After you have successfully cloned Sonic Hedgehog into pSELECT-CCFP-blasti and transfected HEK 293 cells (human embryogenic kidney cells), you observed, using confocal microscopy that the GFP fluorescence displays a reticulate network pattern in the cytoplasm of the cells. Describe an approach you can use to determine the subcellular localization of the Sonic Hedgehog-GFP fusion protein. Nco I (200)Bam HI (450)Sal I (650) Eco47 III (200)ATG (1)TGA (1000)SHFW1SHRV1Figure 1. Schematic representation of Sonic Hedgehog gene showing the size of the full-lengthcoding sequence (1000 bp) and the positions restriction endonuclease recognition sites.NotI, (-1)Sefl (171)Pacl (1906)HindIII (410)Agel (717)Sall (721)BspLU11I (3176)yPaci (3160)PMB1 oriEcod7III (727)PstI (3159)-MEFLHTLV promBamHI (733)Sdal (3158)--Neol (749)MCSPSELECT-CGFP-blasti-AccósI (971)ACMV erbiprom(3925 bp)GFP-CtagSpel (2747)-EM7Sv40 panAsel (2592)-BGlo panВзрHI (2534).Nhel (1472)EcoRI (1706)Figure 2. Schematic representation of the PSELECT-CGFP-blasti mammalian GFP=tagexpression vector conferring Slasticidin S resistance for selection in both bacterial andmammalian cells . Before starting your PCR and cloning, Dr. Strangeclone provided you with a very importantpiece of information below in relation to the cloning strategy using pSELECT-CGFP-blasti.CLONING STRATEGYFor expression of a tagged protein, it is important to ensure to clone yourgene-of-interest into the correct reading frame. In general it isrecommended to use Age I/Bam HI restriction site combination forcloning into plasmids with the C-Tag. For the plasmids with C-Tag, checkwhether the first triplet of your gene of interest and the start codon behindthe C-Tag are in the correct reading frame.MCSAgeIACCGGT..Bam HI C-Tag.GGA TCC' ..TAA AGCTAGCNhe IStopNote: The Bam HI restriction site is compatible with Bgl II.If it is not possible to use the Age I restriction site, it is possible to anotherrestriction site such as Sal I, upstream of Bam HI.Age I'ACCGGT!..MCS Sal IBam HI C-TagNhe I.GTCGAC'AGCGCT GGA TCC...TA A GCTAGCStopIf it is not possible to use the Bam HI restriction site, it is possible to anotherrestriction site such as Nco I upstream of the start codon of your gene of interest.Age I MCS Bam HIACCGGT . GGATCC' GGT GGAGGTG'CCATGG .TAA AGCTAGCNeo IC-TagNhe IStopAlternatively, blunt end cloning can be achieved using the Eco 47 III site,which is upstream of the Bam HI site. Blunt end PCR fragments can beobtained using T4 DNA polymerase or Klenow.Age IMCS Eco47 III Bam HIC-TagNhe IACCGGT!.AGC GCT'GGA TCC'.TAA A GCTAGCStopAs a first step, you have successfully designed a pair of PCR primers, SH1FW1 and SH1RV1 (seeFigure 1), under the guidance of Dr. Strangeclone for amplification of the full-length (1000 bp)coding sequence of Sonic Hedgehog.

Answered: Image /qna-images/answer/772326b5-893d-4f89-8879-6493a400f0a5.jpg

After you have successfully cloned Sonic Hedgehog into pSELECT-CCFP-blasti and transfected HEK 293 cells (human embryogenic kidney cells), you observed, using confocal microscopy that the GFP fluorescence displays a reticulate network pattern in the cytoplasm of the cells. Describe an approach you can use to determine the subcellular localization of the Sonic Hedgehog-GFP fusion protein.

After you have successfully cloned Sonic Hedgehog into pSELECT-CCFP-blasti and transfected HEK 293 cells (human embryogenic kidney cells), you observed, using confocal microscopy that the GFP fluorescence displays a reticulate network pattern in the cytoplasm of the cells. Describe an approach you can use to determine the subcellular localization of the Sonic Hedgehog-GFP fusion protein.

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter30: Protein Synthesis

Section: Chapter Questions

Problem 1P

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70Pre-initiation begins with either TFIIF or TATA binding protein binding to the core promoter. Yesorno 71Exons tend to be non-conserved during evolution, whereas introns usually are conserved. Yesorno 72Transfer RNA molecules fold into L-shaped three-dimensional structures. Yesorno
Multiple Matching. Fill in the blanks with the words below that apply.18. ________ site of protein synthesis19. ________ carries the codon20. ________ carries the anticodon21. ________ a process synonymous with mRNA synthesis22. ________ bacteriophages participate in this transfer23. ________ duplication of the DNA molecule24. ________ process in which transcribed DNA code is deciphered into a polypeptide25. ________ involves plasmidsreplicationtRNAconjugationribosometransductionmRNAtranscriptiontransformationtranslationnone of these PreviousNext
24. A terminator loop is… Group of answer choices a. A tertiary structure in DNA that causes DNA polymerase to detach from the template it is reading. b. A terrible situation caused by Skynet, and only Arnold Schwarzenegger can save us from it. c. A poorly structured region within a protein that is difficult to synthesize and often causes translation to fail spontaneously. d. A tertiary structure in the sgRNA that allows CRISPR-Cas9 to disengage from its substrate after catalysis. e. None of these answers are correct.
88Sequential binding of RNA polymerase II-TFIIF complex, TFIIE, and TFIIH completes ___________________ formation. A.pre-initiation complexb.TF recognition elementc.pre-elongation complexD.TATA binding complex 89Which of the following is the GDP-GTP exchange protein?A.EF-Tub.none of the abovec.EF-TsD.EF-G 90RNA polymerase II has 14 subunits. Yesorno
True or False: 1. The 6X HIS tag is required for a eukaryotic protein to be expressed in E. coli 2. BAC vectors are an appropriate choice for cloning cDNAs 3. Cluster analysis of micro-array data groups together mRNAs of similar sequence 4. In genomic DNA, on average EcoR1 restriction sites are more common than Mse1 restriction sites 5. Tags such as HA that are fused to proteins always compromise their function.
Brenner’s m mutant phages (m1–m6) described inFig. 8.8 were suppressed when grown in suppressor(su−) mutant bacteria; they produced full-length Mproteins that functioned like wild-type M protein.a. What gene do you think was mutant in the su−bacteria?b. When the m− phages were propagated in the su−bacterial strain, not all of the proteins made by themutant m alleles were identical to wild-type Mprotein. How did some of them differ?
QNo.3 Why transgene copy number is a key concern for transgenic studies? Describe an efficient method for the estimation of copy number.(Genetic engineering).
Task #1 Mc1r alleles: In Florida Gulf coast mice, there are two different alleles of the Melanocortin Receptor (Mcr1) gene. The sequence for both alleles is shown below. This is an internal segment of the coding (non-template) sequence of the Mcr1 gene. The reading frame is set and you do not need to find an AUG. M allele 5’...ATC ACC AAA AAC CGC AAC CTG CAC TCG... m allele 5’...ATC ACC AAA AAC TGC AAC CTG CAC TCG... A. Compare these two allele sequences and circle the nucleotides that are different. B. Do you think this change will cause a shift in the codon reading frame? Why or why not? C. Do you think that this change will cause an early stop in translation? Why or why not? Task #2 Flow of information: A codon table is provided above. The 5’ codon nucleotide is in the left column, and second codon nucleotide is on top. The Mcr1 M and m allele sequences are shown again in the central dogma grids below with the reading frame designated. Fill in the grids. In the second column…
NEED ASAP Using the list provided (note you may not need all the steps listed) state the steps in cDNA production 1. Confirm size of bands by comparing to molecular size marker. 2. Ligate oligonucleotides onto blunt ended product. 3. Cut the cDNA with a unique restriction enzyme. 4. Ligate the cDNA and vector followed by transformation of E. coli. 5. Plate cells onto LB-ampicillin plates containing X-gal. 6. Choose colonies that are white in colour. 7. Cut plasmid with unique restriction enzymes. 8. The size of the clone is small and it will ensure that the entire gene will be found in one fragment. 9. Employ replica plating technique and select cells that are ampicillin-sensitive and tetracycline resistant. 10. Blot petri plates with a nitrocellulose filter, lyse the cells and denature the DNA with a dilute sodium hydroxide solution. 11. Blot electrophoresis gel with nitrocellulose filter and denature the DNA with a dilute sodium hydroxide solution. 12. Flood the filters with…
11) Draw a bacterial expression vector with all require vector sequences. Be sure to label any specific promoter elements.
Q 1. Download and read the attached review article https://www.ncbi.nlm.nih.gov/pmc/articles/PMC342244/pdf/nar00470-0155.pdf titled: MspI, an isoschizomer of HpalI which cleaves both unmethylated and methylated Hpall sites and answer the following question: Restriction enzymes have many applications from gene cloning to gene activity and mapping. Use the two above mentioned restriction enzymes to study transcriptional silencing (consider Southern blot method for your example)
35.Draw a bacterial gene (dsDNA and mRNA) showing the promoter (-35 and -10 consensus sense strand sequences), the TC start site (TSS), start of TL, open reading frame (ORF), end of TL, TC termination site (TTS), 5’-UTR, and 3’-UTR. Some of these landmarks are on the dsDNA, some on the mRNA. The TSS and TTS can be drawn on both. Indicate 5’ and 3’ ends on dsDNA and mRNA. You may need to make a messy draft of this drawing as you are working through the details. Draw the TTS as it would fold after it has been transcribed and show basepairing between the mRNA and the template strand that is important for TC termination. Explain how this structure stops transcription.