Water is nearly tetrahedral; it is bonded, on average to 3.

4 other water molecules; even at 37 C, 15% of water molecules are jointed to four others in a short-lived assembly known as a flickering cluster Bottom of ocean is permanently 4 C; pressure of water column keeps bottom of ocean under high pressure and in liquid form The oxygen is the central atom for the water, and the two electron pairs stick out in a tetrahedral configuration, which is what bonds with the hydrogen In ice, each water molecule forms four hydrogen bonds to other water molecules, making a crystal lattice Hydrogen bonds occur between bases in DNA, between peptide bonds in proteins, and between water and proteins; sometimes the formation of H-bonds results in specific biochemical structures such as alpha helices Water competes with biomolecules for H-bonds, and the strength of the H-bond can be increased by shielding the bond from water; this happens in double helical DNA, where the base-base hydrogen bonds are within the helix and not exposed to water Hydrogen bonding occurs in the protein lysozyme, which is in egg whites, and chews up bacteria The reason the H-bonds can occur between amino acids in a protein (which makes 2ndary protein structure), and not with water, is because the strength of the H-bonds depends on the local environement. If there is a lot of water, the water will form the hydrogen bonds instead. Nmethyl acetamide in dioxane, tetrachloromethane and water demonstrates this, since in dioxane, it forms the H-bond with itself, but in water, it makes the H-bond only with water, and there is little H-bonding with itself. H-bonds are strongest when the three atoms lie in a straight line; they are about only 1/20 as strong as a covalent bond, and 0.27 nm in lengthlarger than the normal covalent bond of 0.1 nm Water competes for ionic bonds of salts just as it competes for H-bonds; this results in the solvent properties of water Salts are highly stable structures, since they have positively charges sitting right next to negatively charged ions, but the fact that they dissolve in water and release little heat shows the strength the heat of hydration reaction Outside of DNA has a net negative charge and + ions cluster around it; histones have a net positive charge (therefore, they must have a large proportion of lysine and arginine); DNA folding in chromosomes depends on charge-charge interactions, because the histones neutralize the DNA, and the chromosome is 10,000 times shorter than extended DNA, and that amount of charge would blow up. Thus, folding DNA around nucleosome core particles depends on ionic bonds. Even in water, cations and anions are attracted, which is why DNA and histones are attracted to each other in aqueous environment. However, if there is a large salt concentration in the water, the DNA and histones will be less strongly attracted to each other since the ions will get between the two; this is why separating histones and DNA is done by high salt concentrations. Proteins consist of many amino acids, which are positively and negatively charged. The positive and negative charges depend on the pH; at the pH where there are equal + and charges which cancel each other out, this is called the pI, or isoelectric point. At pH=pI, the protein is least soluble, since it is not charged. At higher or lower than pI, the + or charges accumulate and push away from each other, and are easier to dissolve. As the salt concentration of water in which protein is dissolved increases, the solubility of the protein increases, since the salts interact with the protein Histidine is used a lot in catalysis, because a small change in pH can make it positive or neutral, activating or deactivating it, since it dissociates at neutral pH, in comparison to the carboxylic acid aas which dissociate at low pH and arginine and lysine which dissociate at high pHs

Cysteine oxidizes spontaneously to form Cystine; the disulfide bonds that cysteine forms within a pp or even between two separate peptides can be used in protein engineering by reducing the protein to break the bonds, and then curling the protein and reoxidizing it. Hydrophobic effect is not because of a large affinity of non-polar molecules, but to prevent the disruption of the hydrogen bonds of water Amphipatic molecules form micelles when thrown in water to remove non-polar regions from water; micelles have a non-polar region in the center; liposomes have an aqueous cavity in the center The ionization state of a weak acid or base depends on the pH. At pH values below the pKa the group is mostly protonated; at pH values above the pKa, the group is mainly dissociated. When the pH=pKa, half of the groups are dissociated and half are protonated Weak acids and bases on biomolecules have similar pKa values to the same groups in water but not bound to the biomolecule, but not exactly the same values; this is because on a protein, the pKa of a group can be changed by the local environment. For example, a carboxyl group on a protein with a nearby protonated amino group with a plus charge will favor the dissociation of the carboxyl group and decrease the pKa (as in the case of serine proteases) At pH values near neutrality, an amino acid is a zwitterion, and has both a negative and positive charge Amino acids, except for glycine, have optical isomers, L and D. Proteins consist exclusively of Lamino acids Peptide bonds have partial double bond character which prevents rotation around the C=N partial double bond; the four atoms C=N, and carboxylic O with partial negative charge, and H attached to N, along with alpha carbon are in plane. Rotation of plane occurs around the alpha-carbon Residual amino acid backbone is neutral; the proteins charge comes from the side chains and the N and C terminal residues which is negligible C and N atoms of peptide bond do not have formal charges and are not further protonated or deprotonated at any pH; the O has a partial charge and the N has a partial + charge Peptide bond is very stable and does not hydrolyze as do ether bonds. Only enzymes can cleave them Alpha helix, beta sheet, collagen helixlinear helix created by three peptide chains wrapped around each other and limited flexibility, beta turnsconformations adapted when peptide chain has to sharply change direction in a folded proteinalmost always have proline to force the turn and/or glycine, which bc of its small side chain has larger flexibility Alpha helices such as wool can absorb water and are flexible; beta sheets such as silk do not absorb water and are not flexible Proline has two configurations because of its ring structuretrans (94%) and cis (6%). All other amino acids are almost always trans (99.95%) Examples of protein structure: prealbumin: B-sheet is dominant 2 structure; two B-sheets are adjacent and form a dimer, which is tertiary structure. Two identical subunits= quaternary structure Cytochrome C= monomer, so no quaternary structure, and no common motifs can be seen. It has a prosthetic heme group in the center Lysozyme has groove for substrate; composed of many B-sheets Myoglobin is dominantly a-helix; hemoglobin is tetramer of four myoglobin-like subunits which is quaternary structure; bee venom called melittin normally has the non-polar side chains distributed evenly throughout, but when in contact with a lipid membrane, it forms an alpha-helix type structural motif, so that the side chains of half the helix are non-polar and all on one side so that they can bind to the membrane of the target and destroy it; the other side is positively charged. The melittin forms a tetramer, and makes a large hold in the membrane.

Some proteins have two or more globular regions but on a single peptide chainand these are domainsTroponin C has such domains; in between domains are usually the binding site of substrates, since there is a gap between the two globular regions Ribonuclease has four disulfide bonds between cysteinshas a B-sheet center, and histidines holding a metal (zinc) Because disulfide bonds are so prominent in protein structure, reducing them will destroy the disulfide bonds and denature the proteins. Some common reducing agents used to denature proteins are urea, B-mercaptoethanol (HOCH2CH2SH), and guanidine hydrochloride (see structure before exam) Additional motifs are being identified, many of which are combinations of the well known motifs, such a B-barrel, which is a B-sheet wrapped into a cylinder, B-a-B Loop, a-a Corner, and twisted B-sheet. B-barrel example is green fluorescence protein which oxidizes and fluoresces spontaneously, and can be injected into animals and make them green Protein folding would be too slow if random, which suggests that the primary structure includes information that guides folding Some proteins such as insulin do not spontaneously refold if they are denatured. This is true in proteins that have undergone posttranslational modification. To help such proteins fold are other proteins called chaperone proteins Immunoglbulins: tetramer including four polypeptides, two subunits of two equivalent polypeptides; each pp is composed of two or more flexible domains that helps for binding to antigens and interacting with cell membranes and membrane bound proteins which require flexibility. The quaternary structure is not well defined, as with hemoglobin which has a tetramer structure. The dimains have Serine proteases are used to hydrolyze other proteins and cut them in two. It involves aspartic acid and histidine residues along with serine, which is called the catalytic triad. Because the pKa of serine is very high, it almost never dissociates, so the aspartic acid induces the histidine to remove the hydrogen from the OH group of serine. Serine becomes negatively charged and ready to covalently bond with the protein needing to be cut, thus causing hydration rxn that would chop it in two Only aromatic amino acids absorb light in the UV range, so it can be used to determine protein concentration. In addition, tryptophan (Trp, W) can be used in protein fluorescence experiments After protein is formed, it undergoes posttranslational modification, and some of the aas are changed. Proline can be changed into hydroxyprolinewithout the hydroxyl group, the proline is not stable, and since it is used in connective tissue, scurvy results. Glutamate can be changed into carboxyglutamate which has an extra carboxyl group on the terminal carbon, and binds to calcium well, and is used in blood clotting. In addition, hydroxyl groups in serine can be phosphorylated to make phosphoserine to regulate the enzyme. Flavoproteins are involved in ox/red and have a prosthetic flavin nucleotide in the center that is not covalently bound Zymogens are the inactive precursor of the active enzyme, and they are self-inhibiting, with a length of the pp covering the active site. Other activating enzymes, usually serine proteases come and cleave the pp so that the active site is exposed. This cleavage is irreversible, so other inhibiting proteins are needed to inactivate it after this. In the blood coagulation cascade, each factor (XII, XI, X, IX, prothrombin, etc) are zymogens that become activated into (XIIa, XIa, Xa, IXa, thrombin, etc) and activate the next zymogen in the chain. Each of these activated factors are serine proteases. Maintaining enzymes initially in a zymogen form is important for many enzymes such as chymotrypsin, since they do not eat out of control. Other than zymogens, there exist other ways to regulate enzymes: 1. Allosteric binding sites (i.e. feedback inhibition), 2. Regulation by covalent modification, 3. Induction or repression of enzyme synthesis

3D structure of protein can be determined through NMR and x-ray crystallography Rate limiting step is the highest energy barrier in a free enrgy level diagram

Serine Proteases Example: thrombin (serine protease) cleaves fibrinogen (zymogen) into fibrin II (active form). Different serine proteases cleave different proteins based on the shape and aams at the active site. Although all serine proteases have Ser, His, and Asp at the active site, different serine proteases like different sequences: Trypsin: K,R Chymotrypsin: F,W,Y Thrombin: R,P Elastin: F,G Therefore, thrombin only allows a Arg and Pro sequence, and cleaves between ProArgGly, between ArgGly peptide. This is a hydrolysis rxn and involves water as a reactant. An enzyme is needed to cleave the peptide bond, bc it is extremely stable. The rxn involves an initial burst, which is evidence for an acyl-enzyme intermediate in the reaction mechanism. Drug development of enzyme inhibitors: molecules are searched for by computer that would fit in the active site. Molecules are chosed to maximized H-bonds, hydrophobic interactions, and ionic/ionic interactions. Molecules are tested, and those that have a high Ki are discarded, and low Ki are considered. Since Ki is the dissociation constant of the inhibitor from the enzyme, a low Ki means a tighter bonding. This is the drug design based on shape complimentarity. Opposed to this is a rational drug design which takes into consideration not the shape of molecule that fits in the active site, but the shape of the intermediate structure that fits in the active site; this takes into consideration the mechanism of the reaction the enzyme catalyzes as well, since the enzyme should have an active site that is complentary to the transition state, and not the substrate to be most effective. This is transition state stabilization. The enzyme seeks to destabilize the ES complementarity, otherwise, the free enrgy of the substrate will be so low that even getting to any transition state will be large. Instead, the enzyme seeks to stabilize the transition state by (1) hydrogen bonds from the enzyme to the transition state structure, (2) ionic interactions to neutralize charges, (3) hydrophobic interactions, and (4) chemistry that stabilizes the transition state, such as general acid/base chemistry. In addition, stabilization of the transition state shape is also important. After this the product is released. An example of the acid/base chemistry that stabilizes the transition state is that of the serine proteases. In serine proteases, the general acid/base and transition state stabilization are the three most important parts of catalysis. The general acid/base is histidine in serine proteases, but can be other aas such as Tyr, Cys, Asp, Glu, Lys, Arg, etc. Now that we know this, a drug that inhibits thrombin would have a R, P residues to gain specificity to thrombin; it also needs to attach to a tetrahedral group such as a boron complex. This has a Ki of 26 pM, which is good. However, during the second round of modifications, we notice that in the active site, there is a hydrophobic area, so we attach a Phe residue as well, and the Ki becomes 3.5, which is very good. DONE Rational Drug Design The serine attaches to the carbonyl carbon, forming an acyl-enzyme intermediate. This is the fast step. The rate limiting step is the water attacking and the serine detaching. Enzyme Kinetics

Michaelis-Menten equation ONLY describes initial reaction rates, and therefore, assumes that [P] is low, and therefore, k-2 = 0, since there is little product to go backward. K2 or kcat is the rate limiting step since the product is made from it, and therefore, the v0 of the entire rxn is v0=kcat[ES]. We can assume that [ES] does not change much because, first the [S]>>[E], so [ES] should remain saturated, and second, because this only is initially when ES is all saturated. Note that kcat does not have to equal k2, but is simply the rate limiting step, which can be k3 if it exists, or something else that is more complicated. If the binding of S to E is rapidly reversible relative to catalysis, k-1>>kcat, and Km=k-1/k1=[E][S]/[ES], which is the thermodynamic constant, or the dissociation constant, Kd. This is NOT always true. Low Km values means higher affinity for substrate binding, and high Km values means lower affinity ONLY when Km=Kd; when it does not, then it is not a measure of the affinity of the substrate to the enzyme, but is a more complex constant. Kcat or k2 is Vmax/[E]tot= (number of product molecules/unit of time)/[E]tot, so it is the number of product moleucles formed each unit of time by each enzyme molecule, and thus is called the turnover number. It has the units of 1/s. Because the maximum velocity occurs when the enzyme is saturated, (i.e. when [ES]=[E]tot), Vmax=k2[E]tot, since V0=k2[ES] Human Genetics I Genomics involves the study of ALL genes, not just one; it is the function and interactions of all the genes in the genome.all disease with the esception of trauma is genetic. Types of genetic disease: 1. 2. 3. 4. 5. 6. Single gene disorder Chromosomal disorders Mitochondrial disorders Multifactorial/polygenic disorders Somatic cell genetic disordernot inherited, cancer cells, for insance Other inheritance patterns, such as imprinting, uniparental disomy, etc.

Gene is a functional and physical unit of heredity that is inherited. A locus is a position of a gene on a chromosome. A mutation can occur through substitityuion of one base for another, or deletion of DNA sequence, a duplcication of DNA sequence, or insertion of new DNA material; usually a mutation causes no difference in phenotype, but it can cause mild or severe problems as well. Mutations can cause loss of function of a protein (seen in recessive phenotypes), or gain of function mutation that increases the normal function of protein (frequently dominant phenotypes); the mutation can confer a completely new proprotery on a protein. It can also cause dominant-negative alleles which disrupts the function of the wild-type allele in the same cell (usually seen in dominant)????? Common genetic terms: allele: alternative form of genetic information at a particular locus; polymorphism: at least two relatively common alleles at a locus First cousins first removed, second cousins Single gene traits/disorders Genes are not dominant/recessive, but the phenotype is dominant/recessive/ In addition, there is codominant, where both alleles are expressed, such as the ABO blood groups

Homozygous/heterozygous: idenetical/different alleles at a given locus; compound heterozygote: two different mutant alleles at a given locus Consanguinity: individuals related by blood Degrees of relationships: fisrt degree: parenets, sibs, offsprings; second degree: grandparents, aunts, nieces; third degree: cousins Autosomal dominant inheritance Factors complicating pedigree analysis: new mutations where there is no antecedent in family history; also fitness: if a phenotype causes babies die out, you wont see any of the living children have the disease, even though the parent has it, which might make it seem that the parent does not have it. Germline mosaicism Penetrance is the proportion of individuals with a disease genotype who express the disease phenotype. Some diseases are not 100% penetrant, and are present in the Also there is variable expressivity which is that the severity of disease may vary greatlyextent of expression of phenotype Other complications of pedigree analysis: pleitropy: when a gene has multiple, seemilngly unrelated effects; heterogeneity: similar phenotypes caused by different genotypes: this can happen through locus heterogeneity where mutations at different loci can cause heterogeneity, but can also happen through allelic heterogeneity where two different alleles at the same loci cause the same phenotype Linkage: co-inheritance of two or more non-allelic genes at nearby loci Delayed age of onset: genetic disorders wich are not apparent until into adulthood: huntingdon disease, Parkinsons Dominantvertical transmission Recessivehorizontal transmission Cousins share 1/8 of genes in common When a person is a carrier of a recessive autosomal disease, you know she is not diseased, so her chance is 2/3, and not Obligate heterozygote vs obligate carriers Lyon hypothesis : one X chromosome in each somatic cell is randomly inactivated early in embryonic development in females. CHECK ensures dosage compensation????? May lead to manifesting heterozygote???? Random, fixed, and incomplete?? X-linked dominant: affected fathers will pass it to 100% of daughters; and affected mothers will pass it to 50% of sons, and 50% of females. X-linked recessive???? Other factories: sex-limited traits: autosomal genes expressed only in one sexso a disease of the ovaries will only affect females Sex-influenced traits : different proportions of expression depending on sex Phenocopy: environmental factors mimic a genetically determined trait

Multifactorial Inheritance (not single gene defects): Most common adult disease as well as congenital probles are a result of a complex interaction of genetic and environmental factors. Polygenic are traits caused by an additive effects of multiple genes. We know that genetic factos are important in common disorders because there is a familial tendency towards a certain disease, and many people in a family have the same disease. Also concordance studies in MZ and DZ (dizygotic twins) provides evidence. Note that not everuything that is familial is genetic familial are things that do not necessarily have a genetic basis Quantitative traits: some phenotypes that are measured on a continuous scalelike height, skin color; caused by an additive effect of genetic and environmental factors; abnormal is on the extreme sides. Threshold traits: dichotomous traits: applies to multifactorial traits that dichotomouseither present or notassumes that there is an underlying continuous variation in liability distribution for the disease but no clinical effect or phenotype until it exceeds a threshold for the phenotype. Some common multifactiorial malformations are cleft lip, neural tube defects, congenital dislocation of the hip, congenital heart defects, and pyloric stenosis. Characterisitcs of Multifactorial Inheritance: although the disorder is familial, there is no distinct pattern of inheritance in the family; so family members more likely to have genes in common with affected person have a higher risk. Risk to first degree relatives is the square root of the population risk. Recurrence risk is higher when more than one family member is affected. The more severe the malformation in the family member(s), the greater the recurrence risk. Even if the trait is more frequent in one sex, the other sex is still at higher risk for recurrence. So for example, in pyloric stenosis, the threshold is lower in males, and females have a higher threshold. Regardless of which parent is affected, the male offspring is at higher absolute risk, but sons of the affected mothers are at the highest risk, since an affected female must have an extreme liability to be above the threshold. So the best estimate of recurrence risk is the empiric risk which is what is observed in similar families in a population. Although the recurrence risk estimate increase with more affected individuals, actually what is known about the family risk has increasednot that the risk has actually increasedthe risk was always there. Population Genetics: Gene Frequency: proportion of each allele in a population; genotype frequency: proportion of each genotype in a population Hardy Weinberg Principle: in absence of influences that change them, allele and genotype frequencies are constant from one generation to another. This relates gene frequency to genotype frequency. Assumptions of Hardy Weinberg: population must be so large that there is no random fluctuation in the popnot true because of genetic drift (random change in gene frequency), and founder effect?????? 2. Migration is due to gene flow???? 3. No mutations 4. Random matingnot true because of assertative mating (choosing mates that are similar to us phenotypically, culturally, religiously etc.) consanguinity (individuals marrying blood relatives). 5. No selection (no genotype preferred by environment)not true because of fitnesscertain phenotypes produce fitness and others cause problems that kill people, and the frequency should decrease. Autosomal Dominanthomozgotes are so rare that they can be ignored in calcs. Genetic test: analysis of chromosomes to determine problems: diagnostic testing, newborn screening tests, carrier testing, prenatal testing, predictive testing.

Predictive testing: presymptomatic testing: you test to see whether a certain gene is muatated, and if it is, that person will definitely develop it Predispositional testing: check to see whether a gene is present that will dispose a person to a disease Cancer that is familial is rarethey are rare types and the onset is early, and many people in the family have it. Only 5-10% of cancers are familial. In this case, the BRCA1 and BRCA2 genes, which are autosomal dominant, increase the chance of developing breast and ovarian cancers. High reisk familes can undergo prophylactic mastectomy and oovarie????? Mastectomy where breasts and ovaries are chopped off. Genetic testing for BRCA mutations is useful. Clinical Cytogenetics The study of chromosomes; due to DNA folding around histones, the total length of the chromosome is 10,000 times shorter than extended length. Chromomal abnormalities: constitutional abnormalities: present at birth, usually at conception or shortly thereafter; acquired abnormalities: develop in somatic cells, and are associated with various types of cancer. Hematological malignancies vs solid tumors?????? Mitosis: Interphase consists of G1, S and G2. G1 is interval between mitosis and replication. S is when DNA replication occus. G2 is interval between S and mitosis. DNA chromosomes can only be seen in the metaphase of mitosis. N= haploid chromosome number, and c= DNA content; cells can be arrested in metaphase by use of mitotic poisons such as colchicine which disrupts spindle formation and attachment to kinetochores at the centromere Mitotic nondisjunction: abnormal segregation of chromosomes; an extra chromosome goes to one cell, and the other cell gets one less. Usually more chromosomes is more tolerable than less chromosomes, and the lesser chromosome cell dies. But the extra chromosome cell divides and makes an extra cell lineage (along with the normal cells); when there is more than one type of cell populations, it is called mosaicism. Meiosis: occurs during gametogenesis. Comprised of 1 round of DNA replication, but 2 cell divisions. N decreases by two in the first round, and c decreases by two in the second round. Consequences of nondisjunction at Meiosis I and Meiosis II. If nondisjunction happens at Meiosis I, then out of four daughter cells, two cells will have double chromosomes (heterodisomy because the two chromosomes will be different with both paternal and maternal chromosomes), and two cells will have no DNA content. If the nondisjunction happens at Meiosis II, two cells will be normal, and one cell will have no genetic material, and one cell will be homodisomy with two copies of either the maternal or paternal chromosome. Upon fertilization with normal gamete, the no gentetic material cell will become monosmoy, and the disomy will become trisomy. Numerical chromosome abnormalities: aneuploidy: loss/gain of single chromosomes: monosomy=n=45; trisomy=n=47 Euploid: exact multiple of haploid set; haploid is normal amount in germ cells, with n=23. Diploid is normal amount in somatic cells, with 2n=46, but triploidy is 3n=69, and is a problem which is very rare, and 60% are double fertilizations (two sperms fertilize one egg), and 40% are through diploid eggs. Most die, but rare mosaics (with few abnormal cells and many normal cells, with abnormal cells going to the non-vital organs) may survive with moderate mental retardation Speciments for the Detection of Chromosome Abnormalities need to have a nucleus and be actively dividingand since most cells are not actively dividing and are already differentiated, very few cells can be directly used; the others need to be artificially activated to divide:

1. Bone marrow (divides fast, and in 24 hours abnormality if there can be found) 2. Peripheral blood(good for constitutional abnormality detection, but does not divide as fast, so they can be stimulated to grow faster with mitogens such as phytohemagglutinin (PHA); usually lymphocytes are used for analysis 3. Fibroblasts (skin biopsy, or from internal organs such as liver, or kidney); this is used when blood chromosome results are normal but clinical evaluation suggest an abnormal cell line. It is possible for mosaicism to occur in which one tissue is normal and another is abnormal, so different tissues are sometimes used 4. Amniocentesis, and chorionic villus sampling (for prenatal diagnosis). Amniocentesis is done in second trimester with an ultrasound guided with a needle goes intraabdominal or intravaginal to get fluid which has skin cells or urine of baby and withdraw 10-15 mL, add to centrifuge tube, and remove the cells and culture them (so this process takes some time, not one day), and then analyze the chromosomes. See notes for exact procedure. Chorionic villus sampling CVS is done in the first trimester Procedure culture cells by adding mitogen to make them start dividing, and the harvest them with several steps: 1. Add colchicine to stop them in metaphase. 2. Add them in a hypotonic solution of KCl or Na citrate to make them swell. 3. Put them in a methanol/acetic acid 3:1 ratio to make the outside cell membrane brittle. 4. Drop them onto a microscope slide to shatter them 5. Stain them with a banding method, most commonly Trypsin Giemsa banding or G-banding 6. Microscope analysis to report the findings by arranging them on a karyotope Three types of chromosomes: metacentric where centromere is in the middle, submetacentric where centromere is one side, A special class of chromosomes is termed acrocentric (chromosomes 13, 14, 15, 21, and 22) they have a very small (short arm) p arm with satellites that have repeating DNA sequences and stalks that contain multiple repeated copies of the genes for ribosomal RNA (also called NOR for nuclei organizing regions). The acrocentric chromosomes all code for only rRNA from the short arm, the long arm does nothing. Only the acrocentric chromosomes code for rRNA. Long arm is also called the q arm. Check the division of chromosome lecture and text. Not in notes. The trypsin giemsa banding technique bands the chromosomes through a method not fully understood. This allows for recognition of different chromosomes. The landmark region divides both the p and q arms into a region 1 and 2, and the banding further is divided into subregions. Therefore, the term 5q14 is chromosome 5, region 1 on the q arm, subregion 4. Chromosome 21 is smaller than chromosome 22, even though in all other cases, the smaller number chromosome is larger. So the smallest chromosome is 21, and that is why a trisomy of 21 is more viable than other chromosomal trisomies, and is the most common, though trisomy 13 and 18 can survive as well. Turner Syndrome: only viable monosomy, with only one X chromosome, and no other sex chromosome. Klinefelter 47 XXY, Down syndrome 47 XY+21these are numerical abnormalities: when in the nonsex chromosomes (i.e. autosomes) like down syndrome, or in sex chromosomes, like Turner syndrome. There are also structural abnormalities like deletions (terminal deletion and interstitial deletion), duplications, inversions (switching of material), and translocations (balanced, unbalanced, and Robertsonian translocations)most of these are constitutional abnormalities. Usually in the germ cells

when this happens, it is corrected, but when it is not corrected, these structural abnormalies occur. ACENTRIC ???? Unequal crossing over also causes deletions and gains of genetic material. Ring chromosomes usually involve a loss of both the terminal ends parts and the remaingin sticky ends connect to form a ring. Isochromosomes: isop and isoq chromosome how does it happen???? Unequal crossing over due to misaligned chromosomes results in one chromosome have a duplication (partial trisomy), and the other having a deletion (partial monosomy); ring chromosomes are made when the telomeres from both ends of the chromosome are deleted and the ends which are sticky join together to make a ring. Isochromosomes are created when during separation of sister chromatids, the two p arms and two q arms stay together and the plane along the centromere which the chromosome should separate is wrong, so that each chromosome has two p arms and two q arms. Wolf-Hirschhorn Sundrome (4p-): happens due to partial deletion of chromosome 4. 1/25,000 newborns, low birthweight, microcephaly (small head), beaked, prominent nose, profound mental retardation (IQ<35), congenital heart disease, seizures, FISH probe available for diagnosis Inversions: flip of genetic material so it is out of order; includes pericentric inversion (flip includes centromere) and paracentric inversion (does not include centromere); inversions are balanced rearrangements, so they cause no phetypical abnormality to the individual, but they may cause problems for the offspring. In the case of a paracentric inversion, out of four possibilities, one is completely normal, one is a balanced inversion (like parent inversion), and two are inviabledicentric and acentric. So the inviable ones die, and the two normal ones cause no problem: NO PROBLEM; but for pericentric, out of four possiblilities, one is normal, one is balanced rearrangement, but two are viable but have duplicated and deleted portions of the chromosome, which can cause problems. Reciprocal translocation vs Robertsonian translocation Robertsonian translation occurs onlyt between the acrocentric chromosomes, most commonly between the 13 and 14 chromosomes. This monosomy is the only case in which a normal phenotype exists. SRYsex determining region on Yif you have it you are male; if it is not there, the female forms. With SRY, a testis forms; Sertoli cells in the testis inhibits the formation of the Mullerian ducts by mullerian inhibiting substance that prevent the formation of the female genitalia form. Leydig cells form androgens that lead to growth of the Wolffian ducts into the epididymis, vas deferens, and seminal vesicles. With no SRY, an ovary forms, and mullerian ducts become fallopian tues, uterus, and part of the vagina. The wolffian ducts degenerate due to lack of androgen. The SRY lies near the pseudoautosomal boundary, and during recombination, the SRY gene can get aberrantly exchange from Y to X chromosome; if this is fertilized, the Y chromosome will lead to an XY female, and the X chromosome to an XX male. Lyon hypothesis: the X chromosome on females is inactivated to maintain dosage compensation through methylation to suppress expression, and all tissues in female are germline mosaic. However, there are some genes that are analogs on the Y chromosome, and these are not inactivated, so there is incomplete inactivation. That is why XXY and XXXY, where the extra Xs are inactivated still have problem, since the extra Xs still have some expression. Therefore, the more Xs there are, the more severe the disorder . X inactivation is normallyu random, and either the maternal or paternal chromosome can be inactivated. However, when there is a structurally abnormal chromosome involving X, the inacativation becomes nonrandom. This can involve deletion, duplication, etc., the abnormal X is inactivated, for maximum benefit. When there is a balanced X;autosome translocation, however, the normal X is inactivated; if the abnormal X is inactivated, the genes from the autosome that are now on X would be inactivated to achieve

maximum balance. When there is an unbalanced X;autosome translocation, the abnormal X is inactivated, however. Duchenne muscular Dystrophy (DMD) gene is on X chromosome, and it is very large, so translocation sometimes happens right in the middle of the gene and the gene is split between the X chromosome and autosome. Indications for a Chromosome analysis: 1. 2. 3. 4. 5. 6. 7. 8. 9. Phenotype corresponds to a known chromosomal syndrome Multiple congenital anomalies with mental retardation Unexplained mental and/or growth retardation Ambiguous genitalia Infertility Multiple miscarriage or fetal wastage Family history of chromosome abnormality Prenatal diagnosis for advanced maternal age (>35) Malignancies

Down Syndrome (47,+21) Neonatal hypotonialoose musculature Growth and mental retardation Short neck, flattened face, low ears, open mouth with protruding tongue Congenital heart disease Can happen through nodisjunction, or translocationscan be mosaic

Trisomy 18 (47,+18) Much less frequent in newborns bc many get miscarriage (1/3000-5000) Congenital heart disease Omphalocele and diaphragmatic hernia Limb anomaliesclenched hand High lethality with profound retardation in survivors

Trisomy 13 (47,+13) Chromosome is bigger, so more severe than trisomy 18 or 21 Midline facial clefts Holoprosencephaly (eye in the middle) Congenital heart disease Cystic kidneys Polydactyly, contractures, club feet High lethality with profound retardation in survivors

Turner Syndrome (45,X) 1/5000-10,000 95% die in utero

40% mosaic chromosomes Short stature, webbed neck Underdeveloped ovaries, failure of secondary sex characteristics to appear, sterility

Klinefelter Syndrome (47,XXY) 1/1000 newborns Tall stature IQ 10<other family members Sterile with hypoganidism Diagnosis at amniocentesis More complex karyotypes (XXXY, XXXXY) are shorter and mentally retarded

Prader-Willi Syndrome: defect in paternal genes on chromosome 15 1/15000 births Paternal deficiency 15q11-q13 Hypotoniafloppy babies Feeding difficulty Failure to thrive Food craving and weight gain Learning disabilities Underdeveloped sex organ

Genomic imprinting: certain genes are expressed in a parent of origin manner. Genes in the PWS critical region only express from loss of the paternal chromosome. Therefore, if the maternal 15 chromosome is damaged, the syndrome is not PWS, but Angelman syndrome. PWS comes ONLY from the paternal chromosome deficiency, and this can happen from deletion of the chromosome critical region, or maternal uniparental disomy (both chromosomes come from mother), or a methylation defect of the chromosome that does not allow for expression. Fluroescence In Situ Hybridization (FISH): label the probe DNA with fluorescent dye and denature and hyrbridize. Go over the methodnot in notes; this method allows for 100X higher resolution than banding. This is useful in microdeletions, and also when there are no dividing cells available for metaphase chromosome analysis. Digeorge Syndrome can be diagnosed by FISHdeletion on chromosome 22q11.2. Happens in 1/2500 newborns, and has cardiac defects, developmental delay. This can actually be passed on, since it can be mild enough that a person does not know he/she has it, and mates, and the child has much more severe problem. So everytime a child is diagnosed with DiGeorge syndrome, the parents should be checked as well. Microarray based Comparative Genomic Hybridization (CGH) this does not have to be done at a specific locationif you just think there is a problem with chromosome (structuralbc numerical can be done by banding), then the entire genome of the person can be done. The Patients DNA/control DNA is taken, and if the ratio is from 0.8-1, then it is normal, but if less or more, there is gain or loss of chromosomes, and there is a problem. It is therefore only checking for unbalanced problems (deletions, duplications, etc), since it is checking ratios. FISH remains the choice for balanced chromosome abnormalities (translocations and inversions)

Molecular Mechanisms of Non-Mendelian Inheritance in Humans Mosaicismsomatic and parental, or gonadal, or germline mosaicism. The mosaicism can be in the tissue of the body and not in the gametes (somatic mosaicism), or in the gametes only but not in the cells of the body (germline mosaicism), or in both. This depends if during embryogenesis, the mutation occurred before or after the separation of the germline cells from the somatic cells. If before, both somatic and germline cells will have the mutation, and the mutation has a chance of being passed on to offspring. A mutation occurring later would only be in the germline or a portion of the cells of the body, depending on where the mutation took place. Determining what type of mosaicism a person has (somatic vs germline) is almost impossible since failure to find mutation in a subset of cells from readily accessible somatic tissue does not ensure that the mutation is not present elsewhere in the body, including the germline. Theoretically, every single cell would have to be tested to determine that. Mosaicism can cause a certain disease to become less severe, and in some cases, a lethal disease becomes viable in a human. Partial or segmental presenation of a disorder can occur only in one part of the body where the mutated cell migrated tofor instance in segmental neurofibromatosis. If only there is a mutation in the germline mosaicism, the parent will be completely unaffected, but the child has a chance of inheriting the disorder. If the proportion of the mutated mosaics is high, then the chance that multiple offspring will get the disease is also high. Thus, esp in a dominant disease, if the parents do not have the disease, and multiple children do, then it has to be germline mosaicism, such as osteogenesis imperfect which is characterized by abnormal collagen I, and brittle bones. Causes can result in the rescue of an indicidual from a lethal phenotype, or can be due to a post mitotic non-disjuntion event. Somatic mosicism occurs during embryogenesis We dont know the level of mosaicimand the mosaicism can be in organs that are more or less severe. All mitochondrial genome problems will cause problems in the mitrochondria; however, most mitochondrial problems come from the autosomal chromosomes. Mt chromosomes come only from the mother. In addition, mtDNA has replicative segregation, the mtDNA of the cell in all the mitochondria is randomly divided when a new mitochondria is formed. If there is a mutation in the mtDNA, some of the new mitochondria will have it. Because this is not tightly controlled segregation as nuclear DNA, this can lead to some interesting things: for instance, there may be a mutated mtDNA in a certain mitochondria that due to replicative segregation has now formed in 10% of the mitochondria in the cell. Due to random segregation, a homoplasmy can result where a daughter cell has 0% mutated mtDNA or 100% mutated mtDNA. In addition various proportions of heteroplasmic mtDNA (mosacisim in mtDNA) can form that are different than the starting 10% ratio. The mitochondrial genetic bottleneck does this all over again, where the oocytes, which are the only germ cell that passes on mtDNA (since sperms mtDNA is destroyed), reduce the number of mtDNA molecules randomly and then amplify them, which can lead to even more different proportions. Therefore, the variability of mutant mtDNA in a mother and the child may be much different. All children of an affected female with a mutant mtDNA will be affected, and no children of an affected male will be affected. Males with Lebers Heredtiary Optic Neuropathy are more severely affected than malesso mtDNA disorders have different levels of expressivity within different sexes. Bottleneck theory???? Number of mitohcondria and the severity of mutations???? Mitochondrial genetic bottleneck HeteroplasmyMt DNA have higher error rate than nuclear DNA; Parent-of-origin effects: Genomic Imprinting where the germline of one parent is altered (usually by covalent methylation of cytosine to form 5-methyl cytosine), but not the other, and therefroe there are

differences in gene expression between the allele inherited from the mother and the allele inherited from the father. Imprinting takes place during gametogenesis and marks certain genes as having come from the father. Imprinting is obviously reversible, so that when a father gives his daughter a gene, that gene is originally imprinted as a paternal gene, but when the daughter passes it on, the paternal ID must be removed and made into maternal, which is done by imprinting centers which imprint through epigenetic changes and are located throughout the genome. If the imprinting center is messed up, and a maternal gene is passed on with a paternal ID or vice versa, the result effect on the phenotype of the child can mimic UPD, where the two genes actually are from one parentthis can happen in Anegelman and PWS for instance. Uniparental Disomy (UPD) heterodisomy (both copies of chromosomes from one parent) or isodisomy (a duplicated copy of one of the chromosomes from one parent)this happens from trisomic rescueand randomly a chromosome is thrown out, and if it the parent who donated on chromosome, the other parent will provide both chromosomes. ALL ANGELS MISS THEIR MOMMIES, PRADER MISSES HIS FATHERPrader willis syndrome has a deletion of his father, or maternal UPD. Opposite for Angelman syndrome. Angelman syndrome are always laughing, and are very retarded, though PWS have mild retardation DNA Replication Nucleoside= pentose and base without phosphate; nucleotide includes the nitrogenous base (purine or pyriamdinine), connected to the pentose with a N-glycosylic bond at the 1 carbon; at the 2 carbon of the pentose, if there is a hydroxide, it is RNA, if it has an H, it is DNA. And then on the 5 carbon, the phosphate is attached. Nitrogenous bases of DNA and RNA: C, G, U, A, T. difference of T and U is the tymine is methylated. There are many modified bases in RNA, but one in DNA, with 5-methylcytosine with 4% of Cs are in this form. DNA is a string of 2deoxynucleotides, with phosphodiester bonds; we always read from the 5 to 3 end. 3 O of one to 5 phosphate of next links successive nuelceotides with phosphodiester bond. Nucleic acid backbones are flexible, polar, and negatively charged at physiological pH. DNA and

RNA are highly soluble in water because the phosphate (one negative charge in chain) and pentose chain are hydrophilic; the nitrogenous bases, however, are hydrophobic, and stay inside to limit exposure to water, and stack in a way to increase their interactions with each other. This stacking happens in single and double stranded DNA. Double helixDouble-stranded DNA is a right-handed helix formed by two individual DNA strands aligned in an antiparallel fashion (a right-handed helix, when viewed on end, twists clockwise going away from the viewer). Strands are antiparallel and the strands have opposite polarity, so in one direction it is 5 to 3, and in the other, it is 3 to 5. Hydrogen bonds connects the bases, but shape complementarity is important, so purines and pyramdines bind with each
other and they fit with each other very well. G and C is more stable than A and T since there are 3 Hbonds,vs 2 H-bonds. This stabilizes the bonds through intermolecular interactions, but base stacking interactions also stabilize the DNA within a strand. B-DNA is most common (others are A and Z); B-DNA forms a right-handed helix with wide major and narrow minor grooves of similar depth. Minor groove is line with ordered water molecules and is less accessible. Usually only proteins that do not need to recognize the base sequence, such as repair proteins, bind at the minor groove. Major groove is more accessible to proteins, so for proteins to recognize a certain sequence, they bind at the major groove, since there, the bases are more accessible.

DNA replication in humans in semi-conservative; two replication forks begin at origin and move in opposite directionsso it is bidirectional, on two different strands, however, it should be noted. Two replication machinery or replisomes, therefore, start working. Polymerization reaction is catalyzed by DNA polymerases. Requirements are a template DNA strand obviously, but also a primer with a 3 OH group called the primer terminus for additional since DNA pols only add to preexisting DNA or RNA; also a dNTPs are necessary, since proper balance of nucleotide pool also importantmaintained by cell. DNA polymerases ALWAYS synthesize int eh 5 to 3 directionthe new strand is being formed in the 5 to 3 direction. Solution is Okazaki fragments, which makes DNA replication semi-discontinuous, which is formed in the lagging strand, the other normal strand is the leading strand and the Okazaki fragments connect later. DNA replication is highly accurage. Fidelity (accuracy of DNA relication) is composed of three parts: the polymerization itself makes few mistakes, but there is also a proof reading part and a strand directed mistmatch repair. Things that may contribute to fidelity in DNA replication (polymerization step), since there is only 1 error in 104-5 . DNA polymerases recognizes geometery and stabitlity of AT and CG base pairs; stability of base-pairing depends on H-bonds, steric interactions, and stacking. Mismatched base pairs have altered geometry and stability. Mismatched base pairs makes bringing in the new bases more difficult because of steric hindrance and messed up H-bonds. The proofreading part is important for preventing C-A bonding, since C has a tautomer that happens to base pair with A, but then it shifts back to normal C, and destroys the base pair with A. This is exonuclease activity. ???? LOOK OVER exonuclease activity again Nucleases degrade DNA by cleaving the phosphodiester bond in backbone. Exonucleases degrade nucleic acids at 5 or 3 endsome of them go only in one direction. Endonucleases cleave phosdiester bonds at internal sites of DNA. DNA polymerases: only extend a pre-existing polynucletotide chain and can ONLY catalyze the creation of phospodiester bonds onto a 3 hydroxyltherefore, the new strands can only be 5 to 3, which is the reason for Okazaki fragment; they cannot begin synthesis de novo, and they require a template. DNA polymerases differ in their processivity (which is number of base pairs that it attaches before breaking off????). they have two or more activitiyespolymerizing from 5 to 3 end, and usually exonuclease from 3 to 5 end. A special protein keeps the DNA polymerase attached to the DNA as it repeatedly adds new nucleotides to the growing chain, which is called the sliding clamp or the B-clamp and it forms a ring around the polymerase and the DNA, and without it the DNA would just fall off after a few nucleotide attachments. The clamp loader assembles the sliding clamp to the DNA, and the single strand binding protein (SSB) attaches to the DNA to prevent the DNA from forming the double strand since it is in the way. The DNA polymerase only catalyzes the reaction of the phosphodiester bond if the nitrogenous base correctly pairs, and this is determined by the geometry and stability of the base pairing. However, sometimes, less stable base pairing such as CA and GT form, and the polymerase does not recognize it, and catalyzes the reaction. Therefore, the proofreading portion of the same DNA polymerase kicks in at the same time as synthesis. the proofreading portion of the DNA has a different active site than the polymerization part, although sometimes it can be on the same pp for a DNAP. The DNA polymerase cannot continue base pairing if the previous base is incorrectly paired. Before it moves onto the next nucleotide attachment, the DNA polymerase checks whether the previous already attached nucleotide is correct (thus it checks before attaching and after attaching again); if it is wrong, a different domain of the polymerase cleaves the phosphodiester bond. Thus, the DNA polymerase has 5 to 3 polymerization, and 3 to 5 proofreading, and this explains why there is no 3 to 5 polymerization (which would negate the need for Okazaki fragments)if there was 3 to 5 polymerization, and a mistake were made, then it would be unable to proofread. Sometimes, however, a mistake gets past even the proofreading ability of the DNA polymerase, and in that case the strand-directed mismatch repair is used. When there is a mistake in the pairing, the exonuclease activity of the polymerase can change either the template strand which would be wrong or the new strand; it identifies the new strand in E coli, this is

done by methylation of the DNA a little after it is created, so only the template strand is methylated. In humans, there are nicks in the lagging strand where DNA ligase has not yet connected all the DNA, and the theory is that the leading strand is nicked to identify these nicks from the unnicked template strand. DNAP I has three domains for polymerization, and 5 to 3 exonuclease, and 3 to 5 exonuclease. Normally, most DNAPs do not have 5 to 3 exonuclease, and DNAP I and e uses this for strand displacement synthesis, which degrades RNA or DNA and synthesizes new DNA in the same 5 to 3 direction. DNA Polymerase I: most polymerization of DNA is not done by this since it is too slow; rather DNA polymerase III does most work in prokaryotes. DNAP I performs a host of clean-up functions during replication, recombination, and repair; it has the 5 to 3 exonuclease activity which is used for strand displacement sysnthesis, or nick translation during replication and recombinationunlike DNAP III. The 3 to 5 exonuclease is used for proofreading. It has a slow polymerization rate (20 per/second) and low processitiity (400 nucleotides???), which cannot account for the fas replication of DNA in E coli. Nick translation and the 5 3 exonuclease activity is used, amongst other things, for going in the regular 5 to 3 direction and removing DNA or RNA, like the RNA primers, or mistakes in DNA. The nick between the RNA primer and DNA moves along, and this is the nick translation. DNA polymerase III is much faster (500-1000 nt/s) and high processivity (>500,000) which is why it does most of the work in E coli. It has two core polymerization domeains (aeO) alpha subunit does the polymerase activity, epsilon does the 35 exonuclease activity, and B clamps to the DNA, holding the Polymerase in place to the DNA. The clamp loading complex links two cores and also attaches to the beta-clamp which increases the procesitiy and rate, since the Pol III core alone replicates very slowly and has very low processivity, but with the B clamp, this increase. Replication involves three main phases: initiation, elongation, and termination. Many proteins are required to initiate replication in E coli: DnaA protein, DnaB (helicase), DnaC, Primase (DnaG), singlestranded DNA-binding protein (SSB), and DNA gyrase (DNA topoisomerase II), Dam methylase. In E coli relication begins at a specific site called oriC and this is NOT true in humans, with one location initiating the replication. Five R and thre I sites bind to DnaA, and INITIATION: only phase to be initiated: positions at which DNA is first opened are called replication origins, which are marked by a particular sequence of nucleotides that attract the initiator proteins as well as stretches of DNA that are easy to opensince AT H-bonds are weak, these stretches include many AT rich sequences. These stretches are called DNA unwinding elements (DUE). The replication origin has five repeat units of R sites and three units of I sites. The R sites bind strongly to the initiator protein, DnaA in both its ATP and ADP bound phase, but the I site binds to DnaA only when it is in its ATP phase. This allows for regulation of initiationthe only one that is regulated (elongation and termination are not regulated). 8 copiesof DnaA-ATP bound proteins form a complex, and then DnaC attaches DnaB to come in and begin unwinding, using ATP. In human DNA, there are many replication origins, so that replication can be faster. Once the initiator protein binds to DNA at the replication origin and locally opens up the double helix (opening a few base pairs is easy, but opening the entire helix is difficult due to the combined strength of all the H-bonds), it attracts a group of proteins that comprise of the replication machinery. Because the DNA polymerase can only add nucleotides to the 3 OH of a nucleotide, an RNA primer must be attached by a primase, DnaG, which attaches about 10 nucleotides. Then DNA polymerase takes over. This happens just once on the leading strand, but on the lagging strand, the primase has to attach the RNA primer several times. After it is done, a nuclease (endonuclease since it is breaking an internal bondexonuclease breaks a bond at the end of the chain, such as when DNA polymerase has made a mistake) breaks the RNA bonds with DNA, and a DNA polymerase lays down the new DNA, and a ligase connects the new nucleotides. The primase does not

have a proofreading mechanism, and may make mistakes, so the DNA polymerase checks that the primase had done the correct pairing, before adding its own DNA. DNA helicase (DnaB) continues unzipping the DNA at the replication fork as polymerase moves along. Because the lagging strand needs to attach to an existing 3 OH group, and at the end of the linear chain, called the telomere, there is none, telomerase, which has an RNA portion in the enzyme, adds extra nucleotides to the template strand using its RNA portion as a template by extending the 3 end of the DNA. Then, the DNA polymerase-alpha finishes the new strand. Telomerase activity occurs only in the germ cells and not somatic cells. This need be done only on the lagging strand. Elongation: lagging strand synthesis via Okazaki fragments: there are two subunits of the DNAP III polymerase, and one does the lagging strand and the other does the leading strand. Since they are attached to the same protein, the lagging strand core attaches to protein that has made a ring around the protein and come back for polymerization. As the polymerization of the lagging strand of Okazaki fragment 2 nears the RNA primer of the previously polymerized Okazaki fragment (1), DnaG primase attaches to DnaB helicase and makes a new RNA primer. The B clamp attaches to the new RNA primer, and by now the Okazaki fragment 2 is complete, and the B clamp of that fragment detaches from the core. The core now attaches to the new B-clamp, and begins formation of Okazaki fragment 3. Termination: in E coli, the two replication ofrks move in opposite directions until they meet up at a terminus region containing many copies of a sequence called Ter, wich bind to the Tus protein (terminus utilization substance) and traps the replication fork, not allowing it to exit. The first replication fork to encounter the Ter-Tus complex halts upon collision. The circular DNA will be interlinked or catenated, and so they are separated by topoisomerase IV, a type II topoisomerase. DNA topoisomerases solve the supercoiling problem and coils the positively coiled DNA in a negative direction, since the DnaB helicase causes supercoiling through unwindingthere are two types: Type I and Type IIand both have nuclease and ligase activity to rejoin the DNA; type I cleave just one strand of duplex. Type I does not use energy, and in prokaryotes, relieve positive supercoiling, but in eukaryotes, relieve positive and negative. Type II cuts both chains, passes the DNA through the break to relieve stress, and ligases it. It does use energy, since it can actually induce negative coiling so that later when positive coiling happens, it balances outthis requires energy, and type I cannot induce stress itself. In circular DNA, it is used to separate the two circles of replicated DNA. Eukaryotic replication: PCNA= B clamp that increases preocessivity; two major polymerases (equivalent to DNAP III) are polymerase epsilon which makes the leading strand (has proofreading activity and high processivity), and polymerase delta (does the lagging strand and has high processivity, and has proofreading); the other two dont have porrfreadingalpha and beta; alpha synthesizes short primers for initiating synthesis of Okazaki fragments; beta is involved in DNA repair. RFC is clamp loading complex that attaches the PCNA clamp. RPA = SSB. Mcm= DnaB helicase; Cdc6 and Cdt1=DnaC; these two form a complex with Mcm during G1 before mitosis to make a prereplicative complex. Without this, S cannot start, and this is called licensing. Passage into S is triggered by cyclin dependented kinases (Cdks) that phosphorylate the ORC and replicative proteins, so that the prereplicative complex breaks apart, and Cdc6 and Cdt1 go away, and Mcm begins its helicase activity. Phosphorylation of ORC prevents assembly of second replication fork at the same site. In eurkaryotes, there is no DUE that is easily identifiable, though there are AT rich areas, and here the ORC=DnaA initiator attaches. Replication takes place ONLY in S phase. Many replication sites at once in humans. Reverse transcriptase: produces a DNA-RNA complex, then degrades the RNA and replaces it with DNA. RT is error-prone, leading to a high-mutational frequency for HIV, which limits the efficacy of

drugs. HIV RT has a higher affinity for certain nucleotide analogs than for dNTPs, such as AZT triphosphate, which is an analog of dTTP, but lacks the 3OH primer needed for chain elongation, and incorporation of AZT in a chain can cause chain termination. In addition dideoxyinosine causes a similar effect. Upstream vs downstream???? DNA bases are spontaneously breathingbases are spontaneously flipping out of the duplexnot necessarily 180 degrees completely out of the duplex; at any given time, most bases will be stacked, but some will be flipping DNA Repair DNA is not stable and could get damaged by agents such as Xrays, Oxygen radicals, alkylating agents, UV light, errors in replication, anti-tumor agents, etc. There are many pathways that can repair the DNA. The consequnces of damage is cell-cycle arrest, inhibition of transcription, replication, apoptosis, and mutations. Most carcinogens are also mutagenic (cause mutations in DNA). DNA Damage and its effects: types of DNA damage: mismatched base pairs (imporantant for generating mutations, and corrected by the mismatch repair pathway), base modifications that completely transform the bases into something else, breaks in the backbone, which can be single and double strand, and cross links between and within DNA strands. Mismatched base pairs: replication past a mismatch can cause a mutationa permanent change to the DNA sequenceand can be lethal to cell, and accumulation of mismatches can lead to cancermost carcinogens are also mutagens. Mismatched base pairs must be repaired prior to next replication. Sources of DNA Damage 1. Environmental: UV light, and chemical found in cigarette smoke 2. Agents of normal cellular metabolism: reactive oxygen species (ROS), such as hydroxyl radicals, hydrogen peroxide; reactive nitrogen species cause damage as well as alkylating agents 3. Spontaneous disintegration of covalent bonds: rupture of base-sugar bond (N-glycosylic) bond, and deamination of cytosine, adenine, and guanine 4. Replication errors: mismatched base pairs, and misincorportation of nucleotide tat resembles one of the four canonical bases, but is not. For instance, dUTP can be inserted into DNA instead of dTTP, since it resembles it, and polymerases are bad at differentiating between dU and dT; uracil in DNA is mutagenic, and deamination of cytosine can also create uracil Types of DNA damage 1. Mismatched base pairs: happen almost exclusively during DNA replication, and handled almost completely by Mismatch repair pathway (MMR)the exception being that when a 5methylcytosine is paired with G and is deaminated to T, the pair becomes a TG pair. This is corrected by base excision repair (BER) pathway 2. Abasic or apurinic/apyramidinic (AP) sites in DNA: spontaneous rupture of the N-glycosylic bonds creates an AP site, and this happens 10,000 per cell per day. It is handled by BER pathway 3. Modification of bases in DNA: can happen through deamination which occurs through nitrous acid derivatives such as nitrite, nitrate, nitrosamines. It converts cytosine into U, 5methylcytosine into T, adenine into hypoxanthine, and guanine into xanthine; also alkylation can happen which can make 3-methyladenine and 06-alkylguanine, and this is done by agents such as

dimethylsulfate and nitrogen mustards. Also oxidation can occur to create 8-oxoguanine, which mispairs with adenine to convert GCAT pairs 4. Intra and inter-strand cross links, which happens through exposrure to x-rays, UV, and chemical such as psoralen (which works mainly on tyrosine) and mitomycin C. pyramidine dimer occurs frequently due to exposure to UV, and they are handled by nucleotide excision repair (NER) and direct repair (not in humans) 5. Breakage in DNA backbone (single and double stranded breaks) by X-rays 6. Incorporation of nucleotide analogs: the polymerase adds a similar analog (so this is not happening through chemical reactions like 3), and are incorporated into DNA, fo example dU, 5fluoro dU, 5-bromo-dU, and 2-aminopurine. They are removed by BER. Some enzymes remove these analogs so that they cannot get incorporated into DNA, such as dUTPase converts dUTP into dUMP so that it cannot be incorporated Also, even if there is no mismatche, but if there is a chemical change of the base itself, that can lead to mutations. For instance in a GC match, the guanine can be methylated at the O6, and this would make its bond with C unstable, and it more stably bonds with T; in subsequent replications, the T will pair with A, and the GC will be turned into GT which will be turned into AT. Completely different. This is due to modified bases. DNA base lesions and their damaging effects: alkylation, oxidation, and deamination can happen so that the base is completely changed here as well. Deamination converts cytosine to uracil, which converts a GC match into a GU mismatch. 5-methylcytosine can be turned into a thymine, so that a Gm5C pair leads to a GT mismatch. Replication of GT mismatch will give a AT pair in next round, so GC has now gone to AT. Abasic sites created by spontaneous rupture of base-sugar bond, and this happens spontaneously 10,000 times per cell per day and hydrolysis gets the nitrogenous base completely off of the pentose ring. Crosslink and strand breaks indicued by radiationpyramidine dimers formed by exposure of adjacent pyramidines to UV light. This causes severe distortion of DNA. Repair systems: 1. Mismatch repair: replisome is very accurate in that both the polymerization and proofreading is very accurate1 mistake in 107 base pairs, but this is not good enough, and replication errors still happen through mismatched base pairs, and small insertion or insertion/deletion loops???? It recognizes the template strand in E coli by methylation of template strand by Dam methylase onto all adenines on the 6-NH2 group in the sequence GATC. Not all mismatches are repaired with the same efficiency. In E coli, a MutH remains bound to the hemimethylated (one strand methylated) GATC site. MutS (homodimer) and MutL (homodimer) combine together to form a heterodimer and bind to the mismatched pair or insertion/deletion loop (IDLs) and the DNA is threaded through the MutL/MutS complex until the MutH is reached. Here MutH is activated, and it nicks (hydrolyses) the DNA backbone of the unmethylated strand, which may be hundreds of bp away from the mismatch. The strand from the GATC incision site through to the mismatch site is removed (EXPENSIVE to remove so much for one mistake). The nuecleases involved depend on which side of the mispair the nicked GATC site is found, because it requires either 35 or 53 exonuclease activity. The dupels is unwound by DNA helicase II and stabilized by SSB and the new strand is synthesized by DNA polymerase III and sealed by ligase. In eukaryotes, there is no analog for MutH, since there is no methylation of DNA. Instead, the nicks that were present during replication is identified by PCNA and RFC, which stays there. Then the MutSalpha and MutL-alpha which are both heterodimers get together. The MutS-alpha is used to

2.

3.

4.

5.

identify the mismatch and short IDLs (MutS-beta identifies larger IDLs), and the MutL-alpha with its endonuclease activity nicks the new strand on each side of the mismatch. Exoonucleases (5 to 3) get rid of the strand in between the two nicks, and DNAP sigma puts back the new strand. Human hereditary nonpolyposis colon cancer (HNPCC) is caused by defects in one of several MMR proteins, and the mutations are usually found in MSH1 and MSH2 which are monomers of MutSalpha and MutSbeta. Base excision Repair: corrects modification of the bases with the protein DNA glycosylase which removes the chemically modified basebecause there are so many potential lesions, many of these proteins need to recognize more than one lesion DNA. 12 known human DNA glycosylases, each specific for certain types of damage. Some a highly selective (i.e. uracil DNA glycosylase removes only uracil) while others remove many. Some damaged bases, such as uracil, are removed by more than only glycosylase for backup. AP endonuclease nicks the backbone at the 5 site of the problem and removes the abasic nucleotide (since the glycosylase only removes the base, and not the entire nucleotide), and then DNAP adds the new nucleotide, and DNA ligase connects the two nucleotides. DNA glycolysases use base flipping to recognize and remove lesions. Many base lesions are subtle and dont perturb DNA structure, so the base needs to be flipped into the active site of the DNA glycosylase, which can then remove a problematic base. Nucleotide excision repair (NER) pathwayremoves bulky lesions that distort DNA helix, such as pyramidine dimers (PD) and damage due to exogenous compounds. This is the ONLY repair pathway for pyramidine dimers other than direct repair (in humans there is no direct repair for dimers). In E coli, a trimer protein called Uvr ABC exinuclease cleaves the damaged strand at two stes flanking the lesionUvrB nicks at the 3 side, and UvrC at 5 side. UvrD helicase releases the oligonucleotide chain, and DNAP I resotres the oriinal sequence, and DNA ligase seals it. Mutations in the NER protein genes cause NER defects which cause Xeroderma Pigmentosum (XP) which is extreme light sensitivity, and higher incidence of sun-induced cancer, and increased neurological defects, and an elevated frequency of other cancers. Direct Repair: some damage is directly removeid, without excising the base or nucleotide. This is in the instance of 06 methylguanine, which is methylation. THIS IS EXPENSIVE bc the enzyme methyltransferase that demethylates the guanine and transfers it to the thiol of the cysteine residue and is used up in the processso each enzyme can only catalyze one rxn. Also there can be direct repair of pyramidine dimers in E coli, but not in humans. This is called photoreaction repair and is catalyzed by the enzyme photolyase which uses energy absorbed from light that catalyzes this rxn. In addition, 1-methyladenine and 3-methylcytosine can be turned back into the normal version by the enzyme AlkB which uses direct repair without removing the damaged nucleotide. These can get mistakenly methylated when there is only a single strand of DNA. Error prone polymerases and translesion synthesis (TLS): some lesions are so significant that they can halt the replication fork. Cell can switch to polymerases that can synthesize past lesions but usually with lower fidelity since they dont have 3 5 exonuclease proofreading activity. However, they have low processivity, so they dont make too many errors. In prokaryotes, this is DNA Polymerase V. The lesion will be repaired at a later time. That is why these are also referred to as error prone polymerase but they act on short stretches only, and therefore, their low fidelity usually does not cause much damage. If the TLS gene is mutated, this can kill the cell, since the cell cannot get past lesions, and replication fork will just halt. In humans, the TLS polymerase to allow translesion synthesis across dT-dT dimers is DNAP etaand replicates past TT dimers, and adds AA in new strandGOOD. This does not make as many mistakes as other TLS polymerases. Mutations in gene encoding DNAP eta can cause XP like illness called XP variant or XP-V

DNA recombination

Rearrangement of genetic information withing and among DNA molecules (within and between chromosomes); recombination is important in many ways: meiosis, DNA repair, regulation of Gene expression, viral infections, generating diversity in immune system (antibodies), movement of genetic elements with and between organisms, and diversity amongst organisms 1. Non homologous end joining: (NHEJ) double strand break repair (DSRalong with HR); there is loss of nucletoides, hence a mutation, thought most of genome does not code for protein; NHEJ is prominent mechanism of DSB repair in humans, and HR is used only during and shortly after DNA replication (S and G2 phase); involves a heterodimer Ku 70/80 heterodimer protein that forms a ring and binds the torn DNA ends, and then DNA-PKcs (DNA protein kinsases) phosphorylate Artemis which processes the DNA ends by 53 exonuclease activity. Then DNA ligase IV connects the two. Damage to the NHEJ pathway results in hypersensitivity to ionizing radiation and agents that cause ds breaks, and are immunocompromised due to defective V(D)J recombination since the proteins of this pathway are involved in V(D)J recombination as well. 2. Homologous Recombination (HR) see notes written: exchange between two DNA molecules with long region nearly identical sequence (hundreds of bp), and no specific DNA sequence is required, and major recombination and break repair pathways 3. Site-specific recombinationrequires a specific sequence; found in the virus bacteriophage lambda and uses the enzyme recombinase that has both nuclease and ligase activity. These mobile genetic elements require a specific sequence at both the donor and recipient DNA. The recombinase recognizes the sites on both DHNA molecules, and then cleaves the recipient DNA, forming a covalent DNA-protein complex; then it rejoins the new strands with its ligase activity. 4. DNA transpositiongenetic elements with ability to move one location to another. Requires transposase enzyme that cleaves the transposon from its ends by recognizing a sequence but the transposon travels to a random spot, and therefore can insert into a gene and cause destruction so no sequence homology needed, and target site is random3 types of transposonsDNA only, retroviral like retrotransposons, and nonretroviral retrotransposons. Humans have all three, and the first two are inactive, but the last, nonretroviral retrotransposons, some of them are active. DNA only predominate in bacteria and confer antibiotic resistance. Two types: insertion sequences and composite elements or complex transposons, the first which codes only for the sequence required for transpositionthat is the gene for transposase and the short inverted repeat sequences at each end that are recognized by the transposase and are required for excision of the transposon; the transposon may also encode a resolvase which separates the different genes; the composite elements have other genes as wellsuch as antibiotic resistance. The mechanism for this is done through a cut and paste methodtwo transposase enzymes connect at both ends of the short inverted repeat sequences, and combine to make a loop of DNA that cleaves the DNA. The transposes enzyme then makes a staggered cut at the target DNA, and the transposon inserts; the double strand of target DNA is connected by HR or NHEJ. Retrovirallike retrotransposons are in DNA and they resemble retroviruses, but lack a protein coat, so they cannot exit the cell, and when they are transcribed to RNA and translated to proteins, they yield reverse transcriptase and integrase that converts the RNA back into dsDNA. The integrase cuts the viral DNA and the viral DNA attacks the target DNA and inserts into it. There are no active retroviral like retrotransposons in humans. This is like copy and paste since a new DNA is created from the original one. Nonretroviral retrotransposons also have a copy and paste method, and the DNA to RNA to protein is similarproteins are endonuclease and reverse transcriptasesingle protein. Relics of these elements (LINEs and SINEs) are 1/3 of genome but inactive, but there is some movement of Alu element (which is member of the SINE) but it is 1/200 birth, and contributes minimally to overall mutational burden, though active LINEs may contribute to disease.

5. V(D)J recombinationused to generate diversity in immune system. There are two chains light and heavy. Both have variable and constant regions. 3000 possibilities for the light, 5000 for the heavy, and product for total possibilities. The line that connects them is the J region, and on the light chain there are 4 possible genes that encode for them; then there is the V region, and in the light, there are 300 possible genes that encode for them. The RAG proteins cut at 3 side of V and the 5 side of J segment, and intervening DNA is discarded, and the two V and J segments are connected by NHEJ. This is transcribed into RNA and processed so other stuff is removedthe stuff on the sides that was not discarded. Translation of processed mRNA gives light chain polypeptide. Heavy chain involves similar process. Defects in RAG protein or NHEJ protein leads to vulnerability to infection because there is no V(D)J recombination, and no functional B and T cells, and this is called severe combined immunodeficiency disease (SCID)

RNA metabolism RNA transcripts may be used for protein synthesis or as RNA for diverse cellular functions. Like DNA joined by a 35 phosphdiester linkage. RNA contains a OH group at the 2 carbon of the pentose. A big structural difference between DNA is that RNA is ALWAYS synthesized as a single strand polymerbut if the appropriate strand is available, it can form the double strand version which is an A form helix. There is a lot of heterogeneity in structure; if it bonds with another strand, it forms almost always an A form helix (DNA usually has Z); the structure can include a strand that loops around and pair with itself hairpin loop and a bulge can include where a base does not pair anything and introduces a kink in the 3D structure. Also there is an internal loop where the two strands bases are unpaired and they can readily make contact with proteins and other moleculesso the 3D structure is highly variable. Vast majority 80-85% of RNA in cell is rRNA which is part of ribosome4 fours: 28S, 18S, and 5.8S and 5S in eukaryotes; in prokaryotes, there are only three23S, 16S, 5S; S refers to Svedber Unit which is measure of size based upon the molecular sedmentation rate mRNA makes up only 2-4% of RNA in cell tRNA: 10-12% Other forms: snRNA (small nuclearinvolved in splicing of mRNA); snoRNA (small nucleolar RNAs that participate in rRNA methylation, maturation and processing), and miRNA (microRNA that is involved in gene expression). In general, if DNA strand is shown, it is from 53 order (upstream to downstream), and it is the nontemplate (coding) strand that is going to be exactly the same sequence as the RNA that will be coded from it, except for T replaced with Uthe template strand will have the opposite pair as the RNA, and it is the one that is actually read by RNAP which moves from 5 3. All RNA synthesis is done by a family of enzymes called DNA dependent RNA polymerases that require DNA as a template and these polymerases needs ATP,CTP, GTP, UTP, and Magnesium as a cofactor; it does NOT need a primer like DNA synthesis. therefore the first nucleotide that is attached will have a triphosphate attached to it. General features of a gene: the transcribed region is the part that is actually transcribed and it starts at the initiator site +1. The promoters are upstream and dictate the efficiency with which the RNAP binds to it and how often the transcribed region is transcribed and also makes sure that RNA sits on the right part and only transcribes the correct gene. There are termination signals that are near the end so that RNA stops coding and knows when to stop. Intergenic regions are the regions between genes. General mechanism of RNA synthesis; RNA adds NTPs to the 3 terminus. Note that the first phosphate attached to the pentose is alpha, then beta, then gamma is the last phosphate at the last. So in a NTP

molecule, the phosphate is attached to the 5 carbon of the pentose sugar, and the first phosphate attached is the alpha carbon, and ONLY that phosphate will be incorporated into the RNA. The 3OH acts as a nucleophile, attacking the alpha-phosphate of the incoming NTP and releasing pyrophosphate. So the OH group on the pentose attacks the alpha phosphate. Mechanism is the same for DNA. ssDNA can be used as template for RNA transcription, but dsDNA is more efficient. DNA template can be on both strands; RNA polymerases do not posses 35 proofreading activities, and makes a lot of mistakes1 in 10^4-10^5. But the RNA transcripts are not passed on except with retroviruses. Also, each copy makes a different error since it is a random error, and most copies will have no error. RNA polymerases need to make proteins fast, so the speed sacrifices fidelity. Transcription bubble: about 17 bp of DNA are pulled apart so that transcription can take place. Unwinding takes place ahead of the transcription and rewinding takes place behind the replication. Because of it, there are positive supercoils are ahead of it, and negative supercoils behind it. The reason there is supercoiling in prokaryotes is that the DNA is circular, and opening the helix, and pushing the helix to any side increases the coils. In eukaryotes, although the DNA is linear, it is wound around histones, and therefore is kind of fixed. The RNA-DNA hybrid has about 8 bp, and as one more is added at the 3 end, the one at the 5 end detaches. E coli RNA polymerase: 5 subunits: two alphas, and BBw subunits makes up the core enzyme. Alpha subunits have DNA binding activity, and the BB has active site wehre RNA synthesis actually occurs WHAT??? Sigma is specificity factor and makes the full holoenzyme with the core enzyme, and makes recruitment of RNAP with the promotor good so that it makes sure that only the correct gene is transcribed. In the absence of the sigma unit, the RNAP is promiscuous and will initiate RNA synthesis randomly on a DNA template; in the presence of sigma, the polymerase becomes specific and initiates only at specific sites called promotors. Identification of protein-binding sites on DNA by nuclease footprintingdetermine where the RNA polymerase binds on each gene. Use a radioactively labeled at the end DNA, and throw DNase I into it that cuts it at various locationsrun it on a gel electrophoresis, and only radioactive strands show, all at different lengths, since the DNase cuts it at different lengths. Then, throw RNAP into it, and the place where RNAP binds, the DNase cannot cut, so check at what length there is no cutting, and that is where binding location is. Promoter elements are upstream to the gene at -35 and -10 and have consensus sequence with different but similar bases; they have different sequences to control efficencicy of different transcriptions of different genes to prioritize which genes get transcribed most efficiently. If some genes need even more tightly binding and faster transcription, they have an UP element upstream that makes RNAP bind even faster, and these interact with the alpha subunits to allow for stronger affinity, so it is not just sigma but alpha that is binding. Transcription Initiation in E coli: binding of the sigma and perhaps alpha to the promotor, closed complex since DNA is close, open complex when DNA helicase opens the DNA, and then elongation of 8 bp before the RNAP starts moving forward away from the promotor and promotor clearance and release of the specificity factor sigma subunit since it is not needed anymore. Once the sigma subunit has fallen off, the complex is committed to the elongation phase. To stop, there are two pathways: Rho-independent transcriptional factor: does not require an extra factor, but just a DNA stretch with a lot of AU bonds (WEAK) and an RNA with a GC stretch has to be self-complementary so that it can produce hairpin loop near the 3 end. The other part of DNA has a lot of As and the RNA has a lot of Us at the 3 end of the transcript. if the GC stem loop is created before the polymerase has moved on, the GC H-bonds are strong, and it will tear off the RNAP through torsional strain. If the RNAP has moved on out of the AU rich region before the GC H-bonds are made, the bonds wont be that weak for tearing out and polymerization will continue, and this allows a lot of flexibility, since sometimes the RNAP will move on first, and make a longer chain.

Activation and repression of genes: proteins in addition to RNAP holoenzyme can bind near promoters and affect efficienty with which initiation begins. For example cAMP receptor protein (CRP) activates, and Lac repressor protein represses. Rho-dependent transcriptional termination: requires a separate protein factor to terminate. Rho protein, a hexamer, and contains and ATP dependent RNA/DNA helicase activity attaches to its recognition site on RNA called a RUT site. Rho moves along RNA, following the polymerase. If there is are stem loops in the way, it wont stop the Rho, since it has helicase activity. RNA polymerase pauses at terminator, and rho catches up, and Rho unwinds the DNA:RNA hybrid in the transcription bubble, and RNA polymerase and Rho is released. Presence of Rho factor is the difference between the Rhodependent and rho independent. In eukaryotes, there are three RNAPs: RNAPII has many types of variations, since it needs to recognize many different types of genes; the RNAP I and III are simple and constant 1. RNAP I makes rRNAs except for 5S; the three units (28S, 18S, and 5.8S) are formed from one molecule so that the ratio remains the same; there are two promoters: Upstream control element (UCE) more upstream from the gene and core promoter element (CPE) that is just 1 bp upstream from gene. The upstream binding factor protein (UBF) can bind to either promoter, and loops the DNA to bring both the promoters next to it, and this initiates a cascade which brings all the transcription factors and eventually RNAP I. 2. RNAP II makes mRNA and some small RNAs; has to recognize a broad diversity of promotors. There is a TATA box premotor at -30 and an Inr (initator) sequence near the start site +1. The Pol II promoters require the basal transcription factors such as TATA binding protein (TBP) and TFIIA-H; initiation begins when the TFIIH protein phosphorylates the C-terminal end of the RNAP II; elongation occurs when the first 60-70 (as compared to 17 for prokaryotes) bp have transcribed and the TFIIE and H are released and elongation factors attach to the transcription complex. Termination happens with polyadenylation to the 3 end and this requires the cleavage of the transcript at specific consensus sites. The RNAP II is dephosphorylated and released, ready to be used again. TFIIH has three functions: helicase activity which makes the closed complex and open complex, phosphorylation activity where the C terminal domain of the RNAP II is phosphorylated so that initiation can begin, and transcription coupled DNA repair where it recruits the nucleotide excision repair system to fix lesions in DNA since it is easier to repair DNA when it is being transcribed than when it is nottherefore if this is damaged, XP can happen. 3. RNAP III makes tRNAS, 5S, and additional small RNAs. Interesting is that the prmoter for Pol III can be within the genei.e. downstream to the initiation site, and the information is within the transcribed region. There are two promoter sitesthe A box and B box, and the TFIIIC (transcription factor IIIC) binds to both of them, which recruits TFIIIB and binds upstream the initiation site, which calls in Pol III. Unlike in prokaryotes, each of these require a number of additional proteins called transcription factors in order to specidfically bind to a promotor and initiate transcription. The eukaryotic promoter is more complex and composed of cis sequence of the DNA recruits these trans acting factors through DNAprotein interactions. Protein-protein interactions cause a lot of the machinery needed for transcription to come down. THE MAJOR PROMOTOR in eukaryotes is called the core promotor region and this is absolutely needed to begin transcription. It includes the TATA box, and the initiator region (INR). However, alone, the core promotor region is weak and unregulated, and other premotor regions are needed: proximal promoter region is just a little upstream, distal promoter region is much more upstream, and downstream promoter element. The promoter does not need any of these, not even the TATA or IR. In addition, there may be enhancers that are upstream from the promoter, sometimes over

1000 kb upstream. They enhance basal transcription activity and are tissue specific, and act from far distanced, and do not depend on whether the promoter is up or downstream. TRANSCRIPTION INHIBITORS: 1. Actinomycin D, acridine: has a planar aromatic ring structure that intercalates between G and C successive pairs in a strand of DNA; introduces a kink in the DNA, and does not allow RNAP to transcribe and elongate in both pro and eukaryotes, and is used in cancer treatment. Can be used in lab to determine which cellular processes synthesize protein 2. Rifampicin: binds to the beta subunit of bacterial RNAP, and blocks promoter clearancei.e. elongation. Is a superantibiotic 3. Alpha-amanitin: produced by fungus and potent inhibitor of RNAP II and weak inhibitor RNAP III. Can be fatal to humans. Does not inhibit RNAP I or prokaryotic RNA Initiationa at RNAP I promotor: RNA Processing Major RNA processing mechanisms in eukaryotes rRNA: have to undergo methylation and nucleolytic cleavage mRNA: 5 capping, 3 cleavage because the amount transcribed can be extra, and polyadenylation, and removal of introns through splicing tRNA: cleavage at both ends, and base modification, addition of CCA, few cases of splicing IN Prokaryotes: rRNA: same thing as eukaryotes: methylation and nucleolytic cleavage mRNA: no modification tRNA: both end cleavage, CCA addition, base modificationexcactly the same, but no splicing of introns

mRNA processing in eukaryotes: primary transcript or premRNA, which is longer than mature mRNA because there are introns. Exons are retained in the mature mRNA. Exons are short, but introns have variable length. Splicing is process of removing introns, and there is usually the number of exons1 number of splicing, since the last end is not spliced. How does the mRNA know what is intron and what is exon? If the intron is left in, there is something messed up inside, and if an exon comes out, it messed up too. Most individual introns are U1 introns which all start with GU within a consensus sequence at the 5 end, and at the 3 end, they are AG at the consensus sequence and a polypyramidine (U or C in RNA) sequence in the 3 consensus sequence as well, and also a branch point A at the middleall three parts have to present in that order. These are cis factors just like the DNA had cis factors that called the transcription factors, these also call in transacting factors. for the 3 end, the trans factor is just a normal protein. But for the 5 end and the branch point A, the trans factor is snRNPs, which includes the snRNA that was mentioned before, but attached to a protein. The 5 AG consensus sequence of the intron attaches to the U1 snRNP which has a RNA part at its end that is complimentary to the consensus sequence. The U2 snRNP that attaches to the branch site A is complimentary to that consensus sequence, but the complimentary part is in the middle of the snRNA, and it DOES NOT PAIR with the A, and the A sticks out, particularly the 2 OH.

The majority of introns are spliceosomal introns which require a splicesosome complex to splice out the introns. The U1 and U2 have already been described. Their binding calls in the U4/U6, and U5 complex, and together they form the splicesosome. However at this point it is inactive, and te U4 and U1 fall off. The U6 goes to the 5 splice site and there is a transesterification reaction between the 2 OH of the branch point A that is sticking out and the 5 phosphate end of the intron sequence that forms a lariat which is a loop and the 5 end is cleaved. The U5 and U6 hold on to the upstream exon so that it does not float away; there is another nucleophilic attack now by the 3OH of the released 5 exon splice site to the 3 splice site, and the splicosome is released. The exon sequence is ligated together. This is an ATP dependent process. Thus this involves transesterification and cleavage. Type I and II introns: found in some nuclear, mitochondrial and chloroplast genes; they require no ATP for splicing; they are self-splicing, and no enzymes are required. In Type I intron, transesterification reaction is initiated by guanine nucleotide, but in Type II, it is done by the A branch point generating the normal lariat tRNA have endonucleases come and cleave the introns on both sides, and the exons are ligated together. 5-capping and 3 polyadenylation: the 5 cap is added even while transcript is being formed: it consists of a 7-methyl guanosine molecule attached from a 55 triphosphate attachmentvery rare, and the +1 and +2 nucleotides maybe methylated at the 2 OH. Used to direct transcript to nuclear pores, to identify the RNA for translation, and to protect the RNA from 53 exoribonnuclease activity. 3 polyadenylation happens to all mRNAs except those that code for histones and it involves the RNAP recognizing at the end an AAUAA sequence separated by a GU rich sequence 20 bp downstream. An endoribonuclease cleaves off between the two sequences and polyadenylate polymerase adds on the polyA tail with 80-250 As. Purpose: protect from 35 exonuclease activity, and target RNA for translation through interaction with polyA binding protein, even though it is on wrong side. The polyA binding protein also makes a wall surrounding the polyA tail, and its really tough for exonucleases. Differential mRNA processing: a single gene can give multiple proteins through different mRNA processings: 1. Alternative transcription initiation sites 2. Alternative polyadenylation/cleavage sitesusually results in same sequence but one longer 3. Alternative splicing of the primary transcriptcan result in different order of introns EXAMPLE: the same gene coding for calcitonin also codes for calcitonin gene related peptide (CGRP) based on whether it is in the brain or normal tissue. Can cause problems: thalassemia are manifested through mutations influencing the processing and not the sequence itself. Thalassemia patients have abnormally low hemoglobin levels, and over 50 mutations contribute to this problem. If an exon is not fully excised since there seems to be a more upstream 3 splice site than the true one, some part of the exon will remain on the mature mRNAand 2/3 times this will induce a frameshift mutation, and even if it does not, there is still and extra part of the exon in that will destabilize the protein. In addition, it can also be that it looks to be a new 5 splice site, and an exon will remain upstream. rRNA processing in eukaryotes: the RNAP I has synthesized 3/4 pieces of rRNA in one length (5S is done by Pol III), and the correct pieces (i.e. exons) are methylated at the 2 OH by snoRNPs (snRNA with protein) and then endonucleases and exonucleases get rid of the introns, and you have the final product. In prokaryotes, it is the same, but there is a tRNA piece in the middle for some reason, and snoRNPs do not do the methylation, and it is done directly by methyl transferase.

tRNA processing in eukaryotes: synthexized by RNAP III (along with rRNA 5S which does not undergo major processing); the 5 end is excised by the endonuclease RNase P (ribozyme) and the 3 end by RNase D. sequence CCA is added to the 3 end by nucleotidyltransferase. Base modification (to psuedouracil for instance), and introns are removed (not in prokaryotes mature mRNA transcripts do not start translation right after the 5 cap, but at the sequence AUG and do not end at the polyA tail, but at stop codon that is much more upstream than the polyA tail; the stuff in between is called the 5 untranslated region and 3 untranslated region. The 5 UTR is a translational regulator, and the 3 UTR recruits mRNA stability regulators. mRNA turnover: mRNA that is incorrectly processed is reapidly degraded in the nucleus, but if it gets to the cytoplasm, it has a large chance of being translated, the chance being proportional to its cytoplasmic concentration. Concentration of mRNA is dependent on rates of synthesis versus decay. rRNA and tRNA have stable concentrations, but mRNA concentration depends on the particular gene, with some lasting longer and some shorter. There are two mechanisms of decay: deadenylation-dependent decapping where the polyA tail is shortened by 35 exonuclease activity until it has 20-30 A residues, and then this triggers a decapping enzyme to remove the 5 cap, which exposes the mRNA to both 53 and 35 endonuclease activity; here the deadenylation phase is the rate limiting step. The other method is endonuclease-initiated decay where an endonuclease cleaves at the 3UTR region of the mRNA through a specific binding site, and this is the rate limiting step. Then, the 5 cap is decapped, and both cleavage products are destroyed by both type of endonucleases. NOTE that information regarding decay of mRNA is encoded within the gene and the 3 UTR and these for example may have many AU bases here to destabilize the mRNA so that deadenylation can occur quickly. Ribozymes: examples: RNase P, and self-splicing Group I introns which include both transesterification rxns and cleavage of the phosphodiester bond hydrolysis. They need a correct 3D structure, and active site includes and internal guide sequence that base pairs with substrate. Hammerhead ribozyme: involves self cleavage right next to the part that pairs; tetrahymena group I rRNA intron: places the 5 exon splice site near the internal guide sequence of the RNA itself; catalyzes both transesterification and cleavage rxns. Translation Ribosomes: prokaryotes= 50S + 30S = 70S; Eukaryotes 60S + 40S= 80S. composed of rRNA and proteins, but the catalysis is done by rRNA. A lot of translation factors that are required for each of the 3 stages of translation: initiation (IF, eIF), elongation (EF, eEF), termination (RF, eRFrelease factors) Genetic code: degenerate: single codon is only for Met and Trp, but multiple codons for everything else, including termination. Code is universalsame code for all organisms, with a few exceptions in mitochondria, bacteria, and single celled eukaryotes: exception UGA which is normally stop codon codes for selenocystein for some; code is nonoverlapping so any mRNA has 3 potential reading framesopen reading frames. Silent mutation is change in nucleotide that does not change amino acide. If it changes the amino acid, it is a missense mutation. If it changes into a stop codon, it is nonsense mutations. Unconventional translation: Usually, the two other reading frames contain no other information; exception: programmed ribosomal frameshift: where a movment to another reading frame can code for another protein, and this happens in retroviral gag and pol proitins in HIV that are initially synthesized as a polyprotein (95% of the time, the HIV codes for the gag protein, and ends at a stop codon, but 5% of

the time, it shifts to the base one earlier, and does not have a stop codon, but goes on to code the second half of the protein of which the first half is the same as the gag, and the extra half is shifted one base up or down at the slippery site and this is called the gag/pol) Unconventional translation: RNA editing: covalent change of identity of position of RNA bases after synthesis which can change an amino acid. (RNA processing does not change identiy of base): example: cytosine deaminase converts a C in apoB mRNA to a U which changes a gln to termination codon, so now there is a smaller protein that is called apoB-48 in the intestine since it has 48% of the weight that the full version in the liver. Modification involves RNA enzyme called guide RNA. Protein biosynthesis: 1. Activation of amino acids (tRNA charging by amino acids so they can bring them in)exposed loop called anticodon loop makes pairs with the codon on the mRNA. So 5 on the other end of the tRNA. The anticodon loop is far away from the amino acid arm where the CCA was added at the end. 61 codons code for 20 amino acids, so there should be 61 tRNA, but there are 35, so there is some structural freedom that allows some tRNAs to recognize more than one codon. Some recognize only one codon, some anticodons can only pair with their correct pair: C and A; but a U and G at the 5 position of the anticodon can base pari with AG, and CU on the codon, though if it is on the codon, it wont happen vice versa bc that will mean there should be even more tRNAs than 64; and an inosine which is related to guanine (tRNA has some base modifications, and this is created) can base pair with three bases AUCWobble hypothesis; charging is bringing amino acids to attach to the tRNA and this is done by the enzyme aminoacyl tRNA synthetase and there is one aa-tRNA synthetase for each amino acid, and they attach the aa to the 3 end OH; there is also a proofreading mechanism in place in case the wrong aa is attached. 2. Initiationrate limiting. In prokaryotes starts with two translation factors: IF-1 which binds to the 30S A site to prevent tRNA from binding there during initiation, and IF3 which binds somewhere as well (not one of the sites) on the 30S subunit and prevents premature association of the 30S and 50S and enhances the specificity of the fMet to the P site. Then mRNA binds to the 30S subunit in a way such that the AUG initiation sequence will lie just above the P site. Since there are many AUG sequences in different reading frames, there is a Shine-Dalgarno sequence that is a little upstream of the correct AUG sequence in the right reading frame that base pairs with the 3 end of the 16S rRNA (part of the 30S) such that the AUG sequence downstream is correctly positioned at the P site. So in prokaryotes, there is internal initiation. Then an IF2-GTP complex facilitates binding of fMet-tRNA to the 30S (initiation involves this charged amino acid since only this initator tRNA can actually directly bind to the P sitethe others shift there) and then load the 50S and GTP is hydrolyzed so that the two subunits stay together. Then the three IFs are released, and a 70S unit is created. In eukaryotes, there is no Shine-Dalgarno sequence, and instead the correct reading frame is identified by the cap-binding complex that binds to the 5 cap and then recruits the 40S (small) subunit to form a 43S complex, which then scans mRNA until it reaches AUG (sometimes something else); once it recognizes that, it recruits the tRNAMet (note no formyl in eukaryotes) subsequently the 60S unit. So unlike the prokaryotes, translation does not normally initiate internally, though there are some exceptions where it does (IRESes internal ribosome entry sitesviruses use this). Initiation is enhanced by the 5 cap and the 3 poly-A tail. The mRNA 5 cap is recognized by the eIF4E and this binds to the eIF4G, which binds to the protein that binds to the polyA tail, Pab1p. therefore, eIFE and Pab1p are linked through by eIF4G, and the 5 and 3 ends are brought together in a loop, and the presence of both of them synergistically enhances initiation. 3. Elongation: in prokaryotes: recruitment of aatRNA to the A site (the P site has the ftRNA on it), by EF-Tu-GTP which must be complementary to the codon at A within the rules of Wobble, and

EF-TU checks this and if the pair is correct, the GTP hydrolyzes, and EF-Tu-GDP releases itself. Next is peptide bond formation. Then the amino group of the aa on the tRNA at A site attacks the carbonyl carbon of the aa of the tRNA at P, and the entire chain at P is shifted to the A site. This is catalyzed by the peptidyl transferase activity deep in the 50S structure where there is only rRNA which alone without proteins does catalysis. Next step: ribosome translocation: once the P site tRNA is deacylated, the ribiosome moves one codon toward the 3 end of the mRNA translocation, which is fueled by GTP. The uncharged tRNA from the P site is now in the E site, and the peptidyl-tRNA is now in the P site, and A site is empty awaiting next aminoacylated tRNA. Same in eukaryotes, but factors have different name. 4. Termination and release: when a stop codon comes, no tRNA binds to those three stop codons, so the A site is occupied by a release factor that is structurally similar to tRNA but has no charged aa, so it signals the ribosome to hydrolyze the terminal peptidyl tRNA bond and release the pp, tRNA, and ribosomal subunits. 5. Folding and post translational processing Ribosome: bacterial has two subunits: 50S (2 rRNAs23 and 5S, and 36 proteins) and 30S (16S rRNA and 21 proteins); codon: anticodon recognition and aa-tRNA biding occurs at the 30S/50S interface where the mRNA slithers through. Peptidyl transfer, however, occurs in the 50S subunit, since that is taking place at the top of the tRNA, and the bottom of the tRNA is making contact with the interface. Large/subunit. 3 sites: A site: new aminoacyl-tRNA will be; P site is where the peptidyl tRNA (tRNA with growing chain) will be. Peptidyltransferase center happens deep in the 50S unit, and that is where the tRNAs of the A and P sites are brought into close proximity. E site is exit/escape site and after the P site tRNA has given the A site its stuff (translocation), this empty tRNA at P site is pushed into the E site where it can diffuse away and be recharged for subsequent translation. Polysome: translation of one pp does not have to wait for another: several polypeptides can be created by several ribosomes or polysomes. (can this happen with transcription?) also, in prokaryotes, the translation of a mRNA transcript begins even before transcription is complete, since there is no mRNA processing. Also, using several open reading frames, the prokaryotes make many but short lived (2-5 min) pps from one mRNApolycistronic but most eukaryotes make only one (monocistronic). Nonsense mutation suppression can happen in prokaryotes by a second mutation in the already mutated codon that coded for a stop codon, so that most likely, it codes for an amino acid now, and the pp goes on, or by a mutation in a tRNA gent that can now recognize the termination codon and inserts an amino acid in that positionknown as suppressor tRNAs and are expressed in low levels2nd more common. Translation inhibitors: 1. Puromycin: causes premature chain termination by acting as an analog of aa-TRNA in both prokaryotes and eukaryoteslooks like the A on the 3 end of CCA, but is not attached to the long tRNA molecule, such that if nucleophilic attack by its amine group occurs, it binds to the pp, but since it is not attached to the A site, the pp floats away. 2. Tetracyclins: blocks the A site on the 30S subunit in prokaryotes ONLY 3. Chloramphenicol: inhibits peptidyl transferase in prokaryotes 4. Cyclohexamide: inhibits peptidyl transferase in eukaryotes 5. Streptomycin: inhibits initiation and causes misreading of mRNA in prokaryotes Toxins: diphtheria toxin: inactivates eukaryotic eEF2 (elongation), and ricin which inactivates the 60S subunit of the eukaryotic ribosome. Protein Processing, Targeting, and Degradation

If hydrophobic chains are not buried, they will begin aggregatingamyloid based disease like Alzheimers. Formation of disulfide bonds by cysteine in specific regions through oxidation; proline can form both cis/trans forms, and prolyl isomerase can change conformation. Posttranslational modifications: 1. 2. 3. 4. 5. 6. 7. 8. Enzymatic removal of formyl group in prokaryotes and just the initial Met for eukaryotes Acetylation of amino/carboxy terminal residues Loss of signal sequence (N terminal sequences get cleaved) Phosphorylation of any residue with exposed hydroxyl group Carboxylation Methylation Carbohydrate side chain additions (glycoproteins) Isoprenyl groupshydrocarbon chains multiples of fiveconfer carcinogenic activity of ras isoprene group can covert a soluble protein into membrane associated protein 9. Prosthetic groupscovalent binding of co-factos (biotin, heme) 10. Proteolytic processinginactive precursor form must be trimmed (insulin)zymogens 11. Disulfide cross bridgescan also be inter to other pps Protein targeting: proteins are made in the cytoplasm, yet they show up everywhere, so they have to go to other places. Ribosomes are located in free in the cytoplasm in small clusters or associated with rER. The ER begings processing highway for many proteins. Proteins that have to go to lysosomes, membranes, and those secreted in the extracellular space are translated on rER. Proteins soluble in cytoplasm and end up going to nucleus are translated on cytoplasmic free floating ribosomes. What determines whether it will be translated on rER or not is a signal sequence near the N termini where these lysosomal, membrane, and secreted proteins direct associated ribosomes to the ER. The consensus properties that direct to the ER include a stretch of hydrophobic aas, one or more positively charged residues preceding the hydrophobic sequence, and some polar aas and short side chains near the cleavage site on the unprocessed protein. Proteins targeted to mitochondria or chloroplasts also employ N terminal signal sequences but of different compositions, but proteins that function in the cytosol dont contain signal sequences. Proteins on free ribosomes find the ER with the following mechanism: the mRNA transcript does not directly go to the rER by itself. Rather it initiates synthesis on free ribosomes, such that the first part of the protein that is synthesized is the signal sequence on the N-terminus. Then, signal recognition particles (SRPs) which are composed of 7S RNA and six proteins, bind to the signal sequence once it is fully translated, the ribosomes, and GTP and halts elongation when the nascent peptide is 70 aas long. The GTP-bound SRP directs the ribosome to SRP receptors on the cytosolic face of the ER. Therefore the nascent peptide itself that initiates a cascade of events that brings these translating ribosomes to the ER. Once it is there, the peptide goes through a peptide translocation complex in the ER, and starts going into the lumen of the ER. SRP dissociates from the ribosome and GTP is hydrolyzed, so that elongation of the pp resumes (now that SRP is not halting it) and the pp is coupled to ATP-driven translocation of the pp into the lumen. The signal peptidase cleaves the signal peptide and the ribosomal subunits dissociate. If the protein is a transmembrane protein, it remains in the membrane of the ER, rather than fully being translocated to the lumen. Protein Glycosylation: many cellular proteins have carb chains attached through Asn (N-linked) or Ser, Thr (O-linked) residuesnot all of them, this is determined by the sequence. N-linked glycosylation starts in the ER and finishes in the Golgi, but most O-linked occurs in the Golgi (for ER targeted proteins) or cytoplasm (for proteins that do not enter the ER).

The purpose of glycosylation is to stabilize the protein, to help its function, or to target the protein ot specific cellular organelles. For example, enzymes targeted to the lysosome are modified by the addition of one or more mannose-6-phosphate residues. The ER glycosylation pathway: a core oligosaccharide is attached to dolichol phosphate which is a carrier molecule that has a long greasy hydrocarbon chain that sticks into the ER lumen, and a phosphate group on the cytosolic face. The oligosaccharide is attached to the phosphate group (this is inhibited by tunicamycin); at a certain point, the oligosaccharide is translocated and flipped into the inside of the lumen, and the greasy part comes on the outside. The oligosaccharide has mannose and glucose added to it, and for N-linked proteins, a transferase protein transfers the oligosaccharide to an Asn residue of the pp, and the pp can be further modified. The dolichol phosphate is flipped back and recycled so that the Pi is back on the cytosolic face. The proteins are now moved from the ER to the Golgi through transport vesicles which bud off the ER and fuse with the cis side of the golgi. Sorting occurs on the trans side of the Golgi, and this sorting machinery must distinguish between proteins on the basis of structural features other than the signal sequence (since this was chopped off in the ER). The vesicles going from the ER to the cis have soluble proteins in them, but also membrane proteins that are still in the membrane of the vesicle. The various proteins will go to the lysosome, etc. Default location is the to the plasma membrane outside into extracellular space. Some even go back from trans to the ER, since they needed to function eventually in the ER but first needed to be processed. Transport to the Lysosome: best understood for the lysosomal hydrolase enzyme which requires a Nlinked oligosaccharide with one or more mannose-6-phosphate residue, which are phosphorylated by specific phosphotransferases in the Golgi, though we do not know how the Golgi recognizes which of the mannose to phosphorylate. However, receptors of the Golgi recognize the phosphorylated mannose residues and target them to the lysosome. Transport to the Mitochondria: all Mt proteins are synthesized on free ribosomes. The N terminal signal sequence (which is different from ER sequence in that rather than nonpolar side chains, it has Ser, Thr, and basic residues) it is bound by cytosolic proteins Hsp70 and MSF. It is folded in the cytosol, then delivered to a receptor on the Mt and goes inside, and this translocation is facilitated by ATP and electrochemical potential and during translocation, protein becomes unfolded. The signal sequence is removed once inside, and the protein is refolded. Transport to the Nucleus: proteins headed to nucleus are also synthesized on free ribosomes, and are folded in cytosol. Nuclear proteins also have a signal sequence, called nuclear localization sequence (NLS) which is not restricted to the N-terminus of a protein, since it can be located almost anywhere along a primary sequence as long as it is on the periphery of the protein. The NLS is also not removed to allow repeated nuclear importation following cell division. The NLS consists of several basic residues. Nuclear import is medicated by specific transport proteins and the nuclear pores. Heterodimers of importin alpha and beta function as soluble receptors for proteins targeted to the nucleus. The mechanism for this is that the importin alpha/beta sees the NLS and binds to it and takes it the nuclear pore which is lined with a meshwork of proteins called nucleoporins which form a sieve such that only small proteins below 30 kD and organics can pass through. Larger proteins require a carrier protein like the importins to take them through. The importin takes the pp in although this requires energy. Inside the nucleus, the Ran-GTP complex removes the beta subunit from the pp and both Ran-GTP and CAS (cellular apoptosis susceptibility protein) take off the alpha, so that the pp is now alone and can perform its function; the alpha/beta are recycled.

Protein targeting in prokaryotes: bacterial proteins can be targeted to the inner or outer membrane (no organelles in prokaryotes), the periplasmic space between these membranes, and the extracellular medium. They use N-terminal signal sequences similar to the eukaryotic proteins targeted to the ER, with the normal stretch of hydrophobic aas, etc. the mechanics however, are very different; a soluble SecB protein that binds to the signal sequence of the pp and takes the pp to the membrane. There, it joins with SecA and SecYEG and SecB is released. SecA inserts itself into the membrane, takeing 20 aa with it, and ATP hydrolysis causes a conformational change that withdraws SecA from the membrane. Then SecA binds another ATP and pushes the next 20 aa through the membrane. The entire protein passes through in this fashion and is released in the periplasm. Electrochemical potential is also involved in protein translocation. Most protein exported from bacteria use this pathway, but some follow a path similar to the SRP way in eukaryotes. Protein Degradation: involves an ATP dependent pathway that involves a protein called ubiquitin (UB) which becomes covalently linked to Lys residues of targeted proteins through this mechanism: UB activating enzyme (E1) links to the Gly reisude of the C-terminal of ubiquitin. E1-UB complex is transferred to the UB-conjugating enzyme (E2) which transfers the complex to a Lys residue of the targeted protein. The E2 and Ub protein ligase (U3) are bound to the protein substrate. This cycle is repeated to generate poly-ubiquinated proteins which reaches 4 and gets directed to the proteasome for degradation. Once the polyubiquitin has been added in the slow rate limiting step, the next step is a lot faster, and involves an enormous protein called proteasome which has multiple active sites that face the interior, and act on all sorts of proteins. It is a cylinder, and is present throughout the cel. It only acts on proteins that have been specifically marked for destruction by covalent attachment of polyubiquitin. Cancer Genes and Oncogenes Two key properties that cancer cell adopts in oncogenesis: disrupts processes regulating normal cell division leading to uncontrolled cellular growth, and also invades other tissues either by direct growth into adjacent tissue via local invastion or by implantation into distant sites through metastasis. Cancer Genes 1. Proto-oncogenes and oncogenes 2. Tumor suppressor genes 3. Stability genes Classification by tissue type: 1. carcinoma is 90% of cancersdevelops from epithelial cells, derived mainly from ectoderm and a little endoderm. 2. Sarcoma 3. Leukaemia: circulatory or lymphatic, 8% of cancer, and derviced from mesoderm Classification by type of cells 1. 2. 3. 4. Adenomatous cells Squamous Dfas Dfas

Classification by the site origin of tumor:

1. Breast: carcinoma of ductal 2. Dsfa 3. Afda Differentiation is extent to which neoplastic cells resemble normal cells; opposite is anaplasia is lack of differentiation. Benign tumors are well differentiated; malignant tumors range from well differentiated to undifferentiated. Benign tumor: growth is contained within tissue it comes from and it is encapsulated and looks like the cells of the area of origin; there is no tissue destruction; it has a slow rate of growth, and it is not fatal if untreated, and if it is treated, there is rare recurrentce, and there is poor prognosis only if it cant be taken out. Confined within basal lamina. Malignant tumor: tissue destruction is common, and growth is by infiltration or metastasis, the characterisitcs of these cells are atypical of tissue of origin, unrestrained slow or rapid rate of growth, and recurrence is common, and in ALL cases, it is fatal if left untreated. Cancer starts with multi steps, and it deso not start with a fully invasive cancer cell. It requires specific factors or events that lead to the accumulation of DNA mutations. In all cancers, mutations that alter gene expression are seen. Most mutations are somatic (non-inherited), but a small percentage are inherited gem-line mutations, but there is no clear-cut pattern of inheritance, and usually one mutant allel of a cancer-causing gene is inherite and predisposes the person to cancer. the likelihood of Background rate of spontaneous mutations: baseline rate of cancer where mutations occur spontaneously abve which there are other factors that promote mutation that contribute to cancer (such as environemnetal factors), which mutatnt genes are needed to cause cancer,and how many mutations are needed to cause cancer, what are the differences between normal cells and cancerous cells, so how do these mutations convert normal cells into malignant ones Risk factors for cancer: environmental, random somatic mutations, inherited germ line mutation, infectious agents (viral and bacterial) DNA repair mechanism damage is what directly causes cancer, bc damage is happening all the time, but only when the repair does not work does it remain. DNA damage is therefore necessary but not sufficient to cause cancer; normal cells often develop mutations in their DNA but they have the ability to repair most of these mutation. If they cannot make repairs, they undergo apoptosis. Genes that mutate to cause cancer: 1. Tumor suppressor genes: normal function to regulate cell divisionboth alleles must be mutated or removed to lose the gene activity; most are somatic, not inherited. Second mutation will be a gross event leading to loss of heterozygosity in the surrounding area. Knudsens two hit hypothesis is that once the first mutation has occurred, the second mutation is much more probable. Tumor suppressor genes check at each step of the cell cycle, after G1, S, G2, M. they check if the cell is the correct size, whether DNA is damaged, fully replicated, repaired, if during mitosis spindle fibers have formed, ahave the attached chromosomes separated, etc. Microbiological Techniques RFLP

DNA clonins: plasmids are not part of bacterial chromosome and are vectors for the DNA needing to be multiplied. The plasmid is cleaved by endonuclease, and the DNA fragment to be cloned is inserted into it by DNA ligase and then the bacteria takes it up which is transformation in prokaryotes and transfection in eukaryotes. Transformation occurs by chemical methods or electroprolation.