Genetics - Module 21

7/29/2019 Genetics - Module 21

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Trigger

The clock ticked at exactly 09:00 a.m. A man dressed in barong began to read aloud thenames on a list. All were there. The man then proceeded to begin to read what they allwaited for Mr. Palalas will and testament, the patriarch of the family. The man read

through the whole 5 pages. Whew, its almost done, the 6th

page! they all thought. Thenthe man read the last paragraph which said All who want to receive a piece of myestate must undergo a DNA test. For this purpose, I have left my hair.

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Title: Si Lolo, talaga!

Instructional ObjectivesAt the end of the module, the student is expected to be able to:

1. Discuss the purine/pyrimidine nucleotide:1.1 Identify the descriptions of purine and pyrimidine bases1.2 Identify the descriptions of purine and pyrimidine nucleosides1.3 Identify the descriptions of purine/pyrimidine nucleotides

1.3.1 naturally occurring derivatives1.3.2 synthetic derivatives

2. Discuss the purine/pyrimidine nucleotide metabolism.2.1 Identify the precursors of purine and pyrimidine2.2 Identify the descriptions of purine synthesis de novo to include enzyme, cofactors,

significant intermediate products and its regulation.2.3 Identify the specific enzymes, substrates of the salvage pathway of purine

nucleotides.2.4 Identify the descriptions of de novo synthesis of pyrimidine2.5 Identify the differences between salvage pathways of purine and pyrimidine

nucleotides2.6 Identify the descriptions of de novo synthesis of deoxyribonucleotides2.7 Describe the purine and pyrimidine catabolism

2.7.1 Identify the enzymes and specific substrates involved in purine catabolism2.7.2 Identify the end products of purine catabolism2.7.3 Identify the significant steps in purine catabolism2.7.4 Identify the end products of pyrimidine nucleotide catabolism

2.8 Given specific inherited disorders of purine metabolism, identify the enzymes

involved and its metabolic defect.3. Discuss the nucleic acid structure and function.3.1 Identify the descriptions of deoxyribonucleic acid structure and function3.2 Identify the descriptions of ribonucleic acid structure and function3.3 Identify the difference between RNA and DNA3.4 Identify the different types of RNA

4. Discuss the systematic process involved in DNA replication and repair.4.1 Identify the theories of replication4.2 Identify the enzymes involved in replication as to mode of action4.3 Identify the sequential steps involved in replication4.4 Identify the descriptions of Okazaki fragments4.5 Identify the proofreading device4.6 Identify the repair mechanism during DNA damage4.7 Identify descriptions and examples of mutations

4.7.1 base substitution4.7.2 frame shift mutation

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5. Discuss the processes involved in transcription.5.1 Identify the difference between replication and transcription.5.2 Identify the descriptions of the enzymes DNA-directed RNA polymerase5.3 Identify descriptions of the ff.: promoter, TATA BOX, CAAT BOX, termination

sequence, exons, introns and splicing.

5.4 Identify the sequential steps involve in transcription5.5 Identify the descriptions of RNA processing6. Discuss translation.

6.1 Identify the descriptions of translation, genetic code, codon, anticodon, non-sensecodon

6.2 Identify the following features of the genetic code6.2.1 degeneracy6.2.2 Unambiguous6.2.3 Non-overlapping6.2.4 No punctuation

6.3 Identify the sequential steps & the enzymes involved in protein synthesis.

6.4 Identify the descriptions of post-translational modifications

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Task Sheet

1. Discuss the purine/pyrimidine nucleotides.1.1 Define a nucleotide1.2 Enumerate the importance of nucleotides.

1.3 Describe the chemistry of purine and pyrimidine bases1.4 Describe the purine/pyrimidine nucleosides1.5 Describe the purine and pyrimidine nucleotides1.6 Describe the naturally occurring nucleotide and its derivatives1.7 Describe the synthetic nucleotide derivatives

2. Discuss the metabolism of purine and pyrimidine nucleotides.2.1 State the precursors of purine and pyrimidine nucleotides.2.2 Describe/trace the purine nucleotide synthesis:

2.2.1 Purine nucleotide de novo synthesis2.2.2 Regulation of synthesis2.2.3 Synthesis of deoxyribonucleotides

2.2.4 Salvage pathways of purine nucleotide2.3 Describe/trace pyrimidine nucleotides synthesis2.3.1 De novo synthesis2.3.2 Regulation of synthesis2.3.3 Synthesis of deoxythymidines

2.4 Trace the catabolism of purines and pyrimidines2.5 Describe the inherited disorders of purine metabolism & their associated enzyme

abnormalities3. Discuss nucleic acid structure and functions

3.1 Describe the chemical structure of DNA & its function3.2 Describe the chemical structure of the 3 main types of RNA & state their specific

functions3.3 Differentiate RNA from DNA4. Discuss the systematic process of DNA replication and repair.

4.1 Define replication4.2 Describe the theories of replication4.3 Describe the sequential steps and the enzymes involved in replication4.4 Describe the leading strand, lagging strand, Okazaki fragment, and proofreading

device.4.5 Describe DNA repair in damaged DNA4.6 Describe mutation

4.6.1 Define mutation4.6.2 Enumerate/describe some forms of mutation and give examples

5. Discuss transcription.5.1 Differentiate replication from transcription.5.2 Describe the sequential steps in transcription to include enzymes5.3 Describe the following: promoter site, TATA BOX, CAAT BOX, termination

sequence, exons, introns and splicing5.4 Describe RNA processing

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6. Discuss the process of translation.6.1 Define translation6.2 Describe the sequential steps involved in protein synthesis.6.3 Describe the features of the genetic code.6.4 State some post-translational modifications.

7. Discuss the uses of DNA testing.

Reference:Murray R, et al: Hapers Illustrated Biochemistry, 27th ed, c 2006

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Tutor Guide

1. Discuss purine/pyrimidine nucleotides.1.1 Define a nucleotide.

Nucleotides are ring compound that contain either a purine or pyrimidine base, a

sugar usually pentose (ribose or deoxyribose) and phosphate group.

1.2 Enumerate the importance of nucleotides. Some physicochemicalimportance of nucleotides include:

1. Serves as monomeric unit precursor of the nucleic acids RNA & DNA2. As high energy source that drive otherwise endergonic reactions - Ex. ATP3. High energy intermediate in lipid & carbohydrate metabolism4. Nucleotides form a portion of coenzymes such as: FAD, NAD+, NADP+,

coenzyme A, & S-adenosylmethionine5. As metabolic regulators such as cAMP & cGMP

1.3 Describe the chemistry of purine and pyrimidine bases.Purine & pyrimidine bases are aromatic heterocyclic ring compounds that containboth carbon and non-carbon atoms usually nitrogen. The major purine (adenine &guanine) and pyrimidine (cytosine, uracil & thymine) bases are derived from parentcompound structure of the above bases that is shown in Figure 1.

1.4 Describe the purine and pyrimidine nucleosides.Purine and pyrimidine nucleosides are formed by linking the bases

with sugar usually pentose sugar (contains 5 C atoms) commonly ribose or

deoxyribose. Purines and pyrimidine bases are attached to the sugar via -

N glycosidic linkage (N9 of purine to C1' of the sugar while N1 of pyrimidine

to C1' of the sugar). The atoms of the bases are numbered and designatedwith cardinal number to distinguished from primed number of the atoms ofthe sugar.

1.5 Describe the purine and pyrimidine nucleotides.Nucleotides are nucleosides that are phosphorylated either with one, two or three

phosphate groups. The phosphate is commonly attached to the sugar at its 5' position.It may also be esterified to C2' or C3'. The table below shows the common purine andpyrimidine bases, nucleosides, and nucleotides. These nucleoside and nucleotidenomenclature is associated with ribose sugar only. If the sugar attached to the base isdeoxyribose, the prefix deoxy is attached, e.g. deoxyadenosine, deoxyadenosine

monophosphate (dAMP). Nucleotides that form as units of nucleic acids may be writtenin abbreviation using the initial capital letter of the base it contain.Note that uracil is usually associated with sugar ribose while thymine is usually

associated with sugar deoxyribose.

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1.6 Naturally Nucleotide Derivatives

1.6.1 Adenosine derivatives1.6.1.1 Adenosine diphosphate (ADP) & adenosine triphosphate

(ATP) serve as universal currency of energy in biologic system1.6.1.2 cyclic AMP (cAMP) is a mediator of hormone action

1.6.1.3 S-adenosylmethionine acts as active methionine and amethyl donor in methylation reaction and the source of polyamines inpropylamine synthesis.

1.6.2 Guanosine derivatives1.6.2.1 Guanosine diphosphate (GDP) & guanosine triphosphate

(GTP) are another source of energy1.6.2.2 cGMP is also a mediator of hormone action

1.6.3 Uracil derivatives1.6.3.1 Uridine diphosphate glucose (UDPGlc)is the precursor of

glycogen1.6.3.2 Uridine diphosphoglucuronic acid (UDPGlcUA) serves as the

active glucuronide for conjugation reaction1.6.3.3 Uridine triphosphate (UTP) is the precursor for the

polymerization of uridine mnophosphate into RNA

1.6.4 Cytosine derivatives1.6.4.1 CDP & CTP act as precursor of CMP for polymerization into

RNA CTP is also important for synthesis of some lipid1.7 Synthetic Nucleotides Derivatives

Synthetic derivatives of purine and pyrimidine are now widely use in medicine. Most

of them are utilize by exploiting their role in nucleic acid. They are modified either byaltering the sugar moiety or the heterocylcic rings. These alterations induce toxicwhen the analogue is incorporated into specific cellular component. Examples ofthese synthetic analogues are:1. Arabinosylcytosine - ARA C2. Allopurinol3. Azathioprine4. 5-Fluorouracil5. 6- Mercaptopurine

BASE NUCLEOSIDE NUCLEOTIDE

Adenine Adenosine Adenosine monophosphate (AMP)

Guanine Guanosine Guanosine monophosphate (GMP)

Hypoxanthine Inosine Inosine monophosphate (IMP)Cytosine Cytidine Cytidine monophosphate (CMP)

Uracil Uridine Uridine monophosphate (UMP)

Thymine Thymidine Thymidine monophosphate (TMP)

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2. Discuss the metabolism of purine & pyrimidine nucleotide.2.1 Precursor of Purine/Pyrimidine

The atoms of 2 fused rings of purine are synthesized from 5 differentprecursor molecules. These are glutamine, glycine, aspartate, respiratory CO2, &methenyl tetrahydrofolate (formyltetrahydrofolate). The same precursor molecules

are required for pyrimidine ring synthesis. Both bases requirephosphoribosylpyrrohosphate (PRPP) as the donor of ribose phosphate group ofthe nucleotide.

2.2 Synthesis of Purine Nucleotides2.2.1 De novo Synthesis

Purine rings are specialized products that are synthesized from amino acids.The purine bases adenine and guanine differ only in the substituent group attachedto the rings. Adenine has amino (NH2) group attached to C 6, while guanine hasamino group at C2 & carbonyl O2 at 6.

The step committed to purine synthesis is the formation of 5'

phosphoribosylamine, a reaction catalyzed by glutamine PRPP amidotransferase. Theamide group from glutamine is transferred to C-1' of PRPP, where it replaces thepyrrophosphate. The nitrogen atom of 5'-phosphoribosylamine eventually becomes N9of the purine ring. Through 9 consecutive steps, 5'-phosphoribosylamine is convertedto the parent nucleotide, inosine monophosphate (IMP). The pathway branches at IMP,with separate reactions for AMP & GMP production. The conversion of IMP to both

AMP & GMP involves the addition of an amino group to the ring and requires a sourceof energy. Aspartate is the amino donor to AMP synthesis & GTP supplies the energy.During the addition of the amino group, the remainder of the aspartate is release asfumarate. For GMP synthesis, glutamine is the amino donor & ATP supplies theenergy.

2.2.2 Regulation of SynthesisThe rate-limiting step in purine nucleotide synthesis is the formation of 5-

phosphoribosylamine. The most important factor in regulating the enzymeglutamine:PRPP amidotransferase activity is the intracellular concentration ofPRPP. Small changes on PRPP concentration result in proportional increase in therate of phosphoribosylamine synthesis. The enzyme is also subject to productinhibition that is by IMP, AMP, & GMP. Secondary target of product inhibition are 1)PRPP synthase, which is inhibited by IMP, AMP & GMP, 2) adenosylsuccinatesynthase, the enzyme at the branched point for AMP synthesis is inhibited by AMP,& 3) IM dehydrogenase, the enzyme at the branched point for GMP synthesis whichis inhibited by GMP.

2.2.3 Synthesis of DeoxyribonucleotidesThe synthesis of deoxyribonucleotides is catalyzed by ribonucleotide

reductase, a multienzyme complex containing thioredoxin, and thioredoxinreductase. Ribonucleotide reductase converts all four ribonucleotide diphosphate(NDP) to the corresponding deoxyribonucleotide diphosphate (dNDP). Thioredoxin,a small protein containing cysteine residues, acts as reducing agent in the reaction.The catalytic cycle is completed by thioredoxin reductase, which regenerates thereduced form of thioredoxin. The deoxyribonucleotide diphosphates are

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subsequently phosphorylated to the corresponding triphosphates for use in DNAsynthesis.

Ribonucleotide reductase is subject to a complex pattern of allostericregulation. The enzyme has two types of regulatory sites, one that controls the

overall activity and another that is responsible for maintaining balance in the cellularpool of dATP, dGTP, dCTP, and dTTP that is required for DNA synthesis. Theoverall catalytic activity is inhibited by dATP and activated by ATP.

2.2.4 Salvage Pathway of PurineIn most cells, pathways are available that allow hypoxanthine, guanine, and

adenine to be recycled. The function of these pathways is to avoid the high-energydemand placed on the cell by the de novo synthesis of purine rings. De novosynthesis of each purine nucleoside triphosphate requires nine high-energyphosphate bonds; whereas salvage of a purine base and converting it tocorresponding nucleoside triphosphate requires only four high-energy phosphate

bonds. Rapidly dividing cells, particularly liver and placenta rely heavily on thepathways of the de novo synthesis. Other cells, however, derive most of the purinenucleotides from salvage pathways. The purine bases that are salvage may comefrom either dietary sources or turnover from an intracellular nucleic acid. Twoenzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and adeninephosphoribosyl transferase (APRT), are responsible for salvaging purine rings.Hypoxanthine and guanine are substrate for HGPRT, which transfers ribose-5-phosphate from PRPP to purine ring producing IMP and GMP. APRT transfersribose-5-phosphate to adenine producing AMP. The specific activity of APRT inmost cells is very low compared to that of HGPRT.

2.3 Synthesis of Pyrimidines2.3.1 De novo SynthesisPyrimidine nucleotide synthesis can be describe in 2 stages: 1) the synthesis

of orotidine monophospahte (OMP), the parent nucleotide 2) the conversion of OMPto UTP & CTP. Carbamoyl phosphate, the first intermediate in the pathway, issynthesized from glutamine, bicarbonate, and ATP in a reaction catalyzed bycarbamyl phosphate synthetase-2 (CPS-2). CPS-2 is distinct from CPS-1 amitochondrial enzyme that participate in urea cycle. In contrast CPS-2 is a cytosolicenzyme that is used exclusively for pyrimidine synthesis. Carbamyl phosphate isconverted carbamoyl aspartate a reaction catalyzed by aspartate transcarbomylase(ATCase). The next step is a reaction that converts carbamoyl aspartate to oroticacid, the first complete pyrimidine ring. Transfer of ribose-5-phosphate from PRPPto orotic acid, a reaction catalyzed by orotate phosphoribosytransferase (OPRT)produces OMP, the nucleotide. The reaction converting OMP to UTP & CTP start bydecarboxylation of OMP to UMP, which is followed by 2 sequentialdephosphorylation reactions producing UTP. CTP synthase catalyzed the transferof an amide group from glutamine to C4 of the base producing CTP.

2.3.2 Regulation of Pyrimidine nucleotide SynthesisThe enzyme CPS 2 is the primary regulatory enzyme of pyrimidine

nucleotide synthesis in eukaryotic cells. CPS 2 is allosterically inhibited by UTP. It isactivated by PRPP and ATP. Additionally, eukaryotic cells have most the enzyme in

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the pathway organized as separate domains of the same multifunctional protein, afeature that allows the synthesis of several enzymes to be coordinately controlled.The first three enzymes in the pathway are found in the same multifunctionalprotein, which is known as CAD (from the first letter in the names of the enzyme).Similarly, the 2 enzyme that convert orotic acid to UMP are separate domains on

the same multifunctional protein.

2.3.3 Synthesis of Deoxythmidine MonophosphateThymine rather than uracil is found in DNA. The intermediate precursor of

dTMP is deoxyuridine monophosphate(dUMP). thymidylate synthase catalyzes theaddition of a methyl group to C-5 in the ring of dUMP, resulting in dTMP. Thesource of the methyl group is methylene-THF. During transfer the methylene groupis reduced to a methyl group. This reaction results in the concomitant oxidation ofTHF to DHF. The catalytic cycle is completed by the reduction of DHF to THF, areaction catalyzed by dihydrofolate reductase, followed by the addition of anotherone-carbon fragment to THF.

2.4 Purine & Pyrimidine Catabolism

2.4.1 Purine CatabolismBoth ribonucleotides and deoxyribonucleotides are degraded by the same

pathway. Substituents attached to the ring of AMP, GMP, & IMP are sequentiallyremoved by parallel pathways. The phosphate groups are removed bynucleotidases (kinase). The amino group is then removed from adenosine andguanosine by adenosine deaminase & inosine deaminase. Phosphate is addedacross the bond linking ribose to purine ring, releasing ribose-1-phosphate in areaction catalyzed by purine nucleoside phosphorylase. The sum of these reactions

converts AMP & IMP to hypoxanthine & GMP to guanine. The terminal step inpurine nucleotide degradation is the sequential oxidation of hypoxanthine andxanthine to uric acid, which is excreted in the urine. Both of these reactions arecatalyzed by xanthine oxidase, a flavoprotein that requires molybdenum asessential cofactor.

2.4.2 Pyrimidine Nucleotide CatabolismDegradation of pyrimidine nucleotide starts by removal of amino groups,

phosphate, and ribose from the ring. The ring is then opened up and partially

converted to small soluble molecules, including NH4+, CO2, --alanine, and -

aminoisobutyrate. These highly soluble molecules are excreted without any problem

in the urine.

2.5Inherited disorder of purine metabolism & their associated enzyme abnormalities.2.5.1 Gout - is a group of metabolic disease associated with hyperuricemiaand the deposition of monosodium urate crystals in tissues. The symptoms usuallyappear in the fourth decade in men and after menopause in women. Plasma uricacid levels are usually elevated for so many years before symptoms appear.2.5.2 Lesch Nyhan syndrome2.5.3 Immunodeficiency2.5.4 Renal lithiasis2.5.5 Xanthinuria

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3. Recall nucleic acid structure & function.3.1 Deoxyribonucleic acid structure (DNA) & function

DNA is the chemical basis of heredity and is organized into genes, the

fundamental units of genetic information. DNA is a double stranded polymericmolecule composed of only 4 types of monomeric units. These repeating units:deoxyadenylate, deoxyguanylate, deoxycytidylate, & thymidylate are held inpolymeric form by 3',5' phosphodiester bridge constituting a single strand. Theinformation content of DNA (the genetic code) resides in the sequence in whichthese monomers, purine, & pyrimidine deoxyribonucleotides are ordered. Thepolymer possesses a polarity: one end has a 5' hydroxyl or phosphate terminuswhile the other has a 3'-phosphate or hydroxyl moiety. In a DNA molecule theconcentration of deoxyadenosine nucleotides (A) equals that of thymidine (T)nucleotide (A=T), while the concentration of deoxyguanosine nucleotide (G) equalsthat of deoxycytidine (C) nucleotide (G=C). The 2 strands are held in register by

hydrogen bonds between the purine and pyrimidine bases of the respective linearmolecules. The pairings between the purine & the pyrimidine nucleotides on theopposite strands are very specific. They are dependent upon hydrogen bonding of Awith T, & G with C. Two hydrogen bonds held A=T whereas 3 hydrogen bonds heldG=C. The 2 strands of the double-helical molecule, each of which possesses apolarity, are anti-parallel; ie, one strand runs in the 5' to 3' direction and the other inthe 3' to 5' direction. In the double stranded DNA molecules the genetic informationresides in the sequence of nucleotides on one strand, the template strand; theopposite strand is considered the coding strand because it matches the RNAtranscript that encodes the protein. The 2 strands wind around a central axis in theform of a double helix.

There are major and minor grooves in the DNA molecule. In these grooves,proteins can interact specifically with exposed atoms of the nucleotides (usually byhydrogen bonding) and thereby recognize and bind to specific nucleotidesequences without disrupting the base pairing of the double-helical DNA molecule.Regulatory proteins can control the expression of specific genes via suchinteractions.

The genetic information stored in the nucleotide sequence of DNA serves 2purposes. It is the source of information for the synthesis of all protein molecules ofthe cell and organism, and it provides the information inherited by the daughter cellsor offspring. Both these function require that the DNA molecule serve as a template-in the first case for the transcription of the information into RNA and in the secondcase for the replication of the information into daughter DNA molecules. Replicationof DNA molecule occurs in a semiconservative manner.

3.2 RNA chemical structure & function

Ribonucleic acid (RNA) is a polymer of purine & pyrimidine ribonucleotideslinked together by 3', 5' - phosphodiester bridges analogous to those in DNA.

Although it shares many features with DNA, RNA possesses several specificdifferences:

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1. In RNA, the sugar moiety is ribose rather than deoxyribose2. It does not possess thymine except in rare case. Instead of thymine, RNA

contains the ribonucleotide of uracil3. RNA exist as a single strand however it is capable of folding back upon itself

thus acquiring a double stranded characteristics

4. The purine nucleotides does not necessarily equal to pyrimidine nucleotides5. RNA can be hydrolyzed by alkali to 2', 3' cyclic diesters of the mononucleotides

In all prokaryotic & eukaryotic organism, 3 main classes of RNA moleculesexist: messenger RNA (mRNA), transfer RNA (tRNA), & ribosomal RNA (rRNA).Each differs from the others by size, function and general stability.

3.2.1 Messenger RNA (mRNA) - This class is the most heterogeneous in size andstability. All members of the class function as messenger conveying the informationin a gene to the protein synthesizing machinery where it serves as a template onwhich a specific sequence of amino acids is polymerized to form a specific protein

molecule, the ultimate gene product.It has some unique chemical characteristics. The 5' terminus of mRNA is"capped" by a 7-methyguanosine triphosphate that is linked to adjacent 2' - O -methyl ribonucleoside at its 5' - hydroxyl through the 3 phosphate. The cap isprobably involved in the recognition of mRNA by the translating machinery and ithelps stabilize the m RNA by preventing the attack of 5' - exonucleases. Translationbegins at the 5' or capped terminus. The other end, the 3' hydroxyl terminus has 20-250 adenylate residues (called poly A tail), this prevents exonuclease attacked. Itfrequently contains an internal 6-methyladenylates and other 2' - O - ribosemethylated nucleotides.

3.2.2 Transfer RNA (tRNA)It consists approximately 75 nucleotides. Also generated by nuclearprocessing of a precursor molecule. The tRNA molecule serves as adopters for thetranslation of the information in the sequence of nucleotides of the m RNA intospecific amino acids. There are at least 20 species of tRNA molecules in every cell,at least one (often several) corresponding to each of the 20 amino acids required forprotein synthesis. As a class they have many features in common. There is anextensive folding of its secondary structure that it appears like a cloverleaf.

All tRNA molecules contain 4 main arms. The acceptor arm that can attachedto carboxyl groups of amino acids. The anticodon arm at the end of a base-pairedstem recognizes the triplet nucleotide or codon of the template mRNA. It has anucleotide sequence complementary to the codon (in mRNA) and is responsible forthe specificity of the tRNA. The D arm is named for the presence of the base pair

dihydrouridine, and TC arm for the sequence T, pseudouridine , and C. The extra

arm is the most variable feature and provides a basis for classification.

3.2.3 Ribosomal RNA (rRNA)A ribosome is a cytoplasmic nucleoprotein structure that acts as machinery

for the synthesis of proteins from the mRNA template. On the ribosome the mRNA& tRNA molecules interact to translate into a specific protein molecule informationtranscribed from the gene.

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4. Discuss the systematic processes involved in DNA replication & repair.

4.1 Definition

There are 3 major steps in the processing of genetic information. These are

replication, transcription, & translation.

1. Replication is the copying of the parent DNA to form daughter DNA molecules, havingnucleotide sequences identical to those of the parent DNA.

2. Transcription is the process in which part of the genetic message in DNA is rewritten inthe form of RNA.

3. Translation is the process in which the genetic message coded by RNA is translated bythe ribosomes into the 20-letter alphabet of protein structure.

4.2 Theories of replication

The 2 proposed theories of the manner of replication are the conservative &semiconservative manner.

4.2.1 Conservative replication proposed that each of the heavy strands of parentCNA will be replicated to yield a DNA duplex containing 2 light strands and theoriginal heavy duplex DNA. Continuation of conservative replication will yield (in thenext generation) one heavy DNA (from parent DNA strand) and 3 light DNA's but nohybrid DNA's

4.2.2 Semiconservative replication is the most popular and accepted manner ofreplication. Here, when each strand of the double-stranded parental DNA molecule

separates from its complement during replication, each serves as a template onwhich a new complementary strand is synthesized. The 2 newly formed doublestranded daughter DNA molecules, each containing one strand (but complementaryrather than identical) from the parental double-stranded DNA. Each daughter cellcontains DNA molecules with information identical to that which the parentpossessed; yet in each daughter cell the DNA molecule of the parent cell has onlybeen semiconserved. The next generation will yield 2 hybrid DNA and 2 light DNA.

4.3 The enzymes involved in replication are:

4.3.1 RNA polymerase or primase is responsible for creating a temporarycomplementary RNA primer.

4.3.2 DNA polymerase enzymesPolymerase alpha - present in nucleus & is responsible for chromosomal replication

1. DNA polymerase 1 is the most abundant. It participate in replication is aspecialized manner but it is not the principal enzyme that carries out DNAelongation

2. DNA polymerase II - function is not known3. DNA polymerase III is the principal enzyme responsible for DNA

elongation that is carried out in the 5' ----3' direction.

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Polymerase beta is a lower molecular weight enzyme also present in mammaliannuclei but is not responsible for the usual DNA replication but may function in DNArepairPolymerase gamma is a mitochondrial DNA polymerase responsible for replicationof the mitochondrial genome, another DNA that exists in circular form. Replication

occurs in both directions along the chromosome and both strands are replicatedsimultaneously generating " replication bubbles"

4.3.3 Ligase is responsible for removal of RNA primer & sealing the fragment byfilling the gaps left

4.3.4 Helicase - responsible for unwinding of duplex DNA segments

4.3.5 DNA topoisomerase is the nicking resealing enzyme

4.4 Sequential steps in replication

4.4.1 Replication begins by recognition of origin and unwinding of short segmentsabout10-200 nucleotide long just ahead of replication fork. This is catalyzed by helicase.Unwinding allows DNA replicating enzyme to "read" base sequence of the template

4.4.2 DNA binding protein (DBP) bind tightly to separated strands to prevent baspairing without interfering with the ability of the nucleotides to serve as templates

4.4.3 RNA polymerase (primase) creates temporary complementary RNA primer

4.4.4 Once RNA primer has been synthesized, DNA polymerase located at thereplication fork starts replication (initiation of new daughter strands). Synthesisstarts at the site of unwinding termed replication fork.

4.4.5 Elongation of the daughter strand. On the leading strand, 3'en is open,allowing DNA polymerase to proceed continuously in a 3' - 5' direction starting atthe end of the RNA primer and the primer is then hydrolyzed. On the lagging strand,only 5' end is open. Since DNA polymerase cannot operate in a 5' - 3' direction,lagging strand must be synthesized discontinuously 3' - 5' in pieces known asOkazaki fragments. DNA polymerase removes the primer and fills in the gapsbetween fragments with deoxyribonucleotide. DNA ligase joins the ends of thefragments to create a single DNA molecule.

4.4.6 Rewinding of the DNA molecule

4.4.7 Termination of replication

4.5 Okazaki Fragments are small pieces of newly formed DNA during replication.It is consist of about 200 nucleotides long representing short lengths of DNA replicatedin discontinuous manner and then spliced together by DNA ligase. The direction isopposite the movement of the replication fork and it is located in the lagging strand.

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Synthesis requires short RNA (complementary to DNA template strand) by the sameenzyme, primase that acts on leading strand.

4.6 Proofreading device - 3' exonuclease activity of DNA polymerase I & III

The 3' exonuclease activity of DNA polymerase I & III correct errors made bypolymerase activity. The enzyme can recognize its failure to form correct base pair.It therefore "backs up" and hydrolyzes wrong nucleotide from 3'end thenpolymerase adds the correct nucleotide & resumes replication. Proofreadingguarantees fidelity of replication. Errors in replication places identity of species or itsviability at high risk.

4.7 DNA repair

Enzymes repair damaged RNA. Damage to DNA by environmental, physicaland chemical agents may be classified into 4 types. These are single base

alteration, two-base alteration, chain break, and cross-linking. The damage regionsof DNA may be repaired, replaced by recombination, or retained. Retention leads tomutations and potential cell death. Repair and replacement exploit the redundancyof information inherent in the double helical DNA structure. The defective region inone strand can be returned to its original form by relying on the complementaryinformation stored in the unaffected strand.

The key to all the repair or recombination processes is the initial recognitionof the defect and either repairing it during the recognition step or marking it forfuture attention.

4.8 Describe mutation

Mutation results when changes occur in the nucleotide sequence of DNA.Although the initial change may not occur in the coding strand of the double-stranded DNA molecule for that gene, after replication, daughter DNA moleculeswith mutations in the coding strand will segregate and appear in the populations oforganisms.

4.8.1 Mutation by Base SubstitutionSingle base changes or point mutations may be transitions or transversions.

In the former, a given pyrimidine is changed to the other pyrimidine or a givenpurine is changed to the other purine. Transversions are changes from purine toeither of the 2 pyrimidines or the change of a pyrimidine into either of the 2 purines

T C T A A T

A G C G G C

Transitions Transversions

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If the nucleotide sequence of the gene containing the mutation is transcribedinto an RNA molecule, then the RNA molecule will posses a complementary basechange at this corresponding locus.

Single base changes in the mRNA molecules may have one of severaleffects when translated into protein:

1. There may be no detectable effect because of the degeneracy of the code. Thiswould be more likely if the changed base in the mRNA molecule were to be atthe third nucleotide of a codon. Because of wobble, the translation of the codonis least sensitive to a change at the third position.

2. A missense effect will occur when a different amino acid is incorporated at thecorresponding site in the protein molecule. This mistaken amino acid, ormissense, depending its location in the specific protein, might be acceptable,partially acceptable or unacceptable to the function of that protein molecule.Most single base mutation changes would result in the replacement of oneamino acid by another with rather similar functional groups. This is an effectivemechanism to avoid drastic change in the physical properties of protein

molecule. If an acceptable missense effect occurs, the resulting protein may notbe distinguishable from the normal one. A partially acceptable missense willresult in a protein molecule with partial but abnormal function. If an unacceptablemissense effect occurs, then the protein molecule will not be capable offunctioning in its assigned role.

3. A nonsense codon appear that would then result in the premature termination ofamino acid incorporation into a peptide chain and the production of only afragment of the intended protein molecule. The probability is high that aprematurely terminated protein molecule or peptide fragment would not functionin its assigned role.

Hemoglobin illustrates the effects of single base changes in structural genes.Some mutation have no apparent effect. The lack of effect of a single base changewould only be demonstrable by sequencing the nucleotides in the mRNA moleculesor structural genes for hemoglobin from a large number of humans with normal

hemoglobin molecules. For example, the codon for valine at position 67 of the

chain of hemoglobin is not identical in all persons possessing the normal chain of

hemoglobin.

Hemoglobin Milwaukee has at position 67 glutamic acidHemoglobin Bristol has aspartic acid at position 67

Acceptable missense mutation in the structural gene for the chain ofhemoglobin could be detected by the presence of electrophoretically alteredhemoglobin in the red cells of an apparently healthy individual. For example inHemoglobin Akari, hemoglobin has asparagine substituted for lysine at the 61

position in the chain.

Partially acceptable missense mutation is best exemplified by hemoglobin S,

sickle hemoglobin, in which the normal amino acid in position 6 of the chain

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4.8.2 Frame Shift Mutation

The deletion of a single nucleotide from the coding strand of a gene results inan altered reading frame in the mRNA. The machinery translating the mRNA does

not recognize that a base was missing, since there is no punctuation in the readingof codons.

Examples of the 3 types of missense mutation in abnormal hemoglobin chain

PRO MOLECULE AMINO ACID CODONS

Acceptablemissense

Hb A, chain

Hb Hikari, chain

61 Lysine

Asparagine

AAA or AAG

AAU or AAC

Partiallyacceptablemissence

Hb A, chain

Hb S, chain

6 Glutamate

Valine

GAA or GAG

GUA or GUG

Unacceptable

missense

Hb A, chain

Hb M (Boston),

chain

58 Histidine

Tyrosine

CAU or CAC

UAU or UAC

5. Discuss the processes involved in transcription.

Differences between replication and transcription.

Transcription is the process where an RNA strand having a base sequencecomplementary to one of the DNA strand is synthesized by an enzyme system. It must becarried out faithfully if the cell is to have proteins with their normal genetically determinedamino acid sequence.

Unlike replication where the entire chromosome is copied & both strand acts astemplate to produce 2 daughter DNA, in transcription only a portion of the cell DNA istranscribed. The strand that is transcribed into an RNA molecule is referred to as thetemplate strand. The other is called the coding strand of that gene because with theexception of T for U changes it corresponds exactly to the sequence of the primarytranscript, which encodes the protein product of the gene.

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Promoter site

There are 2 common sequences in promoters used by RNA polymerase II1. TATA (Hogness) box is important in initiation of transcription found in all eukaryote. It is

located 25 base pairs upstream from start point.2. CAAT box is usually found in position -75 to -80. Binding of transcription factors at thissite may influence formation of initiation complexes at other sites.

Describe the enzymes DNA-directed RNA polymerase

It elongates an RNA strand by adding ribonucleotide units to the 3' Oh end of theRNA chain thus builds RNA chains in the 5' to 3' direction. The enzyme requires preformedDNA for activity & the most active enzyme with a natural double-stranded DNA astemplate. ATP, GTP, UTP, CTP is required as precursors & requires Mg++ as cofactor. Itcontains zinc as essential part. There are 3 RNA polymerase identified. These are:

1. RNA polymerase I located in nucleolus 7 form the products: rRNA, 5,8 S,18S,28S2. RNA polymerase II located in chromatin & nucleoplasm that produce mRNA3. RNA polymerase III located in chromatin & nucleoplasm produce tRNA, rRNA &

5S

Steps in Transcription

DNA-dependent RNA polymerase is the main transcription enzyme. The enzymeattaches at a specific site, the promoter site, on the template strand. Once the holoenzyme(RNA polymerase + proteins) is correctly positioned at promoter site and has made a few

phosphodiester bonds, the subunit dissociates from the holoenzyme. Remaining coreenzyme (RNA polymerase) elongates RNA. The process continues until a termination

sequence is reached. The signal is recognized by the rho () factor. Once the signal is

reached, this brings about termination and release of the RNA polymerase from thetemplate DNA. A transcription unit is defined as that region of DNA that extends from thepromoter and the terminator. The RNA product, which is synthesized in the 5' to 3'direction, is the primary transcript. The primary transcripts generated by RNA polymeraseare promptly cap by 7 methylguanosine triphosphate caps that persist and eventuallyappear on the 5' of the mature cytoplasmic mRNA. These caps are necessary for the ff.:

1. processing of primary transcript to mRNA2. for translation of the mRNA

3. for protection of the mRNA against 5' to 3' exonuclease lytic attack.

Processing of RNA molecules

Nearly all eukaryotic RNA primary transcripts undergo extensive processing. Theprocessing occurs primarily within the nucleus. It includes:1. capping2. nucleolytic & ligation reactions3. terminal additions of nucleotides4. nucleoside modifications - splicing

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Eukaryotic genes are mosaics of introns and exons. Exons are sequence of a gene thatis expressed as mRNA. Introns are the long sequence of DNA that does not contribute tothe genetic information. Introns are the untranslated intervening sequences usually splicedout in the information of mature RNA. The introns nearly always begin with GU and endwith an AG that is preceded by a Pyr-rich tract. Splicing is a complex operation carried out

by spliceosomes (assemblies of proteins and small RNA molecules). This enzymemachinery recognizes signals in the nascent RNA that specify the specific sites.

6. Identify descriptions of translation:

Translation is the process in which the genetic message coded by RNA is translatedby the ribosomes into the 20-letter alphabet of protein. The letters A, G, T, C correspond tothe nucleotides found in DNA. They are organized into 3-letter code words called codons,and the collection of these codons makes up the genetic code. A linear array of codons (agene) specifies the synthesis of various RNA molecules, most of which are involved insome aspect of protein synthesis. Protein synthesis occurs in 3 major steps: initiation,

elongation, & termination. This process resembles DNA replication & transcription in itsgeneral features and in the fact that it too, follows a 5' to 3' polarity.

In eukaryotic cells the primary transcript we describe after transcription is called theheterogeneous nuclear RNA (hnRNA) is a much larger molecule than the mature mRNA.The hnRNA is processed within the nucleus, and the introns, which often make up muchmore of the hnRNA than the exons, are removed. Exons are spliced to form maturemRNA, which is transported to the cytoplasm, where it is translated into protein.

mRNA in itself has no affinity for amino acids and, therefore an intermediate adaptermolecule is needed. This adapter molecule, tRNA must recognize a specific nucleotide

sequence on the one hand as well as a specific amino acid on the other. With this tRNA,the cell can direct a specific amino acid into the proper sequential position of a protein asdictated by the nucleotide sequence of the specific mRNA. In fact, the functional groups ofthe amino acids do not themselves actually come into contact with the mRNA templateFeatures of the genetic code

The genetic code is degenerate, unambiguous, non-overlapping, withoutpunctuation, and universal.Degeneracy - multiple codons must decode the same amino acid for example 6 differentcodons specify serine.Unambiguous - given a specific codon only a single amino acid is indicatedReading of the genetic code during the process of protein synthesis does not involveoverlap of codonsNo punctuation - once the reading is commenced at a specific codon, the message is readis a continuing sequence of nucleotide triplets until a nonsense codon is reached.Non-sense codon- refers to the 3 codons that do not code for specific amino acids (UAA,UAG, & UGA). Two of these are utilized in the cell as termination signals.

Protein synthesis (translation)

Stage 1: Activation of amino acid - This takes pale in the cytosol. At least 20 specificenzyme are required for the specific recognition & activation function of the 20 types of

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tRNA. The enzymes are termed aminoacyl-tRNA synthetases that require ATP & Mg++ &the product is aminoacyl-AMP-enzyme complex

Stage 2: Initiation of the polypeptide chain-The initiating amino acid in aminoacyl-AMP-enzyme complex become attached to specific tRNA. The mRNA (bearing code for

polypeptide to be made) is bound to smaller ribosome. Anticodon of tRNA of initiatingamino acid base pairs with codon on mRNA. This process is promoted by initiation factorand requires ATP.

Stage 3: Elongation- This step is promoted by elongation factors & requires 2 molecules ofGTP. The polypeptide is lengthened by covalent attachment of successive amino acidunits catalyzed by peptidyltransferase.

Stage 4: Termination & release- Termination codon signals completion of polypeptidechain. This is followed by release of the protein from the ribosome. The process ispromoted by releasing factors. The releasing factor in conjunction with GTP and the

enzyme peptidyl tansferase promotes the hydrolysis of the bond between the peptide andthe tRNA occupying the P site.

Stage 5: Folding and processing-Once the polypeptide is release it is folded into its 3-dimensional conformation to achieve biologic activity. Further modification by enzymeaction follows. These include removal of initiating amino acid and introduce certain groupsinto amino acid residues such as PO4, CH3, etc.

Post-translational Modification include:1. All polypeptide are begun with Methionine. This residue may be removed by specific

enzyme.

2. Loss of signaling protein by peptidases3. Phosphorylation of hydroxy amino acidOH groups of Ser, Thr, & Tyr are phosphorylated which add negative charges topolypeptide

4. Carboxylation reactions-Extra COOH groups are added to Asp, & Gla5. Methylation of R groups of some Lys residue of some proteins6. Addition of CHO side chains-In many glycoproteins, the CHO side chains are attached

to Asn or Thr7. Addition of prosthetic groups- after it leaves the ribosomes.

Ex. biotin - in acetyl CoA carboxylaseheme - in cytochrome C

8. Formation of S-S cross links to help protect the native conformation of protein fromdenaturation.

7. Discuss the steps to be taken to determine whether or not Mr Ree is thefather of the child

Do blood typing. If the result does not exclude Mr Ree, do DNA testing

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Genetics - Module 21