Biochemistry- Paper II Aug 2011

I. Essay questions ( 2 x 15 = 30 )

1.What is cloning? Mention the various types of cloning. Describe in detail the steps involved in

recombinant DNA technology

Ans.

A clone is a large population of identical molecules, bacteria, or cells that arise from a common ancestor. Molecular cloning allows for the production of a large number of identical DNA molecules, which can then be characterized or used for other purposes. This technique is based on the fact that chimeric or hybrid DNA molecules can be constructed in cloning vectors—typically bacterial plasmids, phages, or cosmids—which then continue to replicate in a host cell under their own control systems. In this way, the chimeric DNA is amplified.

When a cell from an animal is grown to an exact duplicate of that animal, it is known as

somatic animal.

Recombinant DNA technology:

When a gene of one species is transferred to another living organism, it is called

recombinant DNA technology.

1. preparation of specific human gene.

The mRNA of the desired gene is extracted and using reverse transcriptase

complimentary copy of that RNA (cDNA) is produced.

2. preparation of chimeric DNA molecules

Vectors are hosts like bacteria in which the desired gene is inserted into their genome.

Restriction endonuclease is the enzymes which cuts DNA at specific sites. Using a restriction

enzyme like EcoRI both the human DNA and plasmid DNA of the vector is cut. They are cut in

such a way that the ends of both the DNA are sticky and anneal with each other and also with

the help of DNA ligase. The resulting plasmid DNA with the desired gene is called chimeric DNA

3. Cloning of Chimeric DNA:

The plasmid is introduced into the host cell ,a a process called transfection. Host E.coli

cells are incubated with plasmid vectors in a hypertonic medium containing calcium. The

calcium ion channels are opened through which the plasmid is imbibed into the host cell. The

cells are allowed to divide so that a large population of cells with the chimeric DNA is obtained.

Fig. recombinant DNA technology

4. checking the viability of the process:

Plasmid pBR-325 contains Chloramphenicol resistance gene(Cmr), ampicillin resistance and

tetracycline resistance genes. When this plasmid is cut by the EcoRI enzyme it will cut the Cmr

gene. When the foreign DNA is inserted, the will not have Chloramphenicol resistance. This

process functions as a marker for hybrid DNA.

5. selection of colony with the desired gene

After transfection, the bacteria are cultured in a medium containing ampicillin and tetracycline.

Wild bacteria are killed in that medium and the bacteria containing the cloned plasmid will

grow. To check whether the colony containing the desired plasmid, a part of the colony is

subjected to Chloramphenicol media. If the bacteria dies, then it the colony that contains the

hybrid DNA plasmid.

6. Expression vectors:

Vector carrying a foreign gene which can produce the desired protein is called expression

vector. Thus the human proteins like insulin can be harvested from such cultures.

2.Describe the role of plasma and renal buffers in maintaining acid base homeostasis

Ans.

Normal blood pH is btween7.38-7.42. it is maintained by:

I. Role of buffers in body fluids:

Buffers resist changes in pH when small quantities of an acid or an alkali are added.

Various buffers in body are:

1. Bicarbonate buffer system

it is the most important buffer in plasma and is formed by (NaHCO3/H2CO3)

the base HCO3- is the metabolic component as it is regulated by kidney and the acid

H2CO3 is called respiratory component since it is regulated by the lungs.

The normal bicarbonate level in plasma is 24mmol/L. It has a pKa of 6.1-so it is a poor

buffer. But the high blood concentration and the ratio of base to salt is high(20:1), which

makes it a effective buffer.

When acid(H+) is added

o H+ + HCO3- H2CO3 H20 + CO2 excreted by lungs and kidney.

When alkali is (HCO3-) added

o H+ + HCO3- H2CO3 H20 + CO2 excreted by lungs and kidney.

2. phosphate buffer system

It is made of NaHPO4/NaH2PO4. It has a pKa of 6.8.

In acidosis

NaHPO4 + H+ NaH2PO4 - excreted by the kidneys

In alkalosis

NaH2PO4 - NaHPO4 + H+

3. Protein buffer system

The Histidine molecules in albumin acts as a buffer.

In acidosis H+ + Pr- - HPr

In alkalosis HPr H+ + Pr-

II. Kidneys regulate acid base balance by:

1. Excretion of H+ -In PCT cells CO2 combines with water to form carbonic acid using carbonic

anhydrase. Then it becomes H+ and HCO3- . this H+ is then excreted into lumen in exchange for

Na+ .

2. reabsorption of HCO3- - sodium bicarbonate in the lumen becomes sodium and bicarbonate.

Sodium is taken up by PCT cell in exchange of hydrogen ions. H+ combines with HCO3- to form

carbonic acid, which forms CO2 and water and both are reclaimed into the cell and converted

back to carbonic acid and again to H+ and HCO3- . HCO3

- is taken into blood with sodium.

Fig.. Excretion of H+ fig.reabsorption of HCO3-

3. Excretion of titrable acid- The Na2 HPO4 becomes Na+ and NaHPO4 - . sodium is exchanged

with H+ ions and H+ combines with NaHPO4 – to become Na H2PO4 and gets excreted.

Fig.Excretion of titrable acid and ammonium ions

4. Excretion of NH4 + - Glutamine in DCT becomes glutamate and ammonia. This ammonia is

secreted into the lumen which combines with hydrogen ions to become ammonium ions and

gets excreted.

II. Write short notes on: (10 x 5 = 50)

1. Purine salvage pathway

Ans.

- Nucleotides are degraded regularly. Salvage pathway recycles the purines and make

it available for nucleic acid synthesis.

- Adenine is converted to AMP using PRPP by Adenine phosphoribosyl

transferase(APRTase)

- Guanine is converted to GMP using PRPP by Hypoxanthine guanine phosphoribosyl

transferase (HGPRTase)

- The salvage pathway is important for RBC and brain since denovo synthesis of purine

nucleotides are not operative.

- A defect in HGPRTase will lead to Lesch Nyhan syndrome.

Lesch Nyhan syndrome

it is a X-linked inborn error of purine metabolism, incidence 1:10,000

deficiency of HGPRTase which acts in salvage pathway

so the salvage pathway is stopped and PRPP accumulates which will go for catabolism to

uric acid

hyperuricemia leads to nephrolithiasis and gout

it is also characterized by self mutilation, mental retardation

2. Explain the types and functions of IGs

Ans.

Immunoglobulins are γ- globulins which act as antibodies. The antibody has a high

specificity for a particular antigen only. The antibody’s structure is complementary to that

specific antigen.

Classification and structure:

Different types of Igs are A, D, E, G, and M

1. IgG: IgG consist of 2 heavy chains and 2 light chains. VL and CL are variable and constant

regions in light chains and VH and CH in heavy chains. The variable regions are important for

antigen binding and recognition. It is the major antibody. They are produced by B cells and are

involved in secondary immune response and indicate chronicity of the disease. It crosses

placenta and is a reason for Rh isoimmunisation.

Other Igs in comparison with IgG

2. IgM: it has five subunits. It is involved in primary response.

3. IgA: these are secretory antibodies and gives protection to skin, intestine, eyes, urogenital

tract. It is also secreted in breast milk protecting the baby against intestinal infections.

4. IgE: they are produced by mast cells and are the cause for allergy and anaphylaxis.

3. PKU

Ans.

Phenyl ketonuria

-it is an autosomal recessive disease with an incidence of 1:1500 births. It is due to

deficiency of phenylalanine hydroxylase. So phenylalanine is not converted into tyrosine and it

accumulates.

- The excess of phenylalanine is converted to phenyl pyruvate, phenyl lactate, and

phenyl acetate and phenyl acetyl glutamine. Phenyl pyruvate, phenyl lactate, phenyl acetate

are excreted in urine.

- The child is mentally retarded and convulsions, tremors agitation, hyperactivity may

present. The child often has hypo pigmentation due to reduced availability of tyr for melanin

production.

- phenyl lactate causes mousy odor of urine.

- blood level of phenyl alanine is elevated, Guthrie’s test is confirmative. Urine FeCl3 test

is positive.

- tapioca based diet which have less phe is the treatment of choice. Gene therapy is

under trial.

4. Flurosis

Ans.

Fluoride is known to prevent caries. Residual food in the teeth undergoes bacterial

fermentation and leads to acid production which causes dental caries. Fluoride when supplied

in drinking water or tooth paste at about 1 ppm, coats the teeth and exchanges acid and also

kills the bacteria, preventing dental caries.

But when Fluoride in water exceeds more than 2ppm will cause chronic intestinal upset,

gastro-enteritis, loss of appetite and loss of weight. Level more than 5 ppm will cause mottling

of enamel, stratification and discoloration of teeth.

A level more than 20ppm is toxic leading to fluorosis, which causes osteoporosis,

osteosclerosis and brittle bones. Genu valgum is the characteristic feature. Fluorosis leads to

blood concentration of fluoride of 50mcg/dl.

Some districts of andra Pradesh and Punjab has increased ground water contamination

of fluoride leading to a widespread fluorosis endemic.

Fluorosis is also prevalent in areas where jowar is a staple diet. Paan users also affected

by fluorosis due to its fluoride content.

5. Serum protein electrophoresis

Ans. The term electrophoresis refers to the movement of charged particles through an

electrolyte when subjected to an electric field. Cations(positively charged ions) move towards

cathode and anions(negative) to anode.

When a biological mixture is subjected to electrophoresis, the compounds in the

mixture move in relation to their net charge, size, molecular weight and mass and gets

separated according to these characteristics, so that the desired compound can be identified

and isolated.

Factors affecting electrophoresis: rate of migration will depend on:

Net charge of the particles (eg. more negative particles move faster than less negative)

Mass and shape of particles(larger sized particles move slowly)

pH of medium(particles move better in a pH in which they are more ionized)

strength of electrical field(eg. Higher the voltage-faster the movement)

properties of supporting medium

temperature-increased temperature solidifies the support and impairs migration)

The electrophoresis apparatus consists if a tank which contains electrodes connected to a

power supply and buffer. The pH of buffer is selected so that it imparts maximum charge to the

electrophoresed substances(eg. Proteins get separated well in a buffer pH of 8.6

Supporting medium is the surface on which electrophoresis is carried out. It may be agar

gel, agarose gel, cellulose acetate, paper, etc.,

After the run the bands are visualized using naked eye or if needed to be quantified a

densitometer can be used.

Clinical applications:

1. serum protein electrophoresis:

- in nephrotic syndrome – globulin is produced more by liver in compensation of renal

loss of albumin. So alpha 2 band is prominent

- cirrhosis- albumin band is less prominent

- multiple myeloma- light chain immunoglobulins are produced more so there will be a

prominence in gamma globulin region(M band)

Fig serum protein electrophoresis in health and disease

2. hemoglobin electrophoresis

- S band is seen in sickle cell anemia

- various hemoglobinopathies and thalasemias can be diagnosed

6. Cell cycle

Ans.

The term cell cycle refers to the events occurring during the period between two mitotic

divisions. It is divided into G1(gap-1), S (synthesis), G2(gap-2) and M(mitosis) phase.

The cell division is taking place in M phase. The daughter cells then enter either to Go

(dormant/resting) phase or reenter the cell cycle when there is necessity for growth or repair.

In normal cell population most of cells are in G0 phase. Interphase is the period between

the end of M phase and beginning of next mitosis.

In G1 phase protein and RNA content increases. In S phase, DNA is synthesized, only

once becoming tetraploid.

In G2 phase cytoplasmic enlargement takes place.

Fig.Cell cycle

Cell cycle and cyclins:

Cyclins A,B,C,D and cyclin dependent kinases(CDK) 1,2,4,5,6 control cell cycle. CDK2- Cyclin E

directs the cells in G1 phase to enter into S phase. CDK2- Cyclin A pushes the cells to complete S

phase. CDK2-Cyclin A,B make cells complete the G2 phase and enter into M phase.

7. Role of PTH in Ca & P homeostasis

Ans.

PTH is secreted by four parathyroid glands in the thyroid tissue. Decreased serum calcium

leads to release of PTH from parathyroids. PTH activates adenylyl cyclase in target cells and

increases intracellular calcium concentration. A protein kinase is activated which activates

enzyme systems. PTH acts on

1. PTH and bones- PTH causes demineralization in bones. It activates pyrophosphatase in

osteoclasts leading to bone resorption and solubilising calcium. Calcium is released into

the blood stream and increases blood calcium level. This leads to loss of bone matrix.

2. PTH and kidneys: PTH causes decreased renal excretion of calcium and increased

excretion of phosphates and increased reabsorption of calcium leading to increased

blood calcium level.

3. PTH and intestines: PTH stimulates increased production of VIT D3 which acts on

intestine to absorb more calcium leading to increased calcium level in blood.

8. Define xenobiotics and add a note on the various detoxification reactions

Ans.

Xenobiotics are compounds which may be accidentally ingested or taken as drugs or

compounds produced in the body by bacterial metabolism.

Various xenobiotics:

Compounds accidentally ingested like preservatives, food additives and adulterants

Drugs taken for therapeutic purposes

Endogenous compounds which has to be eliminated by body like bilirubin, steroids

Compounds produced by bacterial metabolism

o Histidinehistamine

o Lysinecadaverine

o Ornithineputrescine

Phases of detoxification:

1. phase 1 reactions: it is the alteration of foreign molecule by adding a functional group like

hydroxylation, oxidation, hydrolysis, dealkylation Epoxidation, etc., the main function of phase

1 is to convert it into anon toxic metabolite.

Eg. Toluene Benzyl alcohol by oxidation

Benzene phenol by oxidation

Picric acid picramic acid by reduction

Aspirin acetic acid and salicylic acid by hydrolysis

Sometimes phase one reaction will to production of a toxic product.

Eg. Methanol formic acid

2. phase 2 reactions: conjugation

A xenobiotic that undergone a phase 1 reaction is now a new metabolite that contains a

chemical group like OH, NH2 , COOH groups. Phase 2 reactions lead to conjugation(addition) of

conjugating agents like:

Glucuronic acid: Bilirubin conjugated with glucuronic acid to form Bilirubin

Diglucuronide and excreted in bile.

Sulfate conjugation: phenol converted to phenol sulphate using PAPS(phosphor

adenosyl phosphor sulphate-active sulphate)

Cysteine and Glutathione: alkyl or aryl halide, epoxide are detoxified in this manner.

Acetylation: acetic acid s is conjugated to sulfanilamide, INH

Glycine: Benzoic acid is conjugated with glycine to form hippuric acid

Glutamine: phenyl acetic acid is conjugated to form Phenyl acetyl glutamine

9. Mutations

Ans.

An alteration in genetic material results in mutation. Out of every 106 cell divisions, one

mutation occurs.

Classification:

1. substitution-

-transition- replacement of a purine by purine or pyrimidine by pyrimidine

-transversion-replacement of purine by pyrimidine or vice versa.

2. deletion- removal of one or more bass

3. insertion- addition of one or more base

Effects of mutations:

1. Silent mutations- some mutations cause apparently no effect. Eg. CUA mutated to CUC. But

they both code for leucine.

2. Mis-sense

-acceptable mutation- amino acid may be changed, but it has no effect on protein

function. Eg. Hb sydney

-partially acceptable – Hbs. Here the protein Hb is changed, but it can still do its

function(carrying oxygen)

-unacceptable- here the aminoacid change causes complete loss of protein function- eg.

HbM

3. Non sense mutation- here mutation causes premature termination of protein. Eg. Beta

thalassemia

4. Frameshift mutation: When a base is added or deleted in a nucleotide, the reading frame

shits.

Eg. Addition of A in this strand

AGA CGG TTA GAG CCC becomes

AGA CAG GTT AGA GCC C so that the aminoacid composition of the whole polypeptide is

changed from the region of addition or deletion.

In thalassemia, frame shift mutation leads to premature termination of polypeptide.

10. Secondary structure of proteins

Ans.

Proteins contain primary, secondary, tertiary and sometimes quaternary structure.

Secondary structure of protein denotes the relationship between residues (aminoacids)

which are 3-4 residues apart. secondary structure of proteins is stabilized by non covalent

bonds such as:

1. hydrogen bond

2. electrostatic bond(salt bridges)

3. Hydrophobic bonds

4. Vander waal’s forces

Types of secondary structures:

1. α- helix: it is the most common and stable confirmation in a polypeptide chain. It is more

seen in Hb, Mb, collagen, etc., it has a spiral structure which is stabilized by hydrogen bond

between NH and CO groups of amino acids 4 residues apart forming a spiral. Each turn of helix

contain about 3.6 residues. The distance between each amino acids is about 1.5 Å apart. The

alpha helix is mostly right handed since aminoacids are usually L variety.

Fig.Alpha helix Fig. β-pleated sheet

2.β-pleated sheet: the polypeptide is fully extended. The distance between each residue is

about 3.5 Å. It is stabilized by hydrogen bonds of adjacent strand aminoacids. Adjacent strands

in a sheet can run in same direction (parallel) or in opposite fashion(anti-parallel). It is the major

component of enzymes, etc.,

3. bends and loops: bends are made up of 4 aminoacids where the 1st and 4th are connected by

hydrogen bonds making a U turn. Loops are made of many amino acids and it functions in

connecting two strands of beta sheet or alpha and beta sheet. It also takes part in formation of

active site in many enzymes.

III. Short answer questions ( 10 x 2 = 20 )

1. Urea cycle disorder causes orotic aciduria. Explain

Ans.

In condition like hyperammoninemia type II (ornithine transcarbamylase deficiency)

cabamoyl phosphate is not converted to metabolites of urea cycle. So carbamoyl phosphate

accumulates. Carbamoyl phosphate is also a precursor of pyrimidine synthesis. So it is

channeled to pyrimidine synthesis. Orotic acid is an intermediate in pyrimidine synthesis. So

orotic aciduria occurs.

2. Acidosis causes hyperkalemia why?

Ans.

In acidosis, to reduce serum H+ levels, K+ inside the cells gets exchanged for H+ using H+ -

K+ antiporter. So extracellular K+ increases leading to hyperkalemia. The reverse occurs in

metabolic alkalosis(hypokalemia occurs).

3. Define frame shift mutations with an example

Ans.

When a base is added or deleted in a nucleotide, the reading frame shits.

Eg. Addition of A in this strand

AGA CGG TTA GAG CCC becomes

AGA CAG GTT AGA GCC C so that the aminoacid composition of the whole polypeptide is

changed from the region of addition or deletion.

In thalassemia, frame shift mutation leads to premature termination of polypeptide.

4. We need two primers for PCR. Justify.

Ans.

DNA polymerase is the enzyme needed for synthesis of new strand of DNA using the

template single strand. It synthesizes from 5’ to 3’ direction. A primer is needed for DNA

polymerase to start DNA synthesis(RNAP doesn’t need a primer). A primer is a small strand of

nucleotide which is complementary to the template strand. Since two strands of DNA are

complementary to each other we need a forward primer for one strand and reverse primer for

other strand.

5. Mechanism of action of chloromphenicol

Ans.

Chloramphenicol inhibits peptidyl transferase, which is important in translation process

in bacteria. So it kills bacteria. It does not inhibit human peptidyl transferase. So it can be given

to humans as antibiotic in bacterial infections.

6. Mention the AA which take part of One carbon pool

Ans.

Glycine , serine

7. Mention the enzymes which require Selenium as cofactor

Ans..

1. Thioredoxin reductase(interconversion of deoxyribo nucleotides to ribonucleotides & vice

versa)

2. Glutathione peroxidase(anti oxidant)

3. 5’-deiodinase(thyroid metabolism)

8. Lesch Nyhan syndrome presence with hyperuricemia. Explain?

Ans.

it is a X-linked inborn error of purine metabolism, incidence 1:10,000

deficiency of HGPRTase which acts in salvage pathway

so the salvage pathway is stopped and PRPP accumulates which will go for catabolism to

uric acid

hyperuricemia leads to nephrolithiasis and gout

it is also characterized by self mutilation, mental retardation

9. Hypothyroidism presents with hypercholesterolemia why?

Ans.

Thyroid hormone increases lipoprotein catabolism which is a carrier of cholesterol. So in

hypothyroidism, lipoprotein catabolism is reduced leading to hypercholesterolemia.

10. Histidine load test.

Ans.

It is also known as FIGLU(Formiminoglutamic acid) excretion test. Histidine is normally

metabolized to FIGLU, from which formimino group is removed by tetrahydrofolate. So in folate

deficiency, FIGLU is excreted in urine. So this test is used for diagnosis of folate deficiency.

11. Mention two tumor markers and specify the diagnostic applications

Ans.

1.Carcinoembryogenic antigen- liver cancer

2. Prostate specific antigen- prostate cancer

12. M-band

Ans.

In multiple myeloma, light chain immunoglobulins are produced more so there will be a

prominence in gamma globulin region(M band). It is diagnostic of MM.

Fig. serum protein electrophoresis in MM

13. Beer-Lambert’s law

Ans.

Beer’s law states that the amount of light absorbed by a colored solution is proportional

to the concentration of the solution i.e. Absorbance α concentration

Lambert’s law states that the amount of light absorbed by a colored solution is

proportional to the depth through which the light passes through the solution

Absorbance α length

Combining these two laws C α A x L => C= AxL x absorptivity(an constant)=> absorptivity+ C/(A

X L)

For measuring the concentration of a compound (C1), Absorbance of it (A1) is taken and

known concentration of a standard substance is taken (C2) and its absorbance measured(A2)

Lambert’s law is kept constant by the fixed diameter of the measuring cuvette.

Then according to Beer lambert’s law

C1/A1 = C2/A2

=> C1= C2xA1/A2

This is how colorimetry is used to measure unknown concentration of a substance in a sample

like blood.

14. Mention two transmethylation reactions

Ans.

Transmethylation reaction is acceptance of a methyl group from a donor like S-adenosyl

methionine(SAM) by a compound resulting in another compound.

Transmethylation reaction requires SAM which is obtained by accepting adenosyl group

from ATP by methionine by methionine adenosyl transferase.

The transmethylation reactions are

Methyl acceptor Methylated product

Guanidoacetic acid Creatine

Serine Choline

Epinephrine Metanephrine

Nor epinephrine Epinephrine

tRNA Methylated tRNA

15. Enzyme deficiency in albinism. Mention two clinical features

Ans.

Tyrosinase is the enzyme deficient in albinism. The patient is unable to produce melanin

so there will be universal hypo pigmentation over the skin. Also the patient is more prone for

naevi and skin cancer due to attack by UV rays(melanin protects from UV).