Concept explainers
Chapter 21 describes a technique known as Northern blotting that is used to detect RNA transcribed from a particular gene. In this method, a specific RNA is detected using a short segment of cloned DNA as a probe. The DNA probe, which is labeled, is complementary to the RNA that the researcher wishes to detect. After the probe DNA binds to the RNA, the RNA is run on a gel and then visualized as a labeled (dark) band. As shown here, the method of Northern blotting can be used to determine the amount of a particular RNA transcribed in a given cell type. If one type of cell produces twice as much of a particular mRNA as another type of cell does, the band will appear twice as intense. Also, the method can distinguish whether alternative RNA splicing has occurred to produce an RNA that has a different molecular mass.
Lane 1 is a sample of RNA isolated from nerve cells.
Lane 2 is a sample of RNA isolated from kidney cells. Nerve cells produce twice as much of this RNA as do kidney cells.
Lane 3 is a sample of RNA isolated from spleen cells. Spleen cells produce an alternatively spliced version of this RNA that is about 200 nucleotides longer than the RNA produced in nerve and kidney cells.
Let’s suppose a researcher is interested in the effects of mutations on the expression of a particular protein-encoding gene in eukaryotes. The gene has one intron that is 450 nucleotides long. After this intron is removed from the pre-mRNA, the mRNA transcript is 1100 nucleotides in length. Diploid somatic cells have two copies of this gene. Make a drawing that shows the expected results of a Northern blot using mRNA from the cytosol of somatic cells, which were obtained from the following individuals:
Lane 1: A normal individual
Lane 2: A homozygote for a deletion that removes the
Lane 3: A heterozygote in which one gene is normal and the other gene has a deletion that removes the
Lane 4: A homozygote for a mutation that introduces an early stop codon into the middle of the coding sequence of the gene Lane
5: A homozygote for a two-
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Genetics: Analysis and Principles
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- The following gene sequence of nucleotides is found on the template (non-coding) strand of a molecule of DNA from a bacterial cell. The promoter of the gene is highlighted in bold letters and the +1 is underlined. Use the genetic code at the end of this packet to answer the following questions. 3'-AGGCATATTACGATGCCGGTACTTGATGATGACGGACCCATTATAGGACATATG-5' a) What is the sequence of the mRNA strand that will be transcribed from this piece of DNA? Indicate which is the 5’ and which is the 3’ end of the mRNA. b) What is the amino acid sequence that will be translated from this piece of DNAarrow_forwardRestriction sites are palindromic; that is, they read the same in the5' to 3' direction on each strand of DNA. What is the advantage ofhaving restriction sites organized this way?arrow_forwardIn Northern blot analysis, mRNA samples from tissues are bound to a labeled DNA probe that is complementary to the mRNA, and run on a gel to be visualized. The protein tropomyosin is known to be present in both brain and liver. When brain and liver tissue were assayed for the presence of tropomyosin mature mRNA, bands of two different sizes were seen. Tropomyosin gene diagram (3000 bp total): Shown in attatched image If the band on the Northern blot for mRNA isolated from liver tissue is 2580 bp, whereas from brain tissue the band is 2250 bp, what is most likely? a)The two mRNAs are made from different tropomyosin DNA sequences. b)Exon 2 is alternatively spliced out of the brain mRNA. c)Introns 1 and 2 are spliced out of the brain transcript but not the liver transcript. d)Exons 1 and 3 are spliced out of the brain transcript but not the liver transcript. e)Exon 2 is alternatively spliced out of the liver mRNA.arrow_forward
- Choose one of the strands and transcribe the strand. Show the steps (with proper label) and do a post-transcriptional processing. Once the transcript is made, do the process of translation. Again follow the steps. Use the Wobble Table for reference. DNA strand: 5'-TCGAAACGAGCTGCAGCTAGCTAGCTACGTCAGCTGCATCTGTCTACGAGCGACTGTCTAGCATAGCGTAGCTGC-3 'Complementary strand: 3'-TCGAAACGAGCTGCAGCTAGCTAGCTACGTCAGCTGCATCTGTCTACGAGCGACTGTCTAGCATAGCGTAGCTGC-5'arrow_forwardDescribe the process of cloning a DNA fragment into theBamHI and PstI sites of the vector pUC18. How would youscreen for clones that contain an insert? and explain the process(steps) by drawingarrow_forwardDescribes a method known as Western blotting that can be used to detect a polypeptide that is translated from a particular mRNA. In this method, a particular polypeptide or protein is detected by an antibody that specifically recognizes a segment of its amino acid sequence. After the antibody binds to the polypeptide within a gel, a secondary antibody (which is labeled) is used to visualize the polypeptide as a dark band.For example, an antibody that recognizes α-galactosidase A couldbe used to specifically detect the amount of α-galactosidase A proteinon a gel. The enzyme α-galactosidase A is defective in individuals with Fabry disease, which shows an X-linked recessive pattern of inheritance. Amy, Nan, and Pete are siblings, and Pete has Fabry disease. Aileen, Jason, and Jerry are brothers and sister, and Jerry has Fabry disease. Amy, Nan, and Pete are not related to Aileen, Jason, and Jerry. Amy, Nan, and Aileen are concerned that they could be carriers of a defective…arrow_forward
- Choose one of the strands and transcribe the strand. Show the steps (with proper label) and do a post transcriptional processing Once the transcript is made, do the process of translation. Again follow the steps. Use the Wobble Table for referencearrow_forwardIn producing genetically engineered human insulin in bacteria, why is it important to use the samerestriction enzyme to cut both the human DNA and the bacterial plasmid?arrow_forwardArrange the steps that would be used in a laboratory to engineer a bacterium that could express the human gene coding for factor VIII. (Not all steps will be placed).arrow_forward
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