A researcher is studying the expression pattern of a particular gene. Messenger RNA is isolated from several tissues, subjected to electrophoresis, blotted, and probed with radiolabeled DNA containing sequences from exon 4 from that gene. An x-ray film is then placed over the blotting membrane, with the results of the autoradiogram shown below:
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Which of the following best explains the autoradiogram findings in the different tissues?
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The experiment described above is known as the Northern Blot technique, a procedure used to detect specific mRNA sequences in a sample to assess for gene expression. In this experiment, the Northern Blot identifies three different mRNA transcripts containing exon 4, with varying patterns of expression in the different tissues. This is consistent with alternative splicing, a process whereby the exons of the pre-mRNA produced by transcription of a gene are reconnected in multiple ways during post-transcriptional processing. The resulting finalized mRNAs are then translated into different protein isoforms. Thus, a single gene can code for multiple proteins when the same gene is spliced differently in different tissues.
Alternative splicing is a normal phenomenon in eukaryotes that greatly increases the biodiversity of proteins that can be encoded by the genome. It is thought that at least 70% of the 30,000 genes in the human genome undergo alternative splicing, and that on average, a given gene produces 4 alternatively spliced variants. Thus, the human genome is able to encode a total of 80,000 to 100,000 proteins which differ in their sequence and function.
Abnormal variations in splicing are implicated in many diseases (e.g., beta-thalassemia, cancer). Alternative splicing also plays a prominent role in the lifecycle of many retroviruses. For instance, HIV produces a single primary RNA transcript that is alternatively spliced to produce over 40 different mRNAs.
(Choice B) DNA (gene) rearrangement occurs during the development and maturation of B cells and T cells. VDJ (Variable, Diverse, and Joining) gene recombination is a random process that takes place in the primary lymphoid tissue (the bone marrow for B cells, and Thymus for T cells).
(Choice C) A mutation is a change in the DNA sequence of a gene. While somatic mutations do sporadically occur throughout the body, they do so only in a minority of cells. The vast majority of DNA throughout the body’s tissues consists of identical gene coding sequences.
(Choice D & E) Transcription factors influence RNA polymerase’s affinity for specific genes by binding to DNA promoter sequences or enhancer regions, which can either stimulate or inhibit gene transcription. Transcription factors and enhancer regions affect the expression of pre-mRNA, but they do not influence post-transcriptional processing.
Educational objective:
Alternative splicing is a process where the exons of a gene are reconnected in multiple ways during post-transcriptional processing. This creates different mRNA sequences and subsequently, different protein isoforms. It is a normal phenomenon in eukaryotes that greatly increases the biodiversity of proteins encoded by the genome.