MCB 720 Exam II Answer Key – 2010‐ Key Points 1. Two different strategies ‐reccognize the amino acid sequence corresponds to leptin by using a BLAST search. Thus, you will be able to use a homologous sequence (e.g., gorilla or human leptin cDNA), labelled by the random primer method to screen your cDNA library. Note that since leptin is produced by adipocytes, and secreted into the blood, you will need to use adipocytes for isolation of mRNA in producing your cDNA library. ‐design an oligonucleotide probe corresponding to the amino acid sequence with the least degeneracy. Show the sequence and indicate it will be end‐labeled for use as a probe to screen the library. cDNA library production ‐isolate mRNA from elephant adipocytes using oligo dT cellulose chromatography ‐mention each step of the process including the enzymes involved with each step ‐indicate how the cDNA will be placed in the cloning vector and how the vector will be put in host Verification methods ‐Northern blotting‐ use your selected cDNA as a probe to examine RNA isolated from adipocytes. Does a hybridizing band of ~980nt appear (16,000Da*1AA/100Da*3nt/AA=480 +500 (for 5’&3’UT) ‐DNA sequencing‐sequence your selected cDNA; determine whether it matches the AA sequence ‐Hybrid select translation‐use your selected cDNA to isolate an mRNA that is translated in the presence of 35S‐Met. Does it produce a 16kDa protein band on an SDS‐PAGE gel? 2. Sequencing, assembly, and annotation ‐use massively parallel sequencing which involves attaching digested genomic DNA onto individual beads for pyrosequencing (i.e., 454 sequencing) ‐assembly is facilitated by the use of computer software which recognizes overlaps in the sequenced genomic DNA fragments ‐annotation is facilitated the use of computer algorithms to recognize putative genes and by database searches against know genomes and published literature on homologous sequences in order to arrive at tentative identifications for as many genes as possible Microarray experiment ‐first need to consider which organ(s) you wish to monitor; than isolate mRNAs from this organ(s) from elephants on a high fat diet and those on a normal diet. ‐create an elephant gene microarray chip or slide using gene fragments or oligonucleotides corresponding to the various elephant genes identified by your elephant genome sequencing project ‐produce first strand cDNA from the high fat mRNAs using a red fluorescently labelled nucleotide and produce first strand cDNA from the normal mRNAs using a green fluorescently labelled nucleotide. Hybridize these two mRNAs in equimolar amounts to the microarray. ‐wash the microarray and scan with a laser to identify genes induced (red) or repressed (green) by the high fat diet. Repeat experiment 2 more times to insure accuracy.
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MCB 720 Exam II Answer Key – 2010‐ Key Points
1. Two different strategies ‐reccognize the amino acid sequence corresponds to leptin by using a BLAST search. Thus, you will be able to use a homologous sequence (e.g., gorilla or human leptin cDNA), labelled by the random primer method to screen your cDNA library. Note that since leptin is produced by adipocytes, and secreted into the blood, you will need to use adipocytes for isolation of mRNA in producing your cDNA library. ‐design an oligonucleotide probe corresponding to the amino acid sequence with the least degeneracy. Show the sequence and indicate it will be end‐labeled for use as a probe to screen the library. cDNA library production ‐isolate mRNA from elephant adipocytes using oligo dT cellulose chromatography ‐mention each step of the process including the enzymes involved with each step ‐indicate how the cDNA will be placed in the cloning vector and how the vector will be put in host Verification methods ‐Northern blotting‐ use your selected cDNA as a probe to examine RNA isolated from adipocytes. Does a hybridizing band of ~980nt appear (16,000Da*1AA/100Da*3nt/AA=480 +500 (for 5’&3’UT) ‐DNA sequencing‐sequence your selected cDNA; determine whether it matches the AA sequence ‐Hybrid select translation‐use your selected cDNA to isolate an mRNA that is translated in the presence of 35S‐Met. Does it produce a 16kDa protein band on an SDS‐PAGE gel? 2. Sequencing, assembly, and annotation ‐use massively parallel sequencing which involves attaching digested genomic DNA onto individual beads for pyrosequencing (i.e., 454 sequencing) ‐assembly is facilitated by the use of computer software which recognizes overlaps in the sequenced genomic DNA fragments ‐annotation is facilitated the use of computer algorithms to recognize putative genes and by database searches against know genomes and published literature on homologous sequences in order to arrive at tentative identifications for as many genes as possible Microarray experiment ‐first need to consider which organ(s) you wish to monitor; than isolate mRNAs from this organ(s) from elephants on a high fat diet and those on a normal diet. ‐create an elephant gene microarray chip or slide using gene fragments or oligonucleotides corresponding to the various elephant genes identified by your elephant genome sequencing project ‐produce first strand cDNA from the high fat mRNAs using a red fluorescently labelled nucleotide and produce first strand cDNA from the normal mRNAs using a green fluorescently labelled nucleotide. Hybridize these two mRNAs in equimolar amounts to the microarray. ‐wash the microarray and scan with a laser to identify genes induced (red) or repressed (green) by the high fat diet. Repeat experiment 2 more times to insure accuracy.
3. Construction and screening of a rose genomic library ‐ Isolate genomic DNA from essentially any rose organ (e.g., leaves, stems, roots), do a partial digest of this DNA with Sau3A, and ligate it into the left and right arms of lambda phage following digestion with Bam HI. Then do a packaging reaction and transfect E. coli cells and spread on a petri dish. Library screening involves hybridization of nitrocellulose plaque lifts to your RBC cDNA probe produced by random primer labeling or nick‐translation using an �‐32P‐dNTP.
Restriction enzyme map
S S
B B
E B S E B E S B S E
4. Reverse Transcription PCR: Reverse Transcription PCR involves PCR amplification of the cDNA synthesized from mRNA by the Reverse Transcriptase enzyme and appropriate primer sequences. So, in addition to the normal steps of PCR, reverse transcription PCR involves one extra step of reverse transcription of the mRNA to DNA before amplification. This is an important step as the Taq polymerase cannot use the mRNA as a template. This step utilizes 37 ̊C temperature condition for the reverse transcriptase enzyme works. Once the cDNA is formed, the PCR proceeds as a normal reaction using primers. The amplified DNA is then measured after the reaction using an Agarose‐gel electrophoresis. RT‐PCR tells us whether our RNA of interest is being produced or not. It does not tell us of the relative abundance, but using gels of standard samples, semi‐quantitative results can be obtained. qRT‐PCR(quantitative Real‐time PCR): This technique involves quantitatively measuring the amount of DNA in real time after each cycle of the PCR. The principle difference between this method and other PCR methods is that this method measures amount of DNA formed after each cycle of PCR rather than at the end. The binding of the fluorescent dye/DNA probe to the DNA product gives a fluorescent reading corresponding to the amount of target DNA present in the sample. This principle is used to determine the level of product. The RNA sample is added, along with the enzyme reverse transcriptase, to convert the RNA to cDNA. Reagents then added are the fluorescent DNA probes or DNA binding dyes. PCR is then started. The DNA bound fractions of the probes/dyes fluoresce when incident with a characteristic wavelength which is detected by the instrument after each cycle. The time required for a particular sample to reach a threshold fluorescence with respect to the background is recorded. This method of PCR gives information about the relative expression levels of mRNAs. So, the experimenter can get the relative mRNA copy number for the gene. This enables the experimenter to compare the expression levels of genes. Comparison of RT‐PCR, qRT‐PCR & Northern blot for RNA analysis: RT‐PCR & qRT‐PCR require very minute amounts of RNA sample as compared to Northern blot. They are faster methods than Northern blot. Also, they are much more sensitive than northern blot. qRT‐PCR can give relative expression levels, but the others cannot. So, it is useful to study gene expression. The flip side is qRT‐PCR does not tell anything about the size of the RNA, which can be obtained from Northern Blotting. Note that RT‐PCR does not tell us anything about the mRNA size either. One big advantage of northern blotting is that it can so the size of the transcript and it can reveal if more than one transcript of different sizes are present. One advantage about qRT‐PCR is that it can be used to study si‐RNA. One disadvantage is that the thermocycler for qRT‐PCR is specialized and quite expensive.
c. Full length mRNA or cDNA sequence of the gene: >gi|145359772|ref|NM_126123.4| Arabidopsis thaliana glycosyl transferase family 43 protein (AT5G67230) mRNA, complete cds GTTGAGAAGAAGGAAGAAGAAGAAGACGATGAAGCTCTCTGTGTTTCGATTGAGCTATTGGAACCGTCGA GGAAGTAGTTTCAGATCATCGCCGTCGTTGGATCCATCATTCGATGGCAAATCTCCGTCGTCTGTGTTTT GGTTCGTGATTCATGGTCTCTGCTGCTTGATCAGCTTGATTCTAGGGTTCCGATTCAGCCATTTAGTACT CTTCTTCCTTTTCTCGACTTCCGTCACCAATCTATACACAACGCCATTTCTCTTTGCCGGAAACGGCGGT GTAAGCCAGCTTCTCCGGCTAAAACCTCTGGAAACAGCGACTAACAGCACGGTGAAGAAGAACTCTCGAG TGGTGGTTGGAAGACACGGGATCCGGATCCGTCCATGGCCTCACCCGAATCCGATTGAGGTATTGAGAGC TCATCAGTTGCTTGTGAGAGTACAGAAAGAGCAGAAATCGATGTACGGTGTGAGGAGCCCTAGGACTGTG ATTGTGGTGACGCCGACTTATGTACGGACTTTTCAGGCGCTTCATTTGACCGGAGTTATGCACTCGCTTA TGCTTGTTCCGTACGATTTGGTTTGGATCGTTGTGGAAGCTGGTGGAATCACTAACGAGACTGCTTCGTT TATCGCAAAATCAGGATTAAAGACGATTCACTTAGGATTCGATCAGAAAATGCCTAATACATGGGAAGAT CGTCACAAATTGGAGACCAAAATGAGACTTCACGCCTTGAGAGTTGTGAGAGAGAAGAAGTTAGATGGGA TTGTTATGTTTGCTGATGATAGCAATATGCATAGTATGGAGCTTTTTGATGAGATTCAAACTGTGAAATG GTTTGGTGCTCTATCTGTTGGTATACTTGCTCATTCTGGTAATGCAGATGAATTATCATCGATCTTGAAG AATGAACAAGGGAAGAACAAAGAGAAACCTTCAATGCCAATCCAAGGTCCTAGTTGTAATTCCTCTGAGA AATTAGTGGGTTGGCACATTTTCAACACACAGCCTTATGCCAAGAAGACTGCAGTGTATATCGATGAGAA AGCGCCTGTGATGCCTAGTAAGATGGAATGGTCAGGGTTTGTGTTGAATTCTAGATTGCTCTGGAAGGAA TCTTTAGATGATAAACCAGCATGGGTTAAAGATCTCAGCTTGTTGGATGATGGTTATGCGGAAATTGAGA GTCCTTTGTCTTTGGTGAAGGATCCTTCCATGGTGGAGCCACTTGGAAGCTGTGGCCGTCGTGTCTTGCT TTGGTGGCTTCGAGTTGAAGCTCGAGCTGATAGCAAATTCCCACCTGGCTGGATCATAAAGTCACCTTTA GAAATCACAGTGCCATCAAAGCGGACACCCTGGCCAGACTCTTCCTCAGAGCTCCCAGCGGCGGCGATCA AAGAGGCAAAAAGCAACTCTAAGCCAAGAGTGTCGAAGAGCAAGAGCTATAAGGAGAAACAAGAACCTAA AGCTTTCGATGGTGTCAAAGTGTCAGCAACTAGCTGAAGAAGCTTCTATAGATAGATCGAGTTTCATTTC ATATTCTTTTTCACCGTAAATATCGGAAAATTGTTTTGTGTGCATTAGCTTCATTGTATACAGTACAAAA ATCATATGAGATGTTTTAAGTTCTTCAAATCGATACTTAGCTCT
d. Protein sequence: >gi|15240245|ref|NP_201524.1| glycosyl transferase family 43 protein [Arabidopsis thaliana] MKLSVFRLSYWNRRGSSFRSSPSLDPSFDGKSPSSVFWFVIHGLCCLISLILGFRFSHLVLFFLFSTSVT NLYTTPFLFAGNGGVSQLLRLKPLETATNSTVKKNSRVVVGRHGIRIRPWPHPNPIEVLRAHQLLVRVQK EQKSMYGVRSPRTVIVVTPTYVRTFQALHLTGVMHSLMLVPYDLVWIVVEAGGITNETASFIAKSGLKTI HLGFDQKMPNTWEDRHKLETKMRLHALRVVREKKLDGIVMFADDSNMHSMELFDEIQTVKWFGALSVGIL AHSGNADELSSILKNEQGKNKEKPSMPIQGPSCNSSEKLVGWHIFNTQPYAKKTAVYIDEKAPVMPSKME WSGFVLNSRLLWKESLDDKPAWVKDLSLLDDGYAEIESPLSLVKDPSMVEPLGSCGRRVLLWWLRVEARA DSKFPPGWIIKSPLEITVPSKRTPWPDSSSELPAAAIKEAKSNSKPRVSKSKSYKEKQEPKAFDGVKVSA TS
e. Predicted molecular weight of the protein: The calculated molecular weight of the protein was found to be— 55348.7 f. pI The calculated pI for the protein was found to be 10.2906 g. Protein targeting sequences and likely cellular destinations:
Using neural networks (NN) and hidden Markov models (HMM) trained on eukaryotes >gi_15240245_ref_NP_201524.1_ glycosyl transferase family 43 protein _Arabidopsis thaliana_ SignalP-NN result:
# data
>gi_15240245_ref_NP_2 length = 70 # Measure Position Value Cutoff signal peptide? max. C 18 0.213 0.32 NO max. Y 54 0.211 0.33 NO max. S 44 0.674 0.87 NO mean S 1-53 0.239 0.48 NO D 1-53 0.225 0.43 NO SignalP-HMM result:
# data
>gi_15240245_ref_NP_201524.1_ Prediction: Signal anchor Signal peptide probability: 0.002 Signal anchor probability: 0.997 Max cleavage site probability: 0.002 between pos. 53 and 54 In the results of the peptide signal searching software SignalIP 3.0, the signal NN prediction graph found some C sites, but the S score for these sites did not correlate well to be selected as a cleavage site. So, the software predicts that there is no signal sequence in the protein. Also, in the SignalP-HMM prediction plot,the probability of the C-region is very low. The software also predicts that this protein is an anchored protein in the cell membrane.
no data no data no data iPSORT : mitochondrionLOCtree : no data MitoPred : mitochondrionMitoprot 2 : no data MultiLoc : mitochondrionPeroxP : no data Predotar : mitochondrionSubLoc : mitochondrion TargetP : mitochondrion WoLFPSORT : plastid
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Description (TAIR8) protein_coding glycosyl transferase family 43 protein similar to IRX14 (IRREGULAR XYLEM 14), transferase, transferring glycosyl groups / xylosyltransferase [Arabidopsis thaliana] (TAIR:AT4G36890.1); similar to unnamed protein product [Vitis vinifera] (GB:CAO71907.1); contains InterPro domain Glycosyl transferase, family 43; (InterPro:IPR005027)
1.http://jsp.weigelworld.org/expviz/expviz.jsp?experiment=development&normalization=absolute&probesetcsv=At5g67230&action=Run, 02/20/2010 i. 5 most similar DNA sequences related to the gene:
Accession Description
NM_126123.4 Arabidopsis thaliana glycosyl transferase family 43 protein (AT5G67230) mRNA, complete cds
>gi|42454836|emb|BX829393.1| Arabidopsis thaliana Full-length cDNA Complete sequence from clone GSLTFB10ZF05 of Flowers and buds of strain col-0 of Arabidopsis thaliana (thale cress) GACGATGAAGCTCTCTGTGTTTCGATTGAGCTATTGGAACCGTCGAGGAAGTAGTTTCAGATCATCGCCG CTCGTTGGATCCATCATTCGATGGCAAATCTCCGTCGTCTGTGTTTTGGTTCGTGATTCATGGTCTCTGC TGCTTGATCAGCTTGATTCTAGGGTTCCGATTCAGCCATTTAGTACTCTTCTTCCTTTTCTCGACTTCCG
Name of Mutant Location/Insertion position T-DNA.GK-322A11-015946 26839848T-DNA.SAIL_598_F01.v1 26839724T-DNA_LB.T-DNA.SAIL_598_D01.v1 26839724T-DNA.SALK_066961.16.30.x 26840629
6. 5 ́‐ACCCAAGGGTGCGCGCGTGGCTCCTGGCGCGCCGAGGCCCT‐3 ́ A primer complementary to this DNA sequence is used during the sequencing procedure. This means that the sequence which we get at the end of the experiment will be the sequence of the complementary strand to the above strand. 5 ́‐ACCCAAGGGTGCGCGCGTGGCTCCTGGCGCGCCGAGGCCCT‐3 ́‐‐‐DNA sequence 3 ‐TGGGTTCCCA CGCGCGCACCGAGGACCGCGCGGCTCCGGGA‐ 5́‐‐‐complementary sequence formed in 5 ́ 3 ́ direction. By automated dideoxy sequencing method:
5 ‐A G G G C C T C G G C G C G C C A G G A G C C A C G C G C G C A C C C T T G G G T‐ 3 ́
By pyro‐sequencing :
G C T A G C T A G C T A G C T A G C T A G C T A G C T A G C T A G C T A G C T A G C T A G C T A G C T A etc.
A BLAST search with this sequence reveal that is corresponds to the promoter region of the human leptin (ob) gene.
7. Transgenic production of human growth hormone (hGH): Gene construction In tobacco and goats ‐for tobacco, use a leave‐specific promoter like the RBC promoter to drive expression of the hGH cDNA (do not use the 35S CaMV promoter which is expressed everywhere in the plant) ‐for goats, use a mammary gland‐specific promoter like the beta‐lactoglobulin promoter to drive expression of the hGH cDNA Of course you will need a 5’UT, signal sequence, and 3’UT containing the poly A tail addition sequence, but the hGH cDNA sequence should provide these sequences, even in plants Introduction of the transgene construct into tobacco and goats ‐ explain the binary Ti plasmid system, including transformation of E. coli and subsequently transformation of Agrobacterium which already contains the disarmed Ti plasmid ‐explain the Agrobacterium infection process and the tissue culturing of transformed plant tissue to regenerate tobacco plants ‐for goats, need to obtain fertilized egg cells and then use microinjection to deliver your gene construct into one of the two pronuclei in the 1 cell embryo. Subsequently, implant several microinjected embryos into surrogate goat mothers. The use of embryonic stem cells (ESC) is unnecessary and more complicated. Testing for the transgenic DNA, RNA and protein ‐To test for the transgenic DNA, isolate DNA from any tobacco or goat organ/tissue and run a PCR using primers corresponding to the transgene. Alternatively, do a Southern blot with this DNA using the transgene as a probe. ‐To test for transgenic RNA, isolate RNA from tobacco leaves or goat mammary glands and conduct RT‐PCR using primers corresponding to the transcribed transgene. Alternatively, do northern blotting with this RNA using the transgene as a probe. ‐To test for transgenic protein, you would need to have an antibody against hGH. If so, you can isolate protein from tobacco leaves or goat milk and perform an ELISA or western blot using this antibody as your primary antibody and an appropriate secondary antibody conjugated to an enzyme like alkaline phosphatise or horseradish peroxidise. Expression of the transgene Expression will be variable in different transgenic tobacco plants as well as in different transgenic goats. The main reasons for variable expression (of RNA and protein) is because of the random, variable location of the site of insertion (i.e., “position effect”) and because of the variable number of transgene constructs copies inserted (i.e., “copy number”).