Content Module 6 the Basis of Molecular Biology

Part II

Module III. Molecular biology. Biochemistry of intercellular communications. Biochemistry of tissues and physiological functions.

Content module 6 “The basis of molecular biology”

Topic 3.1. Investigation of purine nucleotides synthesis and degradation. Determination 2-4

of final products of their metabolism.

Topic 3.2. Investigation of pyrimidine nucleotides metabolism.Determination of 5-8

nucleic acid chemical composition.

Topic 3.3. Investigation of DNA replication and RNA transcription. Analysis of DNA 9-11

mutation and repair mechanisms.

Topic 3.4. Protein synthesis. Investigation of initiation, elongation and termination. 12-14

Inhibition of protein synthesis by antibiotics.

Content module 7 “Molecular mechanisms of hormone action on the target cells

and biochemistry of hormonal regulation”

Topic 3.5. Investigation of hypothalamus and hypophysis (pituitary gland)hormones. 15-16

Topic 3.6. Investigation of pancreas andgastrointestinal tract hormones. 17-19

Metabolic changes in diabetes mellitus.

Topic 3.7. Endocrine control of glucose concentration in the blood. Glucose tolerance test. 20-24

Sugar curves.Hormones of the adrenal gland.

Topic 3.8. Hormonal regulation of calcium metabolism. Definition of iodine in the thyroid 25-27

gland. Physiologically active eicosanoids

Topic 3.9. Steroid hormones of sex glands. Endocrine control of metabolism in the 28-30

well-fed state. Regulation of metabolism in starvation.

Topic 3.10. Interrelation and regulation of all metabolism pathways. 31-33

Content module 8 “Biochemistry and pathobiochemistry of the Blood”

Topic 3.11.Investigation of chemical composition and acid-base balance of blood. 34-39

The determination of blood rest nitrogen..

Topic 3.12.Investigation of coagulation, anti- coagulation and fibrinolytic system of blood 40-42

Topic 3.13.Investigation of erythrocytes metabolism. Normal and pathological hemoglobin 43-45

varieties. Investigation of heme degradation.

Content module 9 “Biochemistry of tissues and organs”

Topic 3.14. Biochemistry of the liver. Microsomal oxidation, cytochrome P -450 46-50

Topic 3.15. Studies of biological oxidation of different types.The role of fat-soluble

vitamins in functioning of tissues and organs 51-57

Topic 3.16. Investigation of normal and pathological components of urine. 58-61

Topic 3.17. Biochemistry of nerve and connective tissue. 62-66

Topic 2.18. Summarized control of the Module 3

Questions to Summarized control of Module 3. «Molecular biology. Biochemistry 67-68

of intercellular communications. Biochemistry of tissues and physiological functions.

Part II

Module III. Molecular biology. Biochemistry of intercellular communications. Biochemistry of tissues and physiological functions.

Content module 6 “The basis of molecular biology”

Topic 3.1. THE METHODICAL GUIDELINES FOR PRACTICE ACTIVITY ON THE THEME:

Investigation of purine nucleotides synthesis and degradation.

Determination of final products of their degradation.

Biomedical importance:

Even when humans consume a diet rich in nucleoproteins, dietary purine and pyrimidine bases are not incorporated into tissue nucleic acids. Humans biosynthesize the purines and pyrimidines of tissue nucleic acids, ATP, NAD+, coenzyme A, etc, from amphibolic intermediates. However, injected purine or pyrimidine analogs, including potential anticancer drugs, may be incorporated into DNA. The biosyn-theseses of purine and pyrimidine oxy- and deoxyribonucleotides (NTPs and dNTPs) are precisely regulated events coordinated by feedback mechanisms that ensure production in appropriate quantities and at times appropriate to varying physiologic demand (eg, cell division). Human diseases that involve abnormalities in purine metabolism include gout, Lesch-Nyhan syndrome, adenosine deaminase deficiency, and purine nucleoside phosphorylase deficiency. Diseases of pyrimidine biosynthesis, while more rare, include orotic acidurias. Since, unlike the urates, the products of pyrimidine catabolism are highly soluble (carbon dioxide, ammonia, and β-aminoisobutyrate), there are fewer clinically significant disorders of pyrimidine catabolism.

The purpose:To develop skills in interpreting nucleoprotein structure on the basic of qualitative reactions for their constitutive components for the further estimating of these biopolymers role in storage and expression of genetic information.

The applicable materials:

1. The tutorial book"Principles of biochemistry", 2005.p.271-279

2. "Biochemistry", Pamela C. Champe at al.2005.p.289-294, 296-299 (V).

3. Lecture on the theme «The nucleoproteins metabolism»,

The main theoretical questions:

1. General representation about nucleoproteins.

2. Nucleotide structure:

2.1. Purine and pyrimidine nitrogen bases;

2.2. Nucleosides;

2.3. Nucleotides.

2.4. Primary structure of nucleic acids

Digestion and absorption of dietary nucleoproteins in GIT.
Degradation of purine nucleotides. Reactions.
De novo purine nucleotides synthesis:
The sources of nitrogen and carbon atoms of purine ring./Scheme/

5.2. Synthesis of 5-phosphoribisylamine /Reactions/.

5.3. Conversion of IMP to AMP and GMP /Scheme/.

5.4.Conversion of nucleoside monophospates to nucleoside di- and triphosphates.

5.5.Regulation of purine synthesis.

Salvage pathway for purines.
The purine metabolism disorders: gout, Lesch-Nyhan syndrome and adenosine deaminase deficiency.
Сlinical signification of uric acid determination in blood and urin.

Practice instructions

THE QUANTITATIVE URIC ACID DETERMINATION IN URINE

The essence of the method:

The method is based on the ability of uric acid to reduce phosphotungsten reagent in resulting dark blue color product, which color intensity proportionally depends on uric acid concentration. The quantity of this product is determined by titration with K3[Fe(CN)6] to disappearance of blue color.

Sequence of procedure:

Do experiments simultaneously in two glasses.

With urine

With standard uric acid solution.

Pour 1,5 ml of examined solution into the glasses. (Urine into the 1-st, standard solution of uric acid into the 2-nd).
Add 1ml of 20% Na2CO3 and 1 ml phosphotungsten Folin reagent both into the tubes and mix well.
Titrate the solutions with K3[Fe(CN)6] to disappearance of blue color.
Calculate the uric acid concentration by the formula:

A ×B

X = 0.75 ------mg/day, = ------= mg/day

A0 ×1.5

Where:

A, ml – the amount of K3[Fe(CN)6], which was spent on urea titration

A0, ml – the amount of K3[Fe(CN)6], which was spent on standard uric acid solution titration.

0.75– the amount of uric acid in 1.5 ml of standard solution.

B – The daily volume of urea (1500 ml)

1.5 – the volume of solution for experiment.

To obtain results in SE-system (mmol/day) multiply the data on 0.0059.

Normal contents: 1.6 – 3.5 mmol/day

Or 276-600 mg/day

X SE = X (mg/day) × 0/0059 = mmol/day

Conclusions:

Task.

1. Write the structure and show the sources of N atoms of purine ring.

2. Write the 2-nd reaction of the purine de novobiosynthesis. Name activators and inhibitors of enzyme.

M.C.Q.

1. The patient, 55 years old, is admitted to a hospital with a joint pain syndrome. During examination the contents of uric acid in the blood was 2.1 mmol/l (increased), in the urine 0,066 g/l((little increased).The cause of such state can be:

A. podagra (gout)

B.phenylketonuria

C.branched chain aminoaciduria (maple syrup disease)

D.alkaptonuria

E.Homocistinuria

2. The doctor administered allopurinol to a patient with gout. What biochemical mechanism of allopurinol action promotes therapeutic effect in this case?

A. Increased rate of excretion of nitrogen-containing compounds

B. Competitive inhibition of xanthinoxidase

C. Inhibition of reutilization of pyrimidine nucleotides

D. Accelerated biosynthesis of nucleic acids

E. Increased catabolism of pyrimidine nucleotides

3. A patient has increased contents of uric acid in his blood, what is clinically manifested by pain syndrome due to accumulation of urates in his joints.As a result of which process does this acid form in gout?

A. Purine bases re-using

B. Proteolysis

C. Purine nucleotide degradation

D. Heme catabolism

E. Pyrimidine nucleotide degradation

4. The four nitrogen atoms of purines are derived from:

A. Urea and Ammonia,

B. Ammonia, Glycine and Glutamate

C. Ammonia, Aspartate and Glutamate

D. Aspartate, Glulamine and Glycine,

E. Glycine, Ammonia and Aspartate.

Glycine contributes to the following C and N of purine nucleus:

A. C-l, C-2 and N-7

B. C-8, C-6 and N-9

C. C-4, C-5 and N- 7

D. C-3, C-4 and N-1

E. C-4, C-5 and N-9

Inosinic acid is the biological precursor of:
Cytosinic and Uric Acid
Adenylic acid and Guanylic acid
Orotic acid and Uridylic acid
Adenosine and Thymidine
Uracil and Thymidine.

The probable metabolic defect in gout is:

A. A defect in excretion of uric acid by kidney

B. an overproduction of pyrimidines

C. an overproduction of uric acid

D. an underproduction of purines

E. rise in calcium leading to deposition of calcium urate

8. Synthesis of GMP from IMP requires the following:

A. Ammonia, NAD+, ATP

B. Glutamine, NAD+, ATP

C. Ammonia, GTP, NADP+

D. Glutamine, GTP, NADP+

E. Glutamine, UTP, NADP

Topic 2.2. THE METHODICAL GUIDELINES FOR PRACTICE ACTIVITY ON THE THEME:

Investigation of pyrimidine nucleotides metabolism.

Determination of nucleic acid chemical composition.

Biomedical importance:

This theme introduces the aromatic heterocyclic purine and pyrimidine and their major derivatives, the nucleosides and nucleotides, which supply the monomer units or building blocks of nucleic acids and serve additional diverse functions essential for life and health.

Major biochemical functions of purine and pyrimidine nucleotides include the numerous phosphate transfer reactions of ATP and other nucleoside that drive otherwise endergonic reactions. UDP-glucose and UDP-galactose function inbiosynthesis of carbohydrates and CDP-acylglycerol in phospholipid biosynthesis, as "high-energy intermediates". Nucleotides form a portion of coenzymes such as FAD, NAD+, NADP+, coenzyme A. and 5-adenosylmethionine. Nucleotides also serve regulatory functions. ADP levels regulate themitochondriaoxidative phosphorylation. Specific nucleotides act as allosteric regulators of enzyme activity, cAMP and cGMP serve "second messenger” functions. Finally, nucleoside triphosphates serve as the monomer unit precursors of the nucleic acids RNA and DNA.

The purpose: To develop skills in interpreting nucleoprotein structure on the basic of qualitative reactions for their compound components for the further estimating of these biopolymers role in storage and expression of genetic information.

The literature:

The tutorial book"Principles of biochemistry", 2005. p.279-287, p.305-307. 285-287

2. The «The nucleoproteins», Lecture Materials; 299-304,295-296 (IV), 372-373.393-394,413-414

3. The «The nucleoproteins», Lecture Materials;

The main theoretical questions:

1. The biological role of nucleotides and nucleoproteins.

2. Nucleotide structure:

2.1. Purine and pyrimidine nitrogenous bases;

2.2. Nucleosides;

2.3. Nucleotides.

3.Pyrimidine synthesis:

3.1. Syntesis of UMP. (Reactions of orotic acid formation).Regulation.

3.2. Synthesis of UTP and CTP /schem/.

Conversion of ribonucletides to deoxyribinucleotides.
Synthesis of thymidine monophosphate from dUTP.
Degradation of pyrimidine nucleotides. Name final products only.

7. The pyrimidine mеtabolism disorders: orotic aciduria.

8. Structure of nucleic acids: DNA, mRNA, tRNA, rRNA (primary, secondary, tertiary).

9. Structural organization of eukaryotic DNA: histones and formation of nucleosomes.

10. Higher levels of organization, DNA folding in a chromatin and chromosomes.

Practice instructions:

“Qualitative reactions for nucleoproteincomponents”.

The essence of the method: The «The nucleoproteins», Lecture Materials;

The method is based on the qualitative determination of nucleoprotein separate compounds: pentoses, phosphoric acid and protein, which are formed as a result of acid hydrolysis of yeast, reach with nucleoprotein.

Students receive yeast hydrolyzate ready for the work.

Work № 1. BIURETIC TEST FOR PROTEIN:

The essence of the method: Peptide bonds of protein forms in alkaline medium with copper (Cu2+) ions complex of violet color.

Sequence of procedure:

Pour 10 drops of hydrolyzate into the tube.

Add 10 drops 10 % of alkali liquor (NAOH) and 1 drop 1 % of copper sulphate (2+) solution.

In 15 minutes violet color appears if the tube contains protein.

Work № 2. Molish REACTION for PENTOSES:

The essence of the method:

The pentoses are dehydrated by concentrated sulfuric acid to yield furfurol, which gives red color product with thymol.

Sequence of procedure:

Pour 10 drops of hydrolyzate into the tube.

Add 5 drops of methyl-isopropyl phenol alcoholic solution and mix.

On a wall of a test tube cautiously add 5 drops of concentrated sulfuric acid.

If the tube contains pentose, the product of red color is formed at the bottom upon shaking.

Work № 3. REACTION FOR DEOXYRIBOSE AND RIBOSE:

The essence of the method:

The diphenylamine gives dark blue coloring with deoxyribose, and green with ribose.

Sequence of procedure:

Pour 5 drops of hydrolyzate into the tube.

Add 20 drops 1 % of diphenylamine solution and boil the tube on a water bath for 15 minutes.

The blue ore green color is appeared. Look at the coloring and answer, which of the pentoses is present in the hydrolyzate.

Work № 4. MOLYBDENIC TEST FOR THE PHOSPHORIC ACID:

Sequence of procedure:

Pour 10 drops of hydrolyzate into the tube.

Add 20 drops of molybdenic reagent and boil.

The fluid is colored in citric-yellow color.

Cool the test tube with cold water. Crystalline citric-yellow sediment appears at the bottom of the test tube.

H3PO4 + 12 (NH4)2 MoO4+ 21HNO3 → (NH4)2PO4•12MoO3+ 21 NH4NO3+ 12H2O

Phosphomolybdenum ammonia (crystalline yellow sediment)

Results:

№ of the tube / Coloring / Nucleoprotein component
1
2
3
4

Conclusions:

Task.

1. Draw the formulas of dTMP and dGMP, join them by 3,5 phosphodiester bonds. Which type of nucleic acid does this fragment belong?

2. Write the scheme: orotic acid-- -> CTP

3. Draw formulas of AMP and CMP, join them by 3,5 phosphodiester bonds. Which type of nucleic acid does this fragment belong?

4. Write the scheme: GMP--- ->dGTP

M.C.Q.

1. In a DNA molecule guanosine nucleotide is held by the cytosine nucleotide by the
number of hydrogen bonds:

2. Which one of the following is characteristic of orotic aciduria?

Immunodeficiency
Genetic deficiency of the enzyme orotidine phosphate decarboxylase
Self-mutilation
Increased levels of uric acid in blood
Impairment of T-cell function

3.In humans, the principal catabolic product of pyrimidines is:

uric acid
allantoin
hypoxanthine
-Alanine
Urea

4.Two nitrogen atoms of pyrimidine ring are obtained from:

Glutamine and carbamoyl-P.
Aspartate and carbamoyl- P.
Glutamate and ammonia
Glutamine and ammonia
Aspartate and glycine

5. The complementary base sequence in the second strand of DNA for the base sequence CCGATT would be:

A. GGCTAA

B. GGCUAA

C. AATCGG

D. CCGATT

E. GTACCG

6. Synthesis of what substance is blocked by 5- fluorodesoxiuridine, an inhibitor of thymidilatsynthase?

A. DNA

B. tRNA

C. Protein

D. ATP

E. mRNA

7. Why do two DNA strands form a double helix?

A. due to base-pairing phenomenon

B. due to the "anti-parallel" orientation;

C. due to various combinations between its nucleotides;

D. due to ability to make copies of itself;

E. due to phosphate sugar backbone of DNA;

8. In humans, the principal catabolic product of Thymidine is:

A. Allantoin

B. beta-Alanine

C. Urea

D. Uric acid

E. beta-aminoisobutirate

9. A key substance in the committed step of pyrimidine biosynthesis is:

A. ATP

B. carbamoyl -phosphate

C. Ribose-5'-phosphate

D. Thiouracil

E. Glutamine

10. Which one of the following is allosteric inhibitor of the de novo Pyrimidine synthesis?

A. PRPP

B. Glutamine

C. Ribose

D. UTP

E. AMP

11.Deficiency of what enzyme is the cause of orotic aciduria?

Xantineoxidase;
Carbamoil-P-synthetase;
Orotate phosphoribosiltransferase;
GH PRT;
Amidotransferase

Topic 3.3. THE METHODICAL GUIDELINES FOR PRACTICE ACTIVITY ON THE THEME: Investigation of DNA Replication and RNA Transcription. Analysis of mutations, DNA Repair.

Biomedical importance:

Nucleic acids are required for the storage and expression of genetic information. There are two chemically distinct types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is present not only in chromosomes in the nucleus of eukaryotic organisms, but also in mitochondria and in the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have a single chromosome but may also contain nonchromosomal DNA in the form of plastids. The DNA contained in a fertilized egg encodes the information that directs the development of an organism. This development may involve production of billions of cells. Each of these cells is specialized, expressing only those functions that are required for it to perform its role in maintaining the organism. Therefore, the DNA must be able not only to replicate precisely each time a cell divides, but also to have the information that it contains be selectively expressed. RNA participates in the expression of the genetic information stored in the DNA . The genetic master plan of an organism is contained in the sequence of deoxyribonucleotides that constitute the DNA. However, it is through the ribonucleic acid (RNA) "working copies" of the DNA that the master plan is expressed. The copying process, which uses one of the two DNA strands as a template, is called transcription. The messenger RNAs, which are transcripts of certain regions of the DNA, are translated into sequences of amino acids— the polypeptide chains. Ribosomal RNAs, transfer RNAs, and additional small RNA molecules perform specialized structural and regulatory functions without translation.

The purpose:

To develop skills in interpreting of nucleic acid structure and functions for molecular basis of inherited diseases explanation and treatment.

The applicable materials:

1. The tutorial book, 2005. p.289-304

2. "Biochemistry", Pamela C. Champe at al.2005.p.393-428

3. Lecture on the theme «Bases of molecular genetics. Protein biosynthesis and its regulation”

The main theoretical questions:

1. Genetic information: storage, expression and types of transmission.

2. Structure of DNA and RNA

2.1. Primary structure - 3’, 5’-phosphodiester bonds;

2.2 DNA double helix; Base pairing: hydrogen bonds, the complementary rules.

2.3. Organization of eukaryotic DNA, nucleosomes.

3. Structure of mRNA, tRNA and rRNA.

4. Steps in DNA synthesis:

4.1. Semi conservative replication mechanism;

4.2. Components required for replication: substrates for DNA synthesis, enzymes (DNA polymerase III and I, helices, topoisomerases, DNA ligase).

4.3. DNA synthesis initiation: separation of the two complementary DNA strands and replication fork formation; RNA primer synthesis.

4.4. Chain elongation: direction of DNA replication; leading and lagging strands; excision of RNA primer and its replacement by DNA; the joining of Okazaki fragments.

5. DNA damage and repair.

6. Point mutations: missense and nonsense;

7. Transcription of genes:

7.1. Structure of operone.

7.2. Components required for transcription. RNA polymerases.

7.3. Steps in prokaryotic RNA synthesis: initiation, elongation, termination.

7.4. Post-transcriptional modification of mRNA.

M.C.Q.

1.Which of the following is NOT correct about nucleic acids?
A. they contain both phosphorus and nitrogen
B. nucleic acids are useful for buoyancy
C. RNA is a type of nucleic acid
D. nucleotides are subunits of nucleic acids
E. DNA is a type of nucleic acid

2. One important function of nucleic acids is that they:
A. form enzymes
B. are structural molecules
C. repel water
D. store energy
E. hold genetic information

3. RNA polymerase is involved in which of the following processes?

A.Photosynthesis

B.Phagocytosis

C.Transcription

D.Sparging

E.Translational

4.Which process takes place under DNA repair?