Important Tips FOR CSIR NET Life Science Exam 2010

1. Effect of EDTA on Bacterial cell is : 1. Effect of EDTA on Bacterial cell is Remove Mg++ ions digest polymeric compounds Removes lipid molecules removes insolubles cell debris. EDTA (ethylenediaminetetraacetic acid). The EDTA molecule 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. 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 lipoprotein and lipopolysaccharides of the cell wall. Antimicrobials in Food By P. Michael Davidson, John Nikolaos Sofos, Alfred Larry Branen

2. Klenow fragment can : 2. Klenow fragment can only polymerise DNA on a single strand only degrade DNA both none Uses of Klenow fragment Synthesis 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 of Klenow 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.

3. which one of the following acts as inducer of lac operon : 3. which one of the following acts as inducer of lac operon lactose allolactose permease ß galactosidase The tetrameric lac repressor protein, has four binding sites for allolactose. When those sites become 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 of permease to get the lactose into the cell and a low level of ß-galactosidase to convert the lactose to allolactose. Mutants that totally lack either the permease or ß-galactosidase cannot be induced by lactose.

4. Most base substitution in -10 to -35 base pair region causes : 4. Most base substitution in -10 to -35 base pair region causes increase in promoter function decrease promoter function does not affect promoter function 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 six nucleotides 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. Its presence allows a very high transcription rate.

5. Linkers are : 5. Linkers are Double stranded DNA with blunt ends Single stranded DNA with sticky ends Single stranded DNA with blunt ends Single stranded DNA with sticky ends Sticky end and Blunt end are the two possible configurations resulting from the breaking of double-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 genetic Engineering. They are used in cloning Experiments.


6. Leucine Zipper has : Leucine Zipper has Leu residue at every 7th position of a -helix Leu residue at every 4th position of a -helix Leu residue at every 7th position of ß-sheet Leu residue at every 4th position of ß-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 side chains 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.

7. Which one can be used to cut a methionine between two polypeptide : Which one can be used to cut a methionine between two polypeptide Factor Xa Thrombin Cyanogen bromide 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 the cyclization of iminolactone, which is hydrolysed in water resulting in peptide cleavage. Thrombin is a serine protease that possesses trypsin-like behavior in that it prefers to cleave its substrates after arginine residues. Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg. Chemical Reagents for Protein Modification By Roger L. Lundblad thioether

8. Thrombin cuts the polypeptide at : . Thrombin cuts the polypeptide at next to Gly next to Arg next to Met next to Lys The thrombin molecule contains two chains. 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 of prothrombin. 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 its substrates after arginine residues.


9. Primer extension is used for identification of : Primer extension is used for identification of 3' end 5' end both none A primer is a strand of nucleic acid that 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 3’end of the primer, and copies the opposite strand. In primer extension, a short antisense 5' end-labeled DNA primer (usually a synthetic oligonucleotide, 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.

10. S1 nuclease analysis can be used for identification of : . S1 nuclease analysis can be used for identification of 3' end 5' end both 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 analysis Preparations 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

11. Adapters has : 11. Adapters has one sticky end and one blunt end both sticky ends both blunt ends 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'

12. What is the ratio of absorbance of UV light by pure DNA at 260 nm and 280nm wavelength : 12. What is the ratio of absorbance of UV light by pure DNA at 260 nm and 280nm wavelength 1.8 4.8 10.2 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 wavelength is calculated. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or 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 the weighted 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: 1.15 Adenine: 4.50 Cytosine: 1.51 Uracil: 4.00 Thymine: 1.47 260/280 ratio

13. The most abundant enzyme in the biosphere : 13. The most abundant enzyme in the biosphere Amylase Rubisco DNA polymerase RNA polymerase Ribulose-1,5-bisphosphate carboxylase/oxygenase, most commonly known by the shorter name RuBisCO, 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 .

14. Method used for differentiating real genes from chance ORF's is . Method used for differentiating real genes from chance ORF's is CpG island codon bias Homology search and transcript analysis All of above An open reading frame (ORF) is a portion of an organism's genome which contains a sequence of bases that could potentially encode a protein. In a gene, ORFs are located between the start-code sequence (initiation codon) and the stop-code sequence (termination codon). However, short ORFs can also occur by chance outside of genes. Usually ORFs 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 or near the transcription start site of genes, particularly housekeeping genes, in vertebrates. DNA regions >500 bp with a 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 it can be confirmed a real gene. Transcript analysis: For many model organisms the EST’s and cDNA sequences are used.if the sequence of ORF matches the sequence of transcript. Then the ORF is a real gene.

15. Hybrid of trp and lac promoter is called : Hybrid of trp and lac promoter is called lrp lap tac trc Tac is a strong promoter and is the hybrid of the trp and lac promoters, containing -35 region of trp fused 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 of lacUV5.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.

16. Which one is not used in making genetic maps : 16. Which one is not used in making genetic maps RFLP STR SNP EST's Genetic maps depict relative positions of loci based on the degree of recombination. This approach studies the inheritance/assortment of traits by genetic analysis. Physical maps show the actual (physical) distance between loci (in nucleotides). This approach applies techniques of molecular biology. Eg: Restriction mapping, EST mapping A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci, that can be observed. Eg: RFLP, STR, SNP, microsatellites etc An Expressed Sequence Tag is a tiny portion of an entire gene that can be used to help identify unknown genes and to map their positions within a genome. ESTs are small pieces of DNA sequence (usually 200 to 500 nucleotides long) that are generated by sequencing either one or both ends of an expressed gene. The idea is to sequence bits of DNA that represent genes expressed in certain cells, tissues, or organs from different organisms and use these "tags" to fish a gene out of a portion of chromosomal DNA by matching base pairs.

17. which is true for homeodomain : which is true for homeodomain was discovered in homeotic genes has DNA binding segment related to helix-turn-helix motif is enclosed in homeobox All of above Homeotic genes are defined by a DNA sequence known as the homeobox, which is a sequence of 180 nucleotides 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. The homeodomain 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.

18. Plasmids are found in - . Plasmids are found in - bacteria yeast mouse 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 natural populations. 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 one open reading frame (ORF) coding for a DNA polymerase or a reverse transcriptase. Linear plasmids generally have two ORFs, coding for presumptive DNA and RNA polymerases with amino acid motifs showing homology to viral polymerases.

19. Which DNA binding protein motif is present in Lac repressor : Which DNA binding protein motif is present in Lac repressor helix-turn-helix zinc finger homeodomain leucine zipper The helix-turn-helix motif consists of about 20 amino acids. The basic form is that of 2 helices separated by a turn. 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 leucines facing 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 Factor IIIA-DNA complex from the clawed frog Xenopus laevis.

20. Which of the following gene is carried by "F" plasmid ? :. Which of the following gene is carried by "F" plasmid ? yrp yar tra trp 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.

Slide 25 : 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 strand Chromosomal 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.

21. In zinc finger Zn++ ion is co-ordinated to : . In zinc finger Zn++ ion is co-ordinated to 4 Cys 4 His 2 Cys and 2 His 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 four cysteine residues bind to a zinc ion and the last four cysteine residues bind to another zinc ion. Eg Estrogen receptor C6 zinc finger. It has the consensus sequence CX2CX6CX5-6CX2CX6C. The yeast's Gal4 contains such a motif where six cysteine residues interact with two zinc ions. Eg: GAL4

Slide 27 : In Fig 2&3 Green strands – Cysteine residue Orange Balls – Zn ion Red Strands - Receptor Fig 2 Fig 3

22. Which one is not used in making genetically modified crops : . Which one is not used in making genetically modified crops vector based on plant virus various plasmid DNA artificial plasmid of animals 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.

23. E.Coli cannot : E.Coli cannot degrade recombinant protein always fold recombinant protein correctly glycosylate recombinant protein correctly 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

24. Lac promoter can be induced by:- : Lac promoter can be induced by:- Iso propyl thio galactosidase iso methyl thio cyanate tryptophan 3-beta -indole acrylic acid Isopropyl-ß-D-thio-galactoside (IPTG) is frequently used as an inducer of the lac operon as it binds to repressor and inactivates it. Unlike allolactose, the sulfur (S) atom creates a 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 functional lacy and lacz products

26. pBIN 19 binary vector consist of : pBIN 19 binary vector consist of lac z' gene kanamycin resistance gene cloning sites all of above

27. In lac operon the repressor is encoded by : . In lac operon the repressor is encoded by Lac A Lac Y Lac Z Lac I The operon is under the control of the adjacent lacI gene, encoding the lactose repressor. The repressor 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 (residues 1-62), 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.

28. Drawback of using baculovirus in protein production : Drawback of using baculovirus in protein production larger nuclear inclusion bodies makes large amount of protein improper glycosylation polyhedrin gene product Advantages of using baculovirus expression system are Eukaryotic 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 non essential when the virus Is propagated.In its absence BV is secreted by the infected cells. This dispensible nature of polyhedrin gene makes BV suitable for constructing vectors. The desired gene is placed under the control of polyhedrin promoter.

29. A yeast vector carrying A 2 ?m circlular origin of Replication : . A yeast vector carrying A 2 ?m circlular origin of Replication YAC YEp YIp YRp The YpI integrative vectors do not replicate autonomously, but integrate into the genome at low frequencies by homologous 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 2 mm 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 to 3000 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.

30. Factor Xa cuts at . Factor Xa cuts at After Arg of Gly-Arg Before Arg of Gly-Arg Before Gly of Gly0 Leu After Gly of Gly-Leu Factor Xa is a serine endopeptidase composed of two disulfide-linked subunits that converts prothrombin to thrombin in 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.

31. A.tumefaciens causes . A.tumefaciens causes rust wort crown gall disease hairy root disease Agrobacterium tumefaciens causes crown gall disease of a wide range of dicotyledonous (broad-leaved) .The disease 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 galls on 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 rusts attack 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. rhizogenes contains 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

33. Which is not a method of DNA insertion in living cells : . Which is not a method of DNA insertion in living cells Transformation Transfection Microinjection none Transformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of 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 animal cells 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 or macroscopic 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.

34. Use of regulatory protein to protect DNA from endonuclease is done in . Use of regulatory protein to protect DNA from endonuclease is done in DNA footprinting DNA fingerprinting gel retardation chemical degradation DNA Footprinting was developed in 1977 and is an analytical procedure in molecular biology for identifying the specific sequence 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 sometimes called 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 a small 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.


35. Advantage of insulin production by recombinant DNA technique : . Advantage of insulin production by recombinant DNA technique can be modified by addition of sugar molecules after translation insulin is a big protein active insulin is synthesized by bacteria 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 has proven 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.



36. trp promoter is repressed by . trp promoter is repressed by leucine lactose galactose 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.



37. T-DNA is not T-DNA is not between 15-30 Kb in size passed to daughter cells responsible for cancerous properties 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 of auxins and cytokynines. These plant hormones cause the tumourous phenotype that is characterised by the ability of the plant cells to proliferate limitlessly and autonomously, even in the absence of added phytohormones. Crown gall tumours are characterised by the production of opines (derivatives of amino-acids). The biosynthesis of opines 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.

38. which one will affect efficiency of expression study of foreign gene in Ecoli . which one will affect efficiency of expression study of foreign gene in Ecoli presence of introns Sequence acting as termination signals in E.Coli codon bias All of above Introns: The foreign gene might contain introns.Since the E.coli does not have introns the host would not have proper machinery to remove introns from transcripts. The foreign gene might also contain sequences that act as termination signals in E.coli.These sequences might be completely 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 abundant tRNAs and the genes expressed at low levels use codon for less abundant tRNA. Equalization of codon-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 translation efficiency. 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.

39. Promoter used for control of cloned genes in saccharomyces cerevisiae Promoter used for control of cloned genes in saccharomyces cerevisiae gal pho5 cup1 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.

40. Repression of lac operon is not caused due to Repression of lac operon is not caused due to absence of lactose mutation in operator functional I gene on it 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. which one is not encoded by structural gene of lac operon : . which one is not encoded by structural gene of lac operon ß-galactosidase galactosidase acetylase galactosidase permease thio-galactosidase transacetylase lacZ encodes ß-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. lacY encodes ß-galactoside permease, a membrane-bound transport protein that pumps lactose into the cell. lacA encodes ß-galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl-CoA to ß-galactosides

42. Which of the phage never cause cell lysis? . Which of the phage never cause cell lysis? M13 Lambda F-X174 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 F-X174 encodes a single lysis protein, E, a 91 amino acid membrane protein,which is a specific inhibitor of Mra Y gene involved in peptidoglycan synthesis. Filamentous hages like M13 and Ff are not lytic.Some of these phages become prophages by integrating into the genome,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. Examples of regulon are . Examples of regulon are SOS response to DNA damage heat shock gene system sugar metabolism system 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, after Save Our Souls. Heat shock regulons contain a group of heat shock proteins (stress proteins). They are induced when a cell undergoes various 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. In HSP which s subunit mediates RNA polymerase binding to promoter : . In HSP which s subunit mediates RNA polymerase binding to promoter s70 s32 s54 s90 Different ss have different affinities for variant promoters. * s70 - general purpose. * s54 - nitrogen metabolism. * s32 - heat shock proteins. * sS - stationary phase. * sF - flagella.

45. What is the function of Alkaline phosphatase and polynucleotide kinase : . What is the function of Alkaline phosphatase and polynucleotide kinase Removes phosphate group from 5' end and add at 5' end respectively Removes phosphate from 3' end and adds at 3' end respectively Adds phosphate at 5' end and removes from 5' end respectively 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 of a phosphate from ATP to the 5' end of either DNA or RNA.

46. Similarity between chain termination and chemical degradation method of sequencing : . Similarity between chain termination and chemical degradation method of sequencing use double stranded DNA Need Primer Degrades the DNA 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)



47. which will negatively effect lac operon expression : . which will negatively effect lac operon expression lactose less glucose concentration reased binding of cAMP and CRP 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.


48. s subunit is a type of . s subunit is a type of specificity factor repressor activator none Bacterial RNA polymerases are best characterised in Thermus aquaticus. The five subunits are: * aI aII - required for DNA binding and assembly. * ß ß' - needed for DNA binding (contains basic amino acid residues) and catalysis. * ? - stabilises ß' binding. * s - forms holoenzyme. s 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 based on Vector used in first cloning experiment in mammalian cell was based on SV 40 BPV adenovirus 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) was used 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 about 100 copies per cell. BPV molecules are passed on to daughter cells.They do not cause death in mouse.

50. Full form of HART is : Full form of HART is Hybridization and retranslation Hybrid arrest translation Hybridization and retsnscription High affinity restriction technique

51. HRT and HART depend on . HRT and HART depend on bacterial cell translation eukaryptic cell translation both prokaryot and eukaryotic cell translation 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.



52. Shotgun cloning is appropriate for Shotgun cloning is appropriate for prokaryotes eukaryotes both 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