csir net notes

7/31/2019 csir net notes

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NET-JRF Sample Paper 2B

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24-Jul-12 BIOTECNIKA.ORG

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1. Effect of EDTA on Bacterial cell is

1. Remove Mg++ ions2. digest polymeric compounds

3. Removes lipid molecules

4. removes insolubles cell debris.

1. EDTA (ethylenediaminetetraacetic acid). The EDTAmolecule can bind to metal ions by forming six bonds to it- two from nitrogen atoms in amino groups and four from

oxygen atoms in carboxyl groups.

2. Ca and Mg ions form the salt bridges in gram-negative

bacteria binding polysaccharides on the surface of the cell

wall. They also chelate the divalent ions of the lipoproteinand lipopolysaccharides of the cell wall.

Antimicrobials in FoodBy P. Michael Davidson, John Nikolaos Sofos, Alfred Larry

Branen


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2. Klenow fragment can

1. only polymerise DNA on a single strand2. only degrade DNA

3. both

4. none

Uses of Klenow fragmentSynthesis of double-stranded DNA from

single-stranded templates.

Filling in recessed 3' ends of DNA fragments.

Digesting away protruding 3' overhangs.

Preparation of radioactive DNA probes.

In some situations, the 3' -> 5' exonuclease activity ofKlenow fragment is either undesirable or not necessary.

By introducing mutations in the gene that encodes Klenow,

forms of the enzyme can be expressed that retain polymerase

activity, but lack any exonuclease activity. These forms are the

enzyme are usually called exo- Klenow fragment.


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3. which one of the following acts as inducer of lac operon

1. lactose2. allolactose

3. permease

4. galactosidase

The tetrameric lac repressor protein, has four binding sites forallolactose. When those sitesbecome occupied, the protein undergoes a change in shape (allosteric modification), which causes

it to dissociate from the operator site, allowing transcription to proceed. Thus allolactose induces

the lac operon by a process of de-repression. In order for lactose to induce the operon, there must

already be present a low level ofpermease to get the lactose into the cell and alow level of-galactosidase to convert the lactose to allolactose.

Mutants that totally lack either the permease or -galactosidase cannot be induced by lactose.


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4. Most base substitution in -10 to -35 base pair region causes

1. increase in promoter function2. decrease promoter function

3. does not affect promoter function

4. increase operator function

A specific region just upstream from a gene that acts as a binding site for transcription factors and

RNA polymerase during the initiation of transcription.

In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream

from the transcription start site.The sequence at -10 is called the Pribnow box, or the -10 element, and usually consists of the sixnucleotides TATAAT. The Pribnow box is absolutely essential to start transcription in prokaryotes.

The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Itspresence allows a very high transcription rate.
http://en.wikipedia.org/wiki/Prokaryotehttp://en.wikipedia.org/wiki/Prokaryote


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5. Linkers are

1. Double stranded DNA with blunt ends2. Single stranded DNA with sticky ends

3. Single stranded DNA with blunt ends

4. Single stranded DNA with sticky ends

Sticky end and Blunt end are the two possible configurations resulting from the breaking ofdouble-stranded DNA. DNA exhibits a stabilizing interaction between complementary base pairs,

providing specificity to the pairing of two strands of DNA.

If two complementary strands of DNA are of equal length, then they will terminate in a blunt end, as

in the following example. (linkers)5'-CpTpGpApTpCpGpCpTpApGpT-3'

3'-GpApCpTpApGpCpGpApTpCpA-5'

However, if one strand extends beyond the complementary region, then the DNA is said to possess

an overhang or it has a sticky end. (Adapters)5'-ApTpCpTpGpApCpT-3

3'-TpApGpApCpTpGpApCpTpApCpG-5'

These linkers play a vital role in geneticEngineering. They are used in cloning

Experiments.


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6. Leucine Zipper has

1. Leu residue at every 7th position of -helix2. Leu residue at every 4th position of -helix

3. Leu residue at every 7th position of -sheet

4. Leu residue at every 4th position of -sheet D represents Leu residue

A structure, referred to as the 'leucine zipper explain how some

eukaryotic gene regulatory proteins work. The leucine zipper consist

of a periodic repetition of leucine residues at every seventh position.

The segments containing these periodic arrays of leucine residues

seem to exist in an alpha-helical conformation. The leucine sidechains extending from one alpha-helix interact with those from a

similar alpha helix of a second polypeptide, facilitating dimerization;the structure formed by cooperation of these two regions forms a

coiled coil. These leucines form the hydrophobic core of a coiled

coil.
http://en.wikipedia.org/wiki/Hydrophobic_corehttp://en.wikipedia.org/wiki/Coiled_coilhttp://en.wikipedia.org/wiki/Coiled_coilhttp://en.wikipedia.org/wiki/Coiled_coilhttp://en.wikipedia.org/wiki/Coiled_coilhttp://en.wikipedia.org/wiki/Hydrophobic_core


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7. Which one can be used to cut a methionine between two polypeptide

1. Factor Xa2. Thrombin

3. Cyanogen bromide

4. none

Cyanogen bromide attacks on the carboxylate side of methionine,converting it to homoserine.

The chemistry of the reaction involves the nucleophilic attack of

the thioether sulphur on the carbon in CNBr, followed by thecyclization of iminolactone, which is hydrolysed in water resulting

in peptide cleavage.

Thrombin is a serine protease that possesses trypsin-likebehavior in that it prefers to cleave its substrates after arginineresidues.

Factor Xa cleaves after the arginine residue in its preferredcleavage site Ile-(Glu or Asp)-Gly-Arg.

Chemical Reagents for Protein ModificationBy Roger L. Lundblad

thioether


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8. Thrombin cuts the polypeptide at

1. next to Gly2. next to Arg

3. next to Met

4. next to Lys

The thrombin molecule contains twochains.The A chain is composed of 36 residues and is non-essential for proteolytic activities.The B chain is composed of 259 amino acids and is derived from the carboxyl terminal sequence ofprothrombin.

The B chain contains the three active site amino acids, His57, Asp102, and Ser195.

Thrombin is a serine protease that possesses trypsin-like behavior in that it prefers to cleave itssubstrates after arginine residues.


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9. Primer extension is used for identification of

1. 3' end2. 5' end

3. both

4. none

A primer is a strand ofnucleic acidthat serves as a starting point for DNA replication.They are required because the enzymes that catalyze replication, DNA polymerases, can only add

new nucleotides to an existing strand of DNA.

The polymerase starts replication at the 3end of the primer, and copies the opposite strand.

In primer extension, a short antisense 5' end-labeled DNA primer (usually a syntheticoligonucleotide, but sometimes a small restriction fragment) is hybridized to RNA, usually total cellular

RNA, then DNA is synthesized from this primer using reverse transcriptase. RTase will copy the RNA

from the site of primer annealing to the 5'-end of the RNA molecule.The reactions are then analyzed by electrophoresis in sequencing gels in lanes adjacent to Sanger

sequencing reactions of DNA containing the gene of question, using the same primer as that used in

the PE analysis.

The transcription initiation site (usually) can then easily be identified as the band in the sequencing

reaction directly parallel to the run-off reverse transcript.


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10. S1 nuclease analysis can be used for identification of

1. 3' end2. 5' end

3. both

4. none

S1 nuclease is an endonuclease that is active against single-stranded DNA and RNA molecules.It is five times more active on DNA than RNA.

S1 nuclease analysisPreparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded

DNA probe.

At the end of the reaction period, nuclease S1 is used to degrade unhybridized regions of the probe,and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized

by either autoradiography or Southern hybridization.

The method can be used to quantify RNAs, to map the positions of introns and to identify the locations of

5' and 3' ends of mRNAs on cloned DNA templates


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11. Adapters has

1. one sticky end and one blunt end2. both sticky ends

3. both blunt ends

4. none

Adapters are linkers with cohesive ends or a linker digested with RE, before ligation.The most widely used definition is cut linkers also called as adapters.They are not perfectly double stranded non single stranded. By adding adaptors to the ends of a DNA, sequences that are

blunt can be converted into cohesive ends.

Adaptor molecules are synthesized so that the blunt end is the same as natural DNA, but the sticky end is different. The

3'-OH terminus of the sticky end is the same as usual, but the 5'-P terminus is modified; it lacks the phosphate group,and is in fact a 5'-OH terminus. DNA ligase is unable to form a phosphodiester bridge between 5'-OH and 3'-OH ends. The

result is that, although base pairing is always occurring between the sticky ends of adaptor molecules, the association is

never stabilized by ligation.

Adaptors can therefore be ligated to a DNA molecule but not to themselves. After the adaptors have been attached, the

abnormal 5'-OH terminus is converted to the natural 5'-P form by treatment with the enzyme polynucleotide kinase,

producing a sticky-ended fragment that can be inserted into an appropriate vector.

5'-ApTpCpTpGpApCpT-3

3'-TpApGpApCpTpGpApCpTpApCpG-5'


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12. What is the ratio of absorbance of UV light by pure DNA at 260 nm

and 280nm wavelength

1. 1.82. 4.8

3. 10.2

4. 7.8

Since nucleotides, RNA, ssDNA, and dsDNA all absorb at 260 nm, they will contribute to the total

absorbance of the sample. Therefore, to ensure accurate results, of purity, ratio of absorbance of UV

light by DNA at 260 nm and 280nm wavelengthis calculated.A ratio of~1.8is generally accepted as pure forDNA; a ratio of~2.0is generally accepted as pureforRNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenolor other contaminants that absorb strongly at or near 280 nm.

The resultant 260:280 ratio for the nucleic acid being studied will be approximately equal to theweighted average of the 260/280 ratios for the four nucleotides present. The generally accepted

ratios of 1.8 and 2.0 for DNA and RNA respectively, are "rules of thumb". The actual ratio will depend

on the composition of the nucleic acid.

RNA will typically have a higher 260/280 ratio due to the higher ratio of Uracil compared to that of

Thymine.

Guanine: 1.15Adenine: 4.50Cytosine: 1.51Uracil: 4.00Thymine: 1.47

260/280 ratio


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13. The most abundant enzyme in the biosphere

1. Amylase2. Rubisco

3. DNA polymerase

4. RNA polymerase

Ribulose-1,5-bisphosphate carboxylase/oxygenase, most commonly known by the shorter nameRuBisCO, is an enzyme that is used in the Calvin cycle to catalyze the first major step of carbon

fixation, a process by which the atoms of atmospheric carbon dioxide are made available to

organisms in the form of energy-rich molecules such as sucrose. RuBisCO catalyzes either the

carboxylation or the oxygenation of ribulose-1,5-bisphosphate (also known as RuBP) with carbon

dioxide or oxygen.

RuBisCO is very important because it catalyzes the most commonly-used chemical reaction by

which inorganic carbon enters the biosphere. RuBisCO is also the most abundant protein in leaves.

It is found in plants, algae, cyanobacteria, phototropic and chemoautotropic proteobacteria .


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14. Method used for differentiating real genes from chance

ORF's is

1. CpG island2. codon bias

3. Homology search and transcript analysis

4. All of above

An open reading frame (ORF) is a portion of an organism's genome which contains a sequence of bases that couldpotentially encode a protein. In a gene, ORFs are located between the start-code sequence (initiation codon) and thestop-code sequence (termination codon). However, short ORFs can also occur by chance outside of genes. UsuallyORFs outside genes are not very long and terminate after a few codons.

CpG islands are genomic regions that contain a high frequency of CG nucleotides. CpG islands typically occur at ornear the transcription start site of genes, particularly housekeeping genes, in vertebrates. DNA regions >500 bp witha GC content >55% and observed CpG/expected CpG of 0.65 were more likely to be the true CpG islands.

In most genomes not all members of a codon family are used with equal frequency.

Eg: Humans,use GTG four times more frequently than GTA for coding valine. If an ORF contains high frequency of rare

codons then it is probably not a gene.

Homology search : When a test sequence is BLASTed and if 30% of amino acids matches with the database, then itcan be confirmed a real gene.

Transcript analysis: For many model organisms the ESTs and cDNA sequences are used.if the sequence of ORFmatches the sequence of transcript. Then the ORF is a real gene.


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15. Hybrid of trp and lac promoter is called

1. lrp2. lap

3. tac

4. trc

Tac is a strong promoter and is the hybrid of the trp and lac promoters, containing -35 region of trpfused with -10 region of lacUV5(a form where there are 2 mutations in -10 region, leading to enhanced

expression) promoter.It is induced by Lactose and IPTG.It is regulated by lac repressors and is

independent of cAMP regulation. Eg of vectors containing tac promoters are pkk223,pkk233 and

others.

Trc is also a hybrid promoter of trp and lac,containing -35 region of trp fused with -10 region oflacUV5.These 2 regions are separated by 17bp whereas in tac they are separated by 16bp.

Lap is a latency associated prmoter and is not a hybrid promoter.


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17. which is true for homeodomain

1. was discovered in homeotic genes2. has DNA binding segment related to helix-turn-helix motif

3. is enclosed in homeobox

4. All of above

Homeotic genes are defined by a DNA sequence known as the homeobox, which is a sequence of 180nucleotides that code for a protein domain known as the homeodomain.The first genes found to encode homeodomain proteins were Drosophila developmental control genes, in

particular homeotic genes, from which the name homeobox was derived. However, many homeobox

genes are not homeotic genes; the homeobox is a sequence motif, while "homeotic" is a functional

description for genes that cause homeotic transformations.

The protein products of homeotic genes belong to a class of proteins known as transcription factors,all of which are capable of binding to DNA, thereby regulating the transcription of genes. The homeobox

sequence codes for a 60 amino acid helix-turn-helix protein known as the homeodomain. Thehomeodomain acts as an "on/off" switch for gene transcription by binding to specific sequence enhancers

of a gene, which either activates or represses the gene.


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18. Plasmids are found in -

1. bacteria2. yeast

3. mouse

4. both a and b

Small, circular, extrachromosomal DNA molecules. They can replicate independently of the genome,

and are found in numbers ranging from one per cell to hundreds per cell (this is called "copy number").

Plasmids frequently carry genes for antibiotic resistance.Plasmids are considered transferable genetic

elements, or "replicons", capable of autonomous replication within a suitable host. Plasmids can be

found in all three major kingdoms, Archea, Bacteria and Eukaryote.

Among eukaryotes, plasmids have been found in fungi and plants but not in animals. Most plasmids

are mitochondrial. In filamentous fungi, plasmids are commonly encountered in isolates from naturalpopulations. Individual populations may show a predominance of one type, but some plasmids have a

global distribution, often crossing species boundaries. Circular plasmids are common only in

Neurospora spp., but linear plasmids have been found in many fungi. Circular plasmids have oneopen reading frame (ORF) coding for a DNA polymerase or a reverse transcriptase. Linear plasmidsgenerally have two ORFs, coding for presumptive DNA and RNA polymerases with amino acid motifs

showing homology to viral polymerases.


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19. Which DNA binding protein motif is present in Lac repressor

1. helix-turn-helix2. zinc finger

3. homeodomain

4. leucine zipper

The helix-turn-helix motif consists of about 20 amino acids. The basic form is that of 2 helices separated by aturn. One helix, the stabilization helix, is there to stabilize the other helix, the recognition helix. The helix-turn-helix

motif will be illustrated with the lac-repressor-DNA complex.

The leucine zipper is an interesting structure made up of two a-helical segments of protein that have leucinesfacing each other along the length of the helices, allowing them to dimerize and form a symmetric interface that can

bind to the DNA on both sides of the double helix (see figure). The leucine zipper motif will be illustrated with the

GCN4 (protein)-AP1 (DNA) complex, a protein involved in activating transcription in yeast.

The Zinc finger motif includes a metal, Zinc, in the DNA-binding motif. The Zinc helps to stabilize the three-dimensional structure of the Zinc-finger. The Zinc is coordinated either with the sulfur in cysteine, or one of the

nitrogens in the histidine imidazole sidechain. The zinc-finger motif will be illustrated with the Transcription FactorIIIA-DNA complex from the clawed frog Xenopus laevis.


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20. Which of the following gene is carried by "F" plasmid ?

1. yrp

2. yar

3. tra

4. trp

5. lac

Yrp - yeast replication plamid

Yar genes found in Human

Y chromosome

Trp found in trp operon

Lac found in lac operon

The Fertility factor is a bacterial DNA sequence that produces a sex pilus necessary for conjugation.It contains 20 tra (for "transfer") genes and a number of other genetic sequences responsible for

incompatibility, replication, and other functions. The F factor is an episome and can exist as an

independent plasmid or integrate into the bacterial cell's genome.


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F plasmid is responsible for

oriV origin for vegetative replication-replication is bi-directional (like bacterial chromosome)

-replicates in synchrony with bacterial chromosome

oriT origin of transfer- rolling circle replication

- single strand enters recipient which will synthesize the complementary strandChromosomal Transfer by F-plasmid tra - transfer genes- genes on F-plasmid (tra genes) specify formation of sex pilus and conjugation bridge

-usually only F-plasmid is transferred,sometimes chromosomal genes are moved.

http://highered.mcgraw-hill.com/olc/dl/120082/bio_f.swf visit this URL for the video.
http://highered.mcgraw-hill.com/olc/dl/120082/bio_f.swfhttp://highered.mcgraw-hill.com/olc/dl/120082/bio_f.swfhttp://highered.mcgraw-hill.com/olc/dl/120082/bio_f.swfhttp://highered.mcgraw-hill.com/olc/dl/120082/bio_f.swf


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21. In zinc finger Zn++ ion is co-ordinated to

1. 4 Cys

2. 4 His

3. 2 Cys and 2 His

4. Both a and c

The zinc finger motif contains one or more zinc ions which are crucial for the structural stability. It

can be divided into three types:

C2H2 zinc finger: It is characterized by the sequence CX2-4C....HX2-4H, where C = cysteine, H= histidine, X = any amino acid. In the 3D structure, two cysteine residues and two histidine

residues interact with a zinc ion .Eg:S1 transcription factor

C4 zinc finger: Its consensus sequence is CX2CX13CX2CX14-15CX5CX9CX2C. The first fourcysteine residues bind to a zinc ion and the last four cysteine residues bind to another zinc ion. EgEstrogen receptor

C6 zinc finger. It has the consensus sequence CX2CX6CX5-6CX2CX6C. The yeast's Gal4contains such a motif where six cysteine residues interact with two zinc ions. Eg: GAL4


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In Fig 2&3

Green strands Cysteine residue

Orange Balls Zn ion

Red Strands - Receptor

Fig 2

Fig 3


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22. Which one is not used in making genetically modified crops

1. vector based on plant virus

2. various plasmid DNA

3. artificial plasmid of animals

4. natural plasmid of agrobacterium

The agrobacterium transfers its own T-DNA, but if the T-DNA is removed and replaced with another gene, A.

tumefaciens can be used to introduce that gene into the plant genome, thereby providing a vector for scientists to

engineer beneficial genes into plants.

Larger genes can be introduced to plants using bacterial artificial chromosomes (BACs). BACs are synthesized gene

vectors based on a plasmid from E. coli. BACs can have inserts ranging from 50 to 350 kb, allowing for the transfer of

large genes or many small genes at once.

For specialized studies (production of foreign proteins in plants, expression of potentially lethal genes, etc.), virus-

based vectors are used.


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23. E.Coli cannot

1. degrade recombinant protein

2. always fold recombinant protein correctly

3. glycosylate recombinant protein correctly

4. All of above

Advantages of prokaryotic recombinant protein expression systems:Ease of culture

Rapid cell growth

Inducible expression using IPTG

Simple purification

Some of the disadvantages in using E.coli as the host are:Most proteins become insoluble and are very difficult to recover as functional proteins

Post-translational modifications are not added by bacteria

Protein may not be functional.

http://www.exptec.com/Stategies/Problems%20and%20hosts.htm follow this URL for the expression problems due

to hosts
http://www.exptec.com/Stategies/Problems%20and%20hosts.htmhttp://www.exptec.com/Stategies/Problems%20and%20hosts.htm


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24. Lac promoter can be induced by:-

1. Iso propyl thio galactosidase

2. iso methyl thio cyanate

3. tryptophan

4. 3-beta -indole acrylic acid

Isopropyl--D-thio-galactoside (IPTG) is frequently used asan inducer of the lac operon as it binds to repressor and

inactivates it. Unlike allolactose, the sulfur (S) atom createsa chemical bond which is non-hydrolyzable by the cell,

preventing the cell from "eating up" or degrading the inductant;

therefore the IPTG concentration remains constant.

IPTG can enter thye cells without lactose permease,and

hence it can act as an inducer in the absence of functionallacy and lacz products


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26. pBIN 19 binary vector consist of

1. lac z' gene

2. kanamycin resistance gene

3. cloning sites

4. all of above


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27. In lac operon the repressor is encoded by

1. Lac A

2. Lac Y

3. Lac Z

4. Lac I

The operon is under the control of the adjacent lacI gene, encoding the lactose repressor. Therepressor is a regulatory gene. In the absence of allolactose, the inducer of the lac operon, the

repressor tetramer binds to the lac operator (lacO) and prevents RNA polymerase from transcribing

the operon. However, when allolactose is present it binds to the repressor this prevents repressor

from binding to lacO and permits RNA polymerase to bind to lacP and to initiate transcription.

The lacI gene is transcribed constitutively from its own promoter.

The lac repressor is a tetramer, where all four identical components are 360 amino acids in length.

There are three main domains in the lac repressor molecule. The NH2 terminal domain (residues1-62), the coreprotein domain (residues 62-340), and the COOH-terminal domain (residues 340-357)

The NH2 terminal domain of the lac repressor is involved with the DNA binding. The NH2 terminal

ends can actually be subdivided into two separate functioning domains: the DNA-binding region

(residues 1-45) and the hinge region (residues 46-62)http://biology.kenyon.edu/BMB/Chime/Mat/MASTER.HTM follow the link for more details.
http://biology.kenyon.edu/BMB/Chime/Mat/MASTER.HTMhttp://biology.kenyon.edu/BMB/Chime/Mat/MASTER.HTM


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28. Drawback of using baculovirus in protein production

1. larger nuclear inclusion bodies

2. makes large amount of protein

3. improper glycosylation

4. polyhedrin gene product

Advantages of using baculovirus expression system areEukaryotic post-translational modification

Proper protein folding and function

High expression levels

Easy scale up with high-density suspension culture

Disadvantages of using baculovirus expression system are

Low secretion level.Improper glycosylation.

Proteins that occur as aggregates.

Refrerence:http://books.google.com/books?id=QUd1FfElRN4C&pg=PA31&lpg=PA31&dq=polyhedrin+gene+product&source=web&

ots=ge0SjQefy-&sig=2ebOC49QenqMOhdgiDqEXpZQ&hl=en&sa=X&oi=book_result&resnum=9&ct=result#PPA27,M1

Polyhedrin gene product is nonessential when the virus

Is propagated.In its absence BV is

secreted by the infected

cells. This dispensible nature ofpolyhedrin gene makes BV

suitable for constructing vectors. The

desired gene is placedunder the control of polyhedrin

promoter.


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29. A yeast vector carrying A 2 ?m circlular origin of Replication

1. YAC

2. YEp

3. YIp

4. YRp

The YpI integrative vectors do not replicate autonomously, but integrate into the genome at low frequencies byhomologous recombination.The YpI integrative vectors do not replicate autonomously, but integrate into the genome at

low frequencies by homologous recombination.

The YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2mm plasmid that serves as an origin of replication (2 mm ori). The 2 mm ori is responsible for the high copy-number and

high frequency of transformation of YEp vectors.

The YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences,

CEN, and autonomously replicating sequences, ARS. The YCp vectors are typically present at very low copy numbers,from 1 to 3 per cell.

A yeast artificial chromosome (short YAC) is a vector used to clone large DNA fragments (larger than 100 kb and up to3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin

sequences needed for replication and preservation in yeast cells.

The Yrp contain ARS as their origin of replication in yeast. they have very high copy numbers but are extremely unstable.


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30. Factor Xa cuts at

1. After Arg of Gly-Arg

2. Before Arg of Gly-Arg

3. Before Gly of Gly0 Leu

4. After Gly of Gly-Leu

Factor Xa is a serine endopeptidase composed of two disulfide-linked subunits that converts prothrombin to thrombinin the blood coagulation cascade. Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or

Asp)-Gly-Arg and it will occasionally cleave at other basic residues.

However, it will not cleave at a site followed by proline or arginine.

Factor Xa is often used to remove fusion tags, such as the common histidine tag, from expressed proteins. By treating

a purified, 6xHis-tagged protein expressed with a factor Xa cleavage site, it is possible to obtain the protein in its native

form. The purified, expressed protein is incubated with factor Xa protease.


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31. A.tumefaciens causes

1. rust

2. wort

3. crown gall disease

4. hairy root disease

Agrobacterium tumefaciens causes crown gall disease of a wide range of dicotyledonous (broad-leaved) .Thedisease gains its name from the large tumour-like swellings (galls) that typically occur at the crown of the plant,

just above soil level.Crown gall tumors result from the over-production of the phytohormones auxin and cytokinin

specified by A. tumefaciens T-DNA genes.

wart:Synchytrium endobioticum, a primitive parasitic fungus forms wart.The primary symptom consists of gallson tubers, stolons and stems, and occasionally on leaves, but never on roots.

The rusts belong to the Basidiomycota, the division of fungi to which the mushrooms belong. Although the rustsattack a large number of seed plants and even some ferns.The common name rusts is based on the "rusty"

colored blotches that is present on the stems and leaves from the urediospore stage in this group of fungi.Eg:Late

Blight of Potato disease was due to the fungal pathogen, Phytophthora infestans.

Hairy root disease is caused by A.rhizogenes.Although the T-DNA of some Riplasmids of A. rhizogenescontains auxin biosynthetic genes, these loci are not always necessary for hairy root formation. Recent

experiments suggest that hairy root tumors result from the increased sensitivity of transformed cells to

endogenous auxin levels.

1

2

4

3


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33. Which is not a method of DNA insertion in living cells

1. Transformation

2. Transfection

3. Microinjection

4. none

Transformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expressionof foreign genetic material (DNA). Terms used for genetic alterations resulting from introduction of DNA by viruses

(transduction) or by cell-cell contact between bacteria (conjugation). Transformation of eukaryotic cells in tissue

culture is usually called transfection.

Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transfection of animalcells typically involves opening transient pores in the cell plasma membrane, to allow the uptake of material.

Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may

be transfected.

Microinjection refers to the process of using a very fine needle to insert substances at a microscopic ormacroscopic level into a single living cell. It is a simple process in which a needle 0.5 to 5 micrometers in diameter

penetrates the cell membrane and/or the nuclear envelope and the desired contents are then injected into the

desired sub-cellular compartment and the needle is removed.


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34. Use of regulatory protein to protect DNA from endonuclease is

done in

1. DNA footprinting

2. DNA fingerprinting

3. gel retardation

4. chemical degradation

DNA Footprinting was developed in 1977 and is an analytical procedure in molecular biology for identifying the specificsequence of DNA (the binding site) that binds to a particular protein. DNA Footprinting is most commonly performed on

proteins that are thought to play some significant functional role such as gene regulation.

DNA Fingerprinting, method of identification that compares fragments of deoxyribonucleic acid (DNA) It is sometimescalled DNA typing.

Gel retardation is a technique used to find out the DNA-protein interactions by electrophoresing the DNA fragment and

the DNA-protein complex.The DNA protein complex moves slowly in the GEL which shows the presence of the protein.

Maxam Gilbert method requires radioactive labelling at one end of the DNA. Chemical treatment generates breaks at asmall proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of

labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule. The fragments in the

four reactions are arranged side by side in gel electrophoresis for size separation. To visualize the fragments, the gel is

exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA

fragment, from which the sequence may be inferred.


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35. Advantage of insulin production by recombinant DNA technique

1. can be modified by addition of sugar molecules after translation

2. insulin is a big protein

3. active insulin is synthesized by bacteria

4. none

Human insulin is the only animal protein to have been made in bacteria in such a way that its structure is absolutely

identical to that of the natural molecule. This reduces the possibility of complications resulting from antibody

production. In chemical and pharmacological studies, commercially available Recombinant DNA human insulin hasproven indistinguishable from pancreatic human insulin. The only disadvantage was contamination due to host cell

and it requires thorough purification.

Nowadays, the recombinant insulin is produced using Yeast cells as they secrete an almost complete human

insulin molecule with perfect three dimensional structure. This minimises the need for complex and costly

purification procedures.


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36. trp promoter is repressed by

1. leucine

2. lactose

3. galactose

4. Tryptophan

The trp operon is a repressible system.The primary difference between repressible and inducible systems is the result that occurs when

the effector molecule binds to the repressor.

With inducible systems, the binding of the effector molecule to the repressor greatly reduces the affinity of the repressor

for the operator, the repressor is released and transcription proceeds.

The lac operon is an example of an inducible system.With repressible systems, the binding of the effector molecule to the repressor greatly increases the affinity of repressor

for the operator and the repressor binds and stops transcription.Thus, for the trp operon , the addition of tryptophan (the effector molecule) to the E. coli

environment shuts off the system because the repressors binds at the operator.


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37. T-DNA is not

1. between 15-30 Kb in size

2. passed to daughter cells

3. responsible for cancerous properties

4. responsible for opine synthesis

During tumour induction, Agrobacterium attaches onto plant cells and then transfers part of its DNA to some of these

cells. The transferred DNA (T-DNA) which is found on a large Ti (tumour inducing) plasmid is modified within the

bacterium and is transferred to the plant where it becomes integrated into the plant genome.

Proteins which are encoded by the virulence (vir) region of the Ti plasmid regulate the T-DNA modification and transfer.

Expression of genes located on the T-DNA leads to the formation of proteins involved with the production ofauxins andcytokynines. These plant hormones cause the tumourous phenotype that is characterised by the ability of the plantcells to proliferate limitlessly and autonomously, even in the absence of added phytohormones.

Crown gall tumours are characterised by the production ofopines (derivatives of amino-acids). The biosynthesis ofopines is catalysed by opine synthase enzymes, which are encoded by the T-DNA. Opines are formed in the tumours

can be metabolised by the tumourigenic agrobacteria, but not by most other soil organisms. Thus, Agrobacterium

creates for itself a favourable niche by genetic modification plant cells.


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38. which one will affect efficiency of expression study of foreign gene

in Ecoli

1. presence of introns

2. Sequence acting as termination signals in E.Coli

3. codon bias

4. All of above

Introns: The foreign gene might contain introns.Since the E.coli does not have introns the host would not have propermachinery to remove introns from transcripts.

The foreign gene might also contain sequences that act as termination signals in E.coli.These sequences might becompletely innocuous in normal cell but in E.coli results in premature termination and loss of gene expression.

Codon bias: tRNA availabilty is addressed,the highly expressed genes will have codons corresponding to abundanttRNAs and the genes expressed at low levels use codon for less abundant tRNA.

Equalization ofcodon-anticodon hydrogen bonding. In cases where there is a choice of synonymous

codon,selection would be to avoid very strong and weak interaction with anticodon,which decreases the translationefficiency.

A variety of factors affect the expression of foreign proteins in Escherichia coli. These include: promoter strength,

efficiency of ribosome binding, stability of the foreign protein in E. coli, location of the foreign protein in E. coli, the

codons used to encode the foreign protein, the metabolic state of the cell, and the location, stability and copy number

of the foreign gene.


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39. Promoter used for control of cloned genes in saccharomyces

cerevisiae

1. gal

2. pho5

3. cup1

4. All of above

Cloned genes in S. cerevisiae are placed under the control of GAL promoter which is normally

upstream of the gene coding galactose epimerase (an enzyme for galactose metabolism). The GAL

promoter is induced by Galactose.

PHO5 promoters are regulated by the phosphate level in growth medium.

CUP1 is induced by copper.

f l d d


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40. Repression of lac operon is not caused due to

1. absence of lactose

2. mutation in operator

3. functional I gene on it

4. functional I gene on another DNA in cell

cis-acting locus

- a genetic region affecting the activity of genes on that same DNA molecule

-Such a locus usually does not code for a protein but instead acts as a binding site for trans-acting proteins.

Jacob and Monod proposed the "operator element" in the lac operon.

- If mutated this operator element should be dominant in cis in that it only affects the genes on the same chromosome

(directly adjacent to it).

- It will not be dominant in trans.

OC is dominant in the cis position

- no diffusible product.

cis dominance - the ability of locus to influence the expression of one or more adjacent loci on the same chromosome,

as occurs in lac operator mutants of E.coli

41 hi h i d d b l f l


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41. which one is not encoded by structural gene of lac operon

1. -galactosidase

2. galactosidase acetylase

3. galactosidase permease

4. thio-galactosidase transacetylase

lacZencodes -galactosidase, an intracellular enzyme that cleaves the disaccharide lactose intoglucose and galactose.

lacYencodes -galactoside permease, a membrane-bound transport protein that pumps lactoseinto the cell.

lacAencodes -galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl-CoA to -galactosides

42 Whi h f h h ll l i ?


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42. Which of the phage never cause cell lysis?

1. M13

2. Lambda

3. -X174

4. none

Bacteriophage lambda has two different life cycles. It can infect its host, E. coli, replicate and synthesize new phage,then lysing and killing the host as the phage burst from the cell. Or, the phage can infect the cell and enter a

dormant phase within the cell in which the phage is present but its genes are generally not being expressed. Then

under certain circumstances, the phage will leave dormancy and resume lytic growth.

Coliphage -X174 encodes a single lysis protein, E, a 91 amino acid membrane protein,which is a specific inhibitor ofMra Y gene involved in peptidoglycan synthesis.

Filamentous hages like M13 and Ff are not lytic.Some of these phages become prophages by integrating into thegenome,others remain as episomal or plasmid like prophages and establish a pseudolysogenic relationship with

host,when they do not integrate but continuously produce progeny.

43 E l f l


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43. Examples of regulon are

1. SOS response to DNA damage

2. heat shock gene system

3. sugar metabolism system

4. All of above

In eukaryotes, a genetic unit consisting of a noncontiguous group of genes under the control of a single regulator gene.

In bacteria, regulons are global regulatory systems involved in the interplay of pleiotropic regulatory domains and consist

of several OPERONS.

When bacteria are exposed to stress they can produce many defence proteins which genes are normally in a repressed

state and that allow repair of damaged DNA and reactivation of DNA synthesis, and that these processes are connected

with mutation. Since emotionally he was bound with the sea he called this phenomenon the SOS response, afterSaveOur Souls.

Heat shock regulons contain a group of heat shock proteins (stress proteins). They are induced when a cell undergoesvarious types of environmental stresses like heat, cold and oxygen deprivation. For example, HSPs help new or distorted

proteins fold into shape, which is essential for their function.

44 I HSP hi h b it di t RNA l bi di


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44. In HSP which subunit mediates RNA polymerase binding

to promoter

1. 70

2. 32

3. 54

4. 90

Different s have different affinities for

variant promoters.

* 70 - general purpose.

* 54 - nitrogen metabolism.

* 32 - heat shock proteins.

* S - stationary phase.* F - flagella.

45 Wh t i th f ti f Alk li h h t d


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45. What is the function of Alkaline phosphatase and

polynucleotide kinase

1. Removes phosphate group from 5' end and add at 5' end respectively

2. Removes phosphate from 3' end and adds at 3' end respectively

3. Adds phosphate at 5' end and removes from 5' end respectively

4. Adds phosphate at 3' end and removes from 3' end respectively

Alkaline phosphatase removes 5' phosphate groups from DNA and RNA.It will also remove phosphates from nucleotides and proteins.

These enzymes are most active at alkaline pH - hence the name.

Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer ofa phosphate from ATP to the 5' end of either DNA or RNA.

46 Similarity between chain termination and chemical degradation


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46. Similarity between chain termination and chemical degradation

method of sequencing

1. use double stranded DNA

2. Need Primer

3. Degrades the DNA

4. none

See Next slide for the explanation.

use double stranded DNA maxim gilbert method (chemical degradation method)

Need Primer Sanger method (chain termination method)

Degrades the DNA - (chemical degradation method)


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Sanger methodChain termination

47 which will negatively effect lac operon expression


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47. which will negatively effect lac operon expression

1. lactose

2. less glucose concentration

3. reased binding of cAMP and CRP

4. decrease in concentration of c-AMP

The activity of RNA polymerase also depends on the presence of another DNA-binding protein called

catabolite activator protein or CAP.CAP can bind to DNA only when cAMP is bound to CAP. so when

cAMP levels in the cell are low, CAP fails to bind DNA and thus RNA polymerase

cannot begin its work, even in the absence of the repressor.


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48 subunit is a type of


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48. subunit is a type of

1. specificity factor

2. repressor

3. activator

4. none

Bacterial RNA polymerases are best characterised in Thermus aquaticus. The five subunits are:

* I II - required for DNA binding and assembly.

* - needed for DNA binding (contains basic amino acid residues) and catalysis.

* - stabilises binding.

* - forms holoenzyme.

stimulates tight binding between RNA polymerase and a promoter.Hence it is the specificity factor.

A repressor is a DNA-binding protein that regulates the expression of one or more genes by

decreasing the rate of transcription.

An activator is a DNA-binding protein that regulates one or more genes by increasing the rate of

transcription.

49 Vector used in first cloning experiment in mammalian cell was


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49. Vector used in first cloning experiment in mammalian cell was

based on

1. SV 40

2. BPV

3. adenovirus

4. retrovirus

Animal virus vectors also deliver the foreign genes into the cultured cells which get integrated into the host genome.

The expression of foreign genes can also be amplified using the promoters of the virus gene. The cloned genes can

be used in gene therapy, for the synthesis of important proteins etc. A vector based on Simian Virus 40 (SV 40) wasused in the first cloning experiment involving mammalian cells in 1979.

SV40 is capable of infecting several mammalian species following a lytic cycle in some hosts and lysogenic in some.

Adenovirus are bigger viruses and are capable of cloning 8KB fragments.

BPV Bovine papilloma virus has an unusual infection in mouse cells taking the form of multicopy plasmid with about100 copies per cell. BPV molecules are passed on to daughter cells.They do not cause death in mouse.

50 Full form of HART is


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50. Full form of HART is

1. Hybridization and retranslation

2. Hybrid arrest translation

3. Hybridization and retranscription

4. High affinity restriction technique

51 HRT and HART depend on


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51. HRT and HART depend on

1. bacterial cell translation

2. eukaryptic cell translation

3. both prokaryot and eukaryotic cell translation

4. cell free translation

Both HRT and HART rely on hybridising cloned DNA fragments to mRNA of the

cell or tissue from which the clones have been derived.In both cases the identity

Protein, hence the gene can be determined by examining the gels.


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HRTHART

52 Shotgun cloning is appropriate for


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52. Shotgun cloning is appropriate for

1. prokaryotes

2. eukaryotes

3. both

4. none

Prokaryotes contain complete and uninterrupted ORFs - therefore prokaryote genes can be cloned directly from

genomic DNA.

Most eukaryotes have ORFs which are divided into coding (exon) and non-coding (intron) sequences therefore

these genes cannot be cloned directly from genomic DNA - these require cDNA cloning.


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