl bond to the 1' carbon of the pentose, and the phosphate is esterified to the 5' carbon. - The N-β-glycosyl bond is formed by removal of the elements of water (a hydroxyl group from the pentose and hydrogen from the base), as in O-glycosidic bond formation.

Triplex DNA

- Nucleotides participating in a Watson-Crick base pair can form additional hydrogen bonds, particularly with functional grouped arrayed in the major groove. • For example, a cytidine residue (if protonated) can pair with the guanosine residue of a G-C nucleotide pair; a thymine can pair with the adenosine of an A-T pair. - The N-7 O^6 and N^6 of purines, the atoms that participate in the hydrogen bonding of triplex DNA are often referred to as Hoogsteen positions, and the non-Watson-Crick pairing is called Hoogsteen pairing. - Hoogsteen pairing allows the formation of triplex DNAs. - The triplexes also form most readily within long sequences containing only pyrimidines or only purines in a given strand. - Some triplex DNAs contain two pyrimidine strands and one purine strand; others contain two purine strands and one pyrimidine strand. polypyrimidine or polypurine sequences that can form triple helices are found within regions involves in the regulation of expression of some eukaryotic genes.

Isolation of a Fragment of DNA

- Once a DNA molecule has been cleaved into fragments, a particular fragment of known size can be enriched by agarose or acrylamide gel electrophoresis or by HPLC. - For a typical mammalian genome, however, cleavage by a restriction endonuclease usually yields too many different DNA fragments to permit convenient isolation of a particular fragment. A common intermediate step in the cloning of a specific gene or DNA segment is the construction of a DNA library.

D/L Isomers

- One of the two enantiomers is by convention designated the D isomer, the other the L isomer. - The stereoisomers of monosaccharides of each carbon-chain length can be divided into two groups that differ in the configuration about the chiral center most distant from the carbonyl carbon. - Those in which the configuration at this reference carbon is the same as that of D-glyceraldehyde are designated D-isomers and those with the same configuration as L-glyceraldehyde as L isomers. - When the hydroxyl group on the reference carbon is on the right in a projection formula that has the carbonyl carbon at the top, the sugar is the D isomer; when on the left, it is the L isomer. - Most of the hexoses of living organisms are D isomers.

UV Promoted Mutations

- Other reactions are promoted by radiation. UV light induces the condensation of two ethylene groups to form a cyclobutane ring. In the cell, the same reaction between adjacent pyrimidine bases in nucleic acids forms cyclobutane pyrimidine dimers. - This happens most frequently between thymidine residues on the same DNA strand. - A second type of pyrimidine dimer called a 6-4 photoproduct is also formed during UV irradiation. Ionizing radiation (x rays and gamma rays) can cause ring opening and fragmentation of bases as well as breaks in the covalent backbone of nucleic acids.

Blunt Ends

- Other restriction endonucleases cleave both strands of DNA at the opposing phosphodiester bonds, leaving no unpaired bases on the ends, often called blunt ends.

Bacterial Artificial Chromosomes

- Plasmids designed for the cloning of very long segments (100,000 - 300,000 bp) of DNA. They generally include selectable markers such as resistance to the antibiotic chloramphenicol (CmR), as well as a very stable origin or replication that maintains the plasmid at one or two copies per cell. - DNA fragments of several hundred thousand base pairs are cloned into the BAC vector. The large circular DNAs are then introduced into host bacteria by electroporation. - These procedures use host bacteria with mutations that compromise the structure of their cell wall, permitting the uptake of large DNA molecules.

Carbohydrates

- Polyhydroxy aldehydes or ketones or substances that yield such compounds on hydrolysis. - Many, but not all, have the empirical formula (CH2O)n; some also contain nitrogen, phosphorus or sulfur.

Nucleic Acid Hierarchical Levels of Complexity

- Primary structure: covalent structure and nucleotide sequence. - Secondary structure: any regular, stable structure taken up by some or all of the nucleotides. - Tertiary structure: complex folding of large chromosomes within eukaryotic chromatin and bacterial nucleoids, or folding of large tRNA or rRNA molecules.

Lectins

- Proteins found in every organism that bind carbohydrates with high specificity and with moderate to high affinity. - Serve in a wide variety of cell-cell recognition, signaling and adhesion processes and in intracellular targeting of newly synthesized proteins.

Pyranoses v. Furanoses

- Pyranoses are aldoses that exist in cyclic forms having xix-membered ring compounds. - Furanoses are aldoses that exist in cyclic forms having five-membered rings.

3D Structures of RNA

- RNA has no simple, regular secondary structure that serves as a reference point, as does the double helix for DNA. The 3D structures of many RNAs, like those of proteins, are complex and unique. - Weak interactions, especially base-stacking interactions help stabilize RNA structures. - Where complementary sequences are present, the predominant double-stranded sequence is an A-form right-handed double helix. Breaks in the regular A-form helix caused by mismatched or unmatched bases in one or both strands are common and result in bulges or internal loops.

Selectable Marker

- Regardless of the approach, few cells actually take up the plasmid DNA, so a method is needed to select those that do. The usual strategy is to use a plasmid that includes a gene that the host cell requires for growth under specific conditions, such as a gene that confers resistance to an antibiotic. Only cells transformed by the recombinant plasmid can grow in the presence of that antibiotic, making any cell that contains the plasmid selectable under those growth conditions. Such a gene is called a selectable marker.

Renaturation

- Renaturation of a DNA molecule is a rapid one-step process, as long as a double-helical segment of a dozen or more residues still unites the two strands. When the temperature of pH is returned to the range in which strands spontaneously rewind, or anneal, to yield the intact duplex. - However, if the two strands are completely separated, renaturation occurs in two steps. • In the first, relatively slow step, the two strands "find" each other by random collisions and form a short segment of complementary double helix. • The second step is much faster: the remaining unpaired bases successively come into register as base pairs, and the two strands "zipper" themselves together to form the double helix.

Restriction Modification System

- Restriction endonucleases recognize and cleave foreign DNA. - In the host cell's DNA, the sequence that would be recognized by its own recognition endonuclease is protected from digestion by methylation of the DNA, catalyzed by a specific DNA methylase. The restriction endonuclease and the corresponding methylase are sometimes referred to as a restriction-modification system.

Nucleotides with phosphate groups in positions other than on the 5' carbon:

- Ribonucleoside 2',3' - cyclic monophosphates are isolatable intermediates and ribonucleoside 3'-monophosphates are end products of the hydrolysis of RNA by certain ribonucleases. - Other variations are adenosine 3',5' cyclic monophosphate (cAMP) and guanosine 3',5' - cyclic monophosphate (cGMP).

Lectins and Viruses

- Several animal viruses, including the influenza virus, attach to their host cells through interactions with oligosaccharides displayed on the host cell surface. The lectin of the influenza virus, known as the HA (hemagglutinin) protein, is essential for viral entry and infection. - After the virus has entered a host cell and has been replicated, the newly synthesized viral particles bud out of the cell, wrapped in a portion of its plasma membrane. A viral sialidase (neuraminidase) trims the terminal sialic acid residue from the host cell's oligosaccharides, releasing the viral particles from their interaction with the cell and preventing their aggregation with one another. Another round of infection can now begin. - Antiviral drugs are sugar analogs; they inhibit the viral sialidase by competing with the host cell's oligosaccharides for binding. This prevents the release of viruses from the infected cell and also causes viral particles to aggregate, both of which block another cycle of infection.

Deamination

- Several nucleotide bases undergo spontaneous loss of their exocyclic amino groups - deamination. • For example, under typical cellular conditions, deamination of cytosine to uracil occurs in about one of every 10^7 cytidine residues in 24 hours. This corresponds to about 100 spontaneous events per day, on average, in a mammalian cell. - The slow cytosine deamination reactions seems innocuous enough, but is almost certainly the reason why DNA contains thymine rather than uracil. The product of cytosine deamination (uracil) is readily recognized as foreign in DNA and is removed by a repair system. If DNA normally contained uracil, recognition of uracils resulting from cytosine deamination would be more difficult, and unrepaired uracils would lead to permanent sequence changes as they were paired with adenines during replication.

Lectins - Circulatory Half-Life of Pituitary Hormones

- Some peptide hormones that circulate in the blood have oligosaccharide moieties that strongly influence their circulatory half-life. - Luteinizing hormone and thyrotropin (polypeptide hormones produced in the adrenal cortex) have N-linked oligosaccharides that end with the disaccharide GalNAc4S (β1→4) GlcNAc, which is recognized by a lectin (receptor) of hepatocytes. - Receptor-hormone interaction mediates the uptake and destruction of luteinizing hormone and thyrotropin, reducing their concentration in the blood. Thus, the blood levels of these hormones undergo a periodic rise (due to pulsatile secretion by the adrenal cortex) and fall (due to continual destruction by hepatocytes).

Polysaccharides and Oligosaccharides as Information Carriers

- Some provide communication between cells and their extracellular surroundings. Others label proteins for transport to and localization in specific organelles, or for destruction when the protein is malformed or superfluous. Still others serve as recognition sites for extracellular signal molecules (e.g. growth factors) or extracellular parasites (bacteria or viruses). - On almost every eukaryotic cell, specific oligosaccharide chains attached to components of the plasma membrane form a carbohydrate layer (glycocalyx), several nanometers thick, that serves as an information-rich surface that a cell shows to its surroundings. These oligosaccharides are central players in cell-cell recognition and adhesion, cell migration during development, blood clotting, the immune response, wound healing and other cellular processes. - In most of these cases, the information carbohydrate is covalently joined to a protein or lipid to form a glycoconjugate, which is the biologically active molecule.

Sticky Ends

- Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to four nucleotides of one strand unpaired at each resulting end. These unpaired strands are referred to as sticky ends because they can base-pair with each other or with complementary sticky end sof other DNA fragments.

Site-Directed Mutagenesis

- Specific amino acids may be replaced individually by site-directed mutagenesis. This approach to studying protein structure and function changes the amino acid sequence of a protein by altering the DNA sequence of the cloned gene. - If appropriate restriction sites flank the sequence to be altered, researchers can simply remove a DNA segment and replace it with a synthetic one that is identical to the original except for the desired change.

Systems Biology

- Studies the multitude of biochemical changes in a cell, including the changes in the cellular protein population, as a function of environmental or genetic stresses.

Polysaccharides

- Sugar polymers containing more than 20 or so monosaccharide units. - Some such as cellulose are linear chains; others such as glycogen are branched.

A-Form of DNA

- The A form is favored in many solutions that are relatively devoid of water. The DNA is still arranged in a right-handed double helix, but the helix is wider and the number of base pairs per helical turn is 11, rather than 10.5 as in B-DNA. The plane of the base pairs in A-DNA is tilted about 20° with respect to the helix axis. - These structural changes deepen the major groove while making the minor groove shallower. - The reagents used to promote crystallization of DNA tend to rehydrate it, and thus the most short DNA molecules tend to crystallize in the A form.

NS Domain Binding and Signaling - Four Types

- The NS domains bind specifically to extracellular proteins and signaling molecules to alter their activities: 1. The change in activity may result from a conformational change in the protein that is induced by the binding. 2. It may be due to the ability of adjacent domains of heparan sulfate to bind to different proteins, bringing them into close proximity and enhancing protein-protein interactions. 3. The binding of extracellular signal molecules to heparan sulfate increases their local concentrations and enhances their interaction with growth factor receptors in the cell surface. The heparan sulfate acts as a co-receptor. 4. The NS domains interact - electrostatically and otherwise - with a variety of soluble molecules outside the cell, maintaining high local concentrations at the cell surface.

B-Form of DNA

- The Watson-Crick structure is also referred to as B-form DNA, or B-DNA. The B form is the most stable structure for a random-sequence DNA molecule under physiological conditions and is therefore the standard point of reference in any study of the properties of DNA.

DNA Hybridization

- The ability of two complementary DNA strands to pair with one another can be used to detect similar DNA sequences in two different species or within the genome of a single species. - Most of the reannealing occurs between complementary mouse DNA strands to form mouse duplex DNA; similarly, most human DNA strands anneal with complementary human DNA strands. However, some strands of the mouse DNA will associate with human DNA strands to yield hybrid duplexes, in which segments of a mouse DNA strand form base-paired regions with segments of a human DNA strand. This reflects a common evolutionary heritage; different organisms generally have many proteins and RNAs with similar functions and often, similar structures. In many cases, the DNAs encoding these proteins and RNAs have similar sequences. The closer the evolutionary relationship between two species, the more extensively their DNAs will hybridize. - A specific DNA sequence or gene can be detected in the presence of many other sequences if one already has an appropriate complementary DNA strand to hybridize with it.

Agarose

- The agar component with the fewest charged groups (sulfates, pyruvates). The gel-forming property of agarose makes it useful in the biochemical lab. - When a suspension of agarose in water is heated and cooled, the agarose forms a double helix: two molecules in parallel orientation twist together with a helix repeat of three residues; water molecules are trapped in the central cavity. These structures in turn associate with each other to form a gel - a 3D matrix that traps large amounts of water. - Agarose gels are used as inert supports for the electrophoretic separation of nucleic acids, and is also used to form a surface for the growth of bacterial colonies.

Mutarotation

- The alpha and beta anomers of D-glucose interconvert in aqueous solution via mutarotation. - Thus a solution of alpha-D-glucose and solution of beta-D-glucose eventually forms identical equilibrium mixtures having identical optical properties.

Average Size of DNA Fragments Produced by Cleavage

- The average size of the DNA fragments produced by cleaving genomic DNA with a restriction endonuclease depends on the frequency with which a particular restriction site occurs in the DNA molecule; this is turn depends largely on the size of the recognition sequence. - In a DNA molecule with a random sequence in which all four nucleotides were equally abundant, a 6 bp sequence recognized by a restriction endonuclease such as BamHI would occur on average once every 4^6 (4,096) bp, assuming the DNA had a 50% G-C content. - Enzymes that recognize a 4 bp sequence would produce smaller DNA fragments from a random-sequence DNA molecule; a recognition sequence of this size would be expected to occur about once every 4^4 (256) bp. - In natural DNA molecules, particular recognition sequences tend to occur less frequently than this because nucleotide sequences in DNA are not random and the four nucleotides are not equally abundant. - In the lab, the average size of the fragments produced by restriction endonuclease cleavage of a large DNA can be increased by simply terminating the reaction before completion; the result is called a partial digest. - Fragment size can also be increased by using a special class of endonucleases called homing endonucleases. These recognize and cleave much longer DNA sequences (14-20 bp).

Monosaccharide Structure - Aldoses - Ketoses

- The backbones of common monosaccharides are unbranched carbon chains in which all the carbon atoms are linked by single bonds. - In the open chain form, one of the carbon atoms is double-bonded to an oxygen atom to form a carbonyl group; each of the other carbon atoms has a hydroxyl group. - If the carbonyl group is at an end of the carbon chain (in an aldehyde group) the monosaccharide is an aldose. - If the carbonyl is at any other position (in a ketone group), the monosaccharide is a ketose. - Monosaccharides with four, five, six and seven carbon atoms in their backbones are called respectively tetroses, pentoses, hexoses and heptoses. There are aldoses and ketoses of each of these chain lengths, e.g. aldotetroses and ketopentoses.

Naming and Numbering of Monosaccharides

- The carbons of a sugar are numbered beginning at the end of the chain nearest the carbonyl group. - Each of the 8 D-aldohexoses, which differ in the stereochemistry at C-2, C-3 or C-4 has its own name: D-glucose, D-mannose and so forth. - The four and five carbon ketoses are designated by inserting "ul" into the name of a corresponding aldose. For example, D-ribulose is the ketopentose corresponding to the aldopentose D-ribose.

Hypochromic Effect & Hyperchromic Effect

- The close interaction between stacked bases in a nucleic acid has the effect of decreasing its absorption of UV light relative to that of a solution with the same concentration of free nucleotides, and the absorption is decreased further when two complementary nucleic acid strands are paired. - Denaturation of a double-stranded nucleic acid produces the opposite result: an increase in absorption called the hyperchromic effect. - The transition from double-stranded DNA to the single-stranded, denatured form can thus be detected by monitoring UV absorption at 260 nm.

Proteome/Proteomics

- The complement of proteins expressed by a genome is called its proteome. This concept rapidly evolved into a separate field of investigation called proteomics.

Convention for Numbering

- The convention for numbering the pentose ring follows standard rules, but in the pentoses of nucleotides and nucleosides the carbon numbers are given a prime (') designation to distinguish them from the numbered atoms of the nitrogenous bases.

Hydrolysis of RNA under Alkaline Conditions

- The covalent backbone of DNA and RNA is subject to slow, nonenzymatic hydrolysis of the phosphodiester bonds. - In the test tube, RNA is hydrolyzed rapidly under alkaline conditions, but DNA is not; the 2'-hydroxyl groups in RNA (absent in DNA) are directly involved in the process. - Cyclic 2',3' - monophosphate nucleotides are the first products of the action of alkali on RNA and are rapidly hydrolyzed further to yield a mixture of 2' and 3'-nucleoside monophosphates.

Lipopolysaccharides

- The dominant surface feature of the outer membrane of gram-negative bacteria such as E. coli and salmonella. - These molecules are prime targets of the antibodies produced by the vertebrate immune system in response to bacterial infection and are therefore important determinants of the serotype of bacterial strands (serotypes of strains that are distinguished on the basis of antigenic properties).

Phenotypic Function

- The effects of a protein on the entire organism. For example, the loss of the protein may lead to slower growth of the organism, an altered development pattern, or even death.

Extracellular Matrix

- The extracellular space in the tissues of multicellular animals is filled with a gel-like material, the extracellular matrix, also called ground substance, which holds the cells together and provides a porous pathway for the diffusion of nutrients and oxygen to individual cells. - The reticular ECM that surrounds fibroblasts and other connective tissue cells is composed of an interlocking meshwork of heteropolysaccharides and fibrous proteins such as fibrillar collagens, elastin and fibronectin.

How to Prepare a Genomic Library

- The first step in preparing a genomic library is partial digestion of the DNA by restriction endonucleases, such that any given sequence will appear in fragments of a range of sizes - a range that is compatible with the cloning vector and ensures that virtually all sequences are represented among the clones in the library. - Fragments that are too large or too small for cloning are removed by centrifugation or electrophoresis. - The cloning vector such as BAC or YAC plasmid, is cleaved with the same restriction endonuclease and ligated to the genomic DNA fragments. The ligated DNA mixture is then used to transform bacterial or yeast cells to produce a library of cell types, each type harboring a different recombinant DNA molecule. Ideally, all the DNA in the genome under study will be represented in the library. - Each transformed bacterium or yeast cell grows into a colony of identical cells, each cell bearing the same recombinant plasmid, one of many represented in the overall library.

Steric Factors and Hydrogen Bonding Influence Homopolysaccharide Folding

- The folding of polysaccharides in three dimensions follows the same principles as those governing polypeptide structure: subunits with a more-or-less rigid structure dictated by covalent bonds form 3D macromolecular structures that are stabilized by weak interactions within or between molecules: hydrogen bonds and hydrophobic and van der Waals interactions, and for polymers with charged subunits, electrostatic interactions. - Because polysaccharides have so many hydroxyl groups, hydrogen bonding has an especially important influence on their structure.

Hydrogen Bonding in Base Pairs

- The functional groups of pyrimidines and purines are ring nitrogens, carbonyl groups and exocyclic amino groups. - Hydrogen bonds involving the amino and carbonyl groups are the most import mode of interaction between two (and occasionally three or four) complementary strands of nucleic acid. - The most common hydrogen-bonding patterns are those in which A bonds specifically to T or U and G bonds to C. These two types of base pairs predominated in double stranded DNA and RNA.

Glycogen

- The main storage polysaccharide of animal cells. - Like amylopectin, glycogen is a polymer of α 1-4 linked subunits of glucose with α 1-6 linked branches, but glycogen is more extensively branched (every 8-12 residues) and more compact than starch. - Glycogen is especially abundant in the liver and is also present in skeletal muscle. In hepatocytes, glycogen is found in large granules, which are themselves clusters of smaller granules composed of single, highly branched glycogen molecules with an average molecular weight of several million. Such glycogen granules also contain, in tightly bound form, the enzymes responsible for the synthesis and degradation of glycogen.

Oxidative Damage of DNA

- The most important source of mutagenic alterations in DNA is oxidative damage. - Excited-oxygen species such as hydrogen peroxide, hydroxyl radicals and superoxide radicals arise during irradiation or as byproducts of aerobic metabolism. • Of these species, the hydroxyl radicals are responsible for most oxidative DNA damage. - Cells have an elaborate defense system to destroy reactive oxygen species, including enzymes such as catalase and superoxide dismutase that convert reactive oxygen species to harmless products. A fraction of these oxidants inevitably escape cellular defenses, however, and damage to DNA occurs through any of a large complex group of reactions ranging from oxidation of deoxyribose and base moieties to strand breaks.

Starch and Glycogen Overview

- The most important storage polysaccharides are starch in plant cells and glycogen in animal cells. Both occur intracellularly as large clusters or granules. - Starch and glycogen molecules are heavily hydrates because they have many exposed hydroxyl groups available to hydrogen-bond with water.

How to Name Disaccharides

- The name describes the compound with its nonreducing end to the left, and the name can be built in the following order: 1. Give the configuration (alpha or beta) at the anomeric carbon joining the first monosaccharide unit to the second. 2. Name the nonreducing residue. To distinguish five and six membered ring structures, insert furano or pyrano into the name. 3. Indicate in parentheses the two carbon atoms joined by the glycosidic bond, with an arrow connecting the two numbers. 4. Name the second residue. If there is a third residue, describe the second glycosidic bond by the same conventions.

Naming Minor Bases

- The nomenclature for the minor bases can be confusing. Like the major bases, many have common names. When an atom in the purine or pyrimidine ring is substituted, the usual convention is simply to indicate the ring position of the substituent by its number. The element to which the substituent is attached is not identified. - The convention changes when the substituted atom is exocyclic (not in the ring structure), in which case the type of atom is identified and the ring position to which it is attached is denoted with a superscript. For example, the amino nitrogen attached to C-6 of adenine is N^6.

Schematic Representation of Nucleotide Sequences

- The nucleotide sequences of nucleic acids can be represented schematically. - The phosphate groups are symbolized by P and each deoxyribose is symbolized by a vertical line, from C-1' at the top to C-5' at the bottom (but keep in mind that the sugar is always in its closed ring β-furanose form in nucleic acids). The connecting lines between nucleotides (which pass through P) are drawn diagonally from the middle (C-3') of the deoxyribose of one nucleotide to the bottom (C-5') of the next.

Disaccharides & Reducing Sugars

- The oxidation of a sugar by cupric ion occurs only with the linear form, which exists in equilibrium with the cyclic form(s). When the anomeric carbon is involved in a glycosidic bond, that sugar residue cannot take the linear form and therefore becomes a nonreducing sugar. - In describing disaccharides or polysaccharides, the end of a chain with a free anomeric carbon (one not involved in a glycosidic bond) is commonly called the reducing end. - The acetal group in the bond cannot be oxidized and mutarotation does not occur because stereochemistry cannot change. There is no OH at the anomeric position.

Nucleotides as Energy Carriers

- The phosphate group covalently linked at the 5' hydroxyl of a ribonucleotide may have one or two additional phosphates attached. The resulting molecules are referred to as nucleoside mono-, di- and triphosphates. - Starting from the ribose, the three phosphates are generally labeled α, β, and γ. - Hydrolysis of nucleoside triphosphates provides the chemical energy to drive many cellular reactions. ATP is by far the most widely used for this purpose, but UTP, GTP and CTP are also used in some reactions. - Nucleoside triphosphates also serve as the activated precursors of DNA and RNA synthesis. - The energy released by hydrolysis of ATP and the other nucleoside triphosphates is accounted for by the structure of the triphosphate group. The bond between the ribose and the alpha phosphate is an ester linkage. - The α, β and β,γ linkages are phosphoanhydrides. - Hydolysis of the ester linkage yields about 14 kJ/mol under standard conditions, whereas hydrolysis of each anhydride bond yields about 30kJ/mol. - ATP hydrolysis often plays an important thermodynamic role in biosynthesis. When coupled to a reaction with a positive free-energy change, ATP hydrolysis shifts the equilibrium of the overall process to favor product formation.

Molecular Function

- The precise biochemical activity of a protein, including details such as the reactions an enzyme catalyzes or the ligands a receptor binds.

RNA Structure Primary Structure

- The product of transcription of DNA is always single-stranded RNA. The single strand tends to assume a right-handed helical conformation dominated by base stacking interactions, which are stronger between two purines than between a purine and pyrimidine or between two pyrimidines. The purine-purine interaction is so strong that a pyrimidine separating two purines is often displaced from the stacking pattern so that the purines can interact. - Any self-complementary sequences in the molecule produce more complex structures. RNA can base-pair with complementary regions of either RNA or DNA. Base pairing matches the pattern for DNA: G pairs with C and A pairs with U. One difference is that base pairing between G and U residues - unusual in DNA - is fairly common in RNA. The paired strands in RNA or RNA-DNA duplexes are antiparallel, as in DNA.

Base Stacking

- The purine and pyrimidine bases are hydrophobic and relatively insoluble in water at the near-neutral pH of the cell. - At acidic or alkaline pH the bases become charged and their solubility in water increases. - Hydrophobic stacking interactions in which two or more bases are positioned with the planes of their rings parallel are one of the two important modes of interaction between bases in nucleic acids. The stacking also involves a combination of van der Waals and dipole-dipole interactions between the bases. Base stacking helps to minimize contact of the bases with water, and base-stacking interactions are very important in stabilizing the 3D structure of nucleic acids.

Comparative Genomics

- The rapid accumulation of genome sequence information has greatly advanced our understanding of evolution. - Another important reason to sequence many genomes is to provide a database that can be used to assign gene functions by genome comparisons.

Factors that Affect DNA Hybridization

- The rate of DNA annealing is affected by temperature, the length and concentration of the DNA fragments being annealed, the concentration of salts in the reaction mixture and properties of the sequence itself (e.g. complexity and G-C content). - Temperature is especially important. If the temperature is too low, short sequences with coincidental similarity from distant, heterologous parts of the DNA molecules will anneal unproductively and interfere with the more general alignment of complementary DNA strands. Temperatures that are too high will favor denaturation.

How to Prepare a cDNA Library

- The researcher first extracts mRNA from an organism or from specific cells of an organism and then prepared complementary DNAs (cDNAs) from the RNA in a multistep reaction catalyzed by the enzyme reverse transcriptase. The resulting double-stranded DNA fragments are then inserted into a suitable vector and cloned, creating a population of clones called a cDNA library. - The search for a particular gene is made easier by focusing on a cDNA library generated from the mRNAs of a cell known to express that gene.

Lectins - Protection from Degradation

- The residues of Neu5Ac (a sialic acid) situated at the ends of the oligosaccharide chains of many plasma glycoproteins protect those proteins from uptake and degradation in the liver. - The mechanism that removes sialic acid residues from serum glycoproteins is unclear. It may be due to the activity of the enzyme sialidase produced by invading organisms or to a steady slow release by extracellular enzymes. - The plasma membrane of hepatocytes has lectin molecules (asialoglycoprotein receptors; asialo - indicating "without sialic acid") that specifically bind oligosaccharide chains with galactose residues no longer protected by a terminal Neu5Ac residue. - E.g. ceruloplasmin

Peptidoglycan

- The rigid component of bacterial cell walls. - A heteropolymer of alternating beta 1-4 linked N-acetylglucosamine and N-acetylmuranic acid residues. - The linear polymers lie side by side in the cell wall, cross linked by short peptides, the exact structure of which depends on the bacterial species. The peptide cross-links weld the polysaccharide chains into a strong sheath that envelops the entire cell and prevents cellular swelling and lysis due to the osmotic entry of water. - The enzyme lysozyme kills bacteria by hydrolyzing the beta 1-4 glycosidic bond between NAG and NAM. Penicillin and related antibiotics kill bacteria by preventing the synthesis of the cross-links, leaving the cell wall too weak to resist osmotic lysis.

Glycobiology

- The study of the structure and function of glycoconjugates. - Cells use specific oligosaccharides to encode important information about intracellular targeting of proteins, cell-cell interactions, cell differentiation and tissue development and extracellular signals.

Nucleic Acid Backbones & Phosphodiester Linkages

- The successive nucleotides of both DNA and RNA are covalently linked through phosphate group bridges, in which the 5'-phosphate group of one nucleotide unit is joined to the 3'-hydroxyl group of the next nucleotide, creating a phosphodiester linkage. - Thus, the covalent backbones of nucleic acids consist of alternating phosphate and pentose residues, and the nitrogenous bases may be regarded as side groups joined to the backbone at regular intervals. - The backbones of both DNA and RNA are hydrophilic. The hydroxyl groups of the sugar residues form hydrogen bonds with water. - The phosphate groups with a pKa near 0, are completely ionized and negatively charged at pH 7, and the negative charges are generally neutralized by ionic interactions with positive charges on proteins, metal ions and polyamines.

Glycomics

- The systematic characterization of all the carbohydrate components of a given cell or tissue, including those attached to proteins and to lipids. - For glycoproteins, this also means determining which proteins are glycosylated and where in the amino acid sequence each oligosaccharide is attached. - Offers potential insights into normal patterns of glycosylation and the ways in which they are altered during development or in genetic diseases or cancer. - Many of the proteins secreted by eukaryotic cells are glycoproteins, including most of the proteins in the blood. For example, immunoglobulins and certain hormones such as FSH, LH and TSH are glycoproteins. - Many milk proteins including lactalbumin and some of the proteins secreted by the pancreas are glycosylated, as are most of the proteins contained in lysosomes.

DNA Microarray Example

- The total complement of mRNA is isolated from cells at two different stages of development and converted to cDNA using reverse transcriptase and fluorescently labeled deoxynucleotides. The fluorescent cDNAs are then mixed and used as probes, each hybridizing to complementary sequences on the microarray. - For example, the labeled nucleotides used to make the cDNA for each sample could fluoresce in two different colors. - By using a mixture of two sample to measure relative rather than absolute abundance of sequences, the method corrects for variations in the amounts of DNA originally deposited in each spot on the grid and other possible inconsistencies among spots in the microarray. - The spots that fluoresce provide a snapshot of all the genes being expressed in the cells at the moment they were harvested. For a gene of unknown function, the time and circumstances of its expression can provide important clues about its role in the cell.

Different Types of Restriction Endonucleases

- There are three types of restriction endonucleases designated I, II and III. - Types I and III are generally large, multisubunit complexes containing both the endonuclease and methylase activities. - Type I restriction endonucleases cleave DNA at random sites that can be more than 1000 base pairs from the recognition sequence. - Type III restriction endonucleases cleave the DNA about 25 bp from the recognition sequence. - Both types move along the DNA in a reaction that requires the energy of ATP. - Type II restriction endonucleases are simpler, require no ATP and cleave the DNA within the recognition sequence itself. The recognition sequences are usually 4-6 bp long and palindromic.

Watson-Crick Model and Replication

- This structural model immediately suggested a mechanism for the transmission of genetic information. The essential feature of the model is the complementarity of the two DNA strands. This structure could logically be replicated by separating the two strands and synthesizing a complementary strand for each. - Because nucleotides in each new strand are joined in a sequence specified by the base-pairing rules, each preexisting strand functions as a template to guide the synthesis of one complementary strand.

Photolithography

- This technique for preparing a DNA microarray makes use of nucleotide precursors that are activated by light, joining one nucleotide to the next in a photoreaction (as opposed to the chemical process). - A computer is programmed with the oligonucleotide sequences to be synthesized at each point on a solid surface. The reactive groups on that surface are initially rendered inactive because of attached photoactive blocking groups. - A screen covering the surface is opened over the areas programmed to receive a particular nucleotide. A flash of light eliminates the blocking group in the uncovered spots, and then a solution with a particular nucleotide activated to react at its 3'-hydroxyl group (A*) is washed over the surface. - A blocking group on the 5'-hydroxyl group of the nucleotide prevents unwanted reactions and the nucleotide becomes linked to the surface in the illuminated spots via its 3'-hydroxyl group. - The screen is then replaced with another screen that selectively illuminates only spots programmed to receive a G; light removes the 5' blocking groups of previously bound nucleotides and G* is then added to link G to those spots. - The surface is washed successively with solutions containing each remaining type of activated nucleotide (C* & T*) using selective screening and light to ensure that the correct nucleotides are added at each spot in the correct sequence. This continues until the required sequences are built up on each spot on the surface. Many polymers with the same sequence are generated on each spot.

Expressed Sequence Tag

- To aid in the mapping of large genomes, cDNAs in a library can be partially sequenced at random to produce a useful type of STS called an expressed sequence tag (EST). ESTs, ranging in size from a few dozen to several hundred base pairs, can be positioned within the larger genome map, providing markers for expressed genes.

Epimers

- Two sugars that differ only in the configuration around one carbon atom.

PCR Procedure

- Two synthetic oligonucleotides are prepared, complementary to sequences on opposite strands of the target DNA at positions defining the ends of the segment to be amplified. The oligonucleotides serve as replication primers that can be extended by DNA polymerase. - The 3' ends of the hybridized probes are oriented toward each other and positioned to prime DNA synthesis across the desired DNA segment. Isolated DNA containing the segment to be amplified is heated briefly to denature it, and then cooled in the presence of a large excess of the synthetic oligonucleotide primers. The four deoxynucleoside triphosphates are then added, and the primed DNA segment is replicated selectively. - The cycle of heating, cooling and replication is repeated 25-30 times over a few hours in an automated process, amplifying the DNA segment between the primers until it can be readily analyzed or cloned. - PCR uses a heat-stable DNA polymerase, such as the Taq polymerase (derived from a bacterium that lives at 90°C), which remains active after every heating step and does not have to be replenished. - Careful design of the primers used for PCR such as including restriction endonuclease cleavage sites can facilitate the subsequent cloning of the amplified DNA.

Contig

- Using hybridization methods, researchers can order individual clones in a library by identifying clones with overlapping sequences. A set of overlapping clones represents a catalog for a long contiguous segment of a genome, often referred to as a contig.

Tm

- Viral or bacterial DNA molecules in solution denature when they are heated slowly. Each species of DNA has a characteristic denaturation temperature, or melting point (tm; formally, the temperature at which half the DNA is present as separated single strands): the higher its content of G-C base pairs, the higher the melting point of the DNA. This is because G-C base pairs, with three hydrogen bonds, require more heat energy to dissociate than A-T base pairs. - Thus, the melting point of a DNA molecule, determined under fixed conditions of pH and ionic strength, can yield an estimate of its base composition. - If denaturation conditions are carefully controlled, regions that are rich in A-T base pairs will specifically denature while most of the DNA remains double-stranded. Such denatured regions (called bubbles) can be visualized with electron microscopy. - In the strand separation of DNA that occurs in vivo during processes such as DNA replication and transcription, the sites where these processes are initiated are often rich in A-T base pairs.

Oligonucleotide-Directed Mutagenesis

- When suitably located restriction sites are not present, an approach called oligonucleotide-directed mutagenesis can create a specific DNA sequence change. - A short synthetic DNA strand with a specific base change is annealed to a single-stranded copy of the cloned gene within a suitable vector. The mismatch of a single base pair in 15 to 20 bp does not prevent annealing if it is done at an appropriate temp. - The annealed strand serves as a primer for the synthesis of a strand complementary to the plasmid vector. This slightly mismatched duplex recombinant plasmid is then used to transform bacteria, where the mismatch is repaired by cellular DNA repair enzymes. - About half of the repair events will remove and replace the altered base and restore the gene to its original sequence; the other half will remove and replace the normal base, retaining the desired mutations. - Transformants are screened (often by sequencing their plasmid DNA) until a bacterial colony containing a plasmid with the altered sequence is found.

Z-Form of DNA

- Z-form DNA is a more radical departure from the B structure; the most obvious distinction is the left-handed helical rotation. There are 12 base pairs per helical turn, and the structure appears more slender and elongated. The DNA backbone takes on a zigzag appearance. - Certain nucleotide sequences fold into left-handed Z helices much more readily than others. Prominent examples are sequences in which pyrimidines alternate with purines, especially alternating C and G or 5-methyl C and G residues. To form the left-handed helix in Z-DNA, the purine residues flip to the syn conformation, alternating with pyrimidines in the anti conformation. - The major groove is barely apparent in Z-DNA and the minor groove is narrow and deep.

Fischer Projections

- in Fischer projections, horizontal bonds project out of the plane of the paper and vertical bonds project behind the plane of the paper.

Steps in DNA Cloning

1. Cutting DNA in precise locations. Sequence-specific endonucleases (restriction endonucleases) provide the necessary molecule scissors. 2. Selecting a small molecule of DNA capable of self-replication. These DNAs are called cloning vectors (a vector is a delivery agent). They are typically plasmids or viral DNAs. 3. Joining two DNA fragments covalently. The enzyme DNA ligase links the cloning vector and DNA to be cloned. Composite DNA molecules comprising covalently linked segments from two or more sources are called recombinant DNAs. 4. Moving recombinant DNA from the test tube to a host cell that will provide the enzymatic machinery for DNA replication. 5. Selecting or identifying host cells that contain recombinant DNA.

How to Convert a Fischer Projection to a Haworth Perspective Formula

1. Draw 6-membered ring with O atom at the upper right. 2. Number carbons in a clockwise direction beginning with anomeric carbon. 3. Place the hydroxyl groups. a) If a hydroxyl group is to the right in the Fischer projection, place it pointing down. b) If a hydroxyl group is to the left, place it pointed up.

Two Major Families of Heparan Sulfate (Membrane) Proteoglycans:

1. Syndecans - have a single transmembrane domain and an extracellular domain bearing 3-5 chains of heparan sulfate and in some cases, chondroitin sulfate. 2. Glypicans - attached to the membrane by a lipid anchor, a derivative of the membrane lipid phosphatidylinositol. - Both syndecans and glypicans can be shed into the extracellular space. A protease in the ECM that cuts close to the membrane surface releases syndecan ectodomains and a phospholipidase that breaks the connection the membrane lipid releases glypicans.

How do other glycosaminoglycans differ from hyaluronan?

1. They are generally much shorter polymers. 2. They are covalently linked to specific proteins (proteoglycans). 3. One or both monomeric units differ from those of hyaluronan.