Passage
In the central nervous system, myelin produced by oligodendrocytes (glial cells) functions as an insulating sheath surrounding certain nerve fibers. To identify candidate genes involved in the myelination process, researchers collected oligodendrocytes from zebrafish and purified the messenger RNA (mRNA) transcripts expressed by these glial cells. The mRNA was converted to complementary DNA (cDNA) by reverse transcriptase, and the cDNA was fluorescently labeled and assessed for hybridization on a microarray. The expression of mRNA in other zebrafish cells was similarly measured.Transcripts detected in zebrafish oligodendrocytes at levels greater than 3 times those found in other cells were selected as candidate myelination genes. Some of the detected genes, including plp1a, Sox10, and mbp, were previously known to be specific to oligodendrocytes, validating the procedure. The cDNA of newly identified candidates was amplified by PCR, and the ends were digested with the restriction enzymes EcoRI and XhoI. Genes were then ligated into the multiple cloning site (MCS) of the pSKII vector, shown in Figures 1 and 2, which had also been digested by EcoRI and XhoI.
Figure 1 Overview of the pSKII vector (plasmid)
Figure 2 The pSKII MCS, showing the restriction sites of several restriction enzymesTable 1 shows the functional parameters of several restriction enzymes.
To confirm that the candidate genes originated in oligodendrocytes, complementary RNA (cRNA) strands were synthesized from the cloned plasmids and hybridized to fixed zebrafish sections. One of the genes that localized to oligodendrocytes, known as cldnk, produces a 915 base pair mRNA transcript from two exons. The cldnk gene is involved in tight junction formation and may be required for myelin sheath development around axons.
-How would expression of cloned cldnk differ from expression of the endogenous cldnk gene?

Question

Passage
In the central nervous system, myelin produced by oligodendrocytes (glial cells) functions as an insulating sheath surrounding certain nerve fibers. To identify candidate genes involved in the myelination process, researchers collected oligodendrocytes from zebrafish and purified the messenger RNA (mRNA) transcripts expressed by these glial cells. The mRNA was converted to complementary DNA (cDNA) by reverse transcriptase, and the cDNA was fluorescently labeled and assessed for hybridization on a microarray. The expression of mRNA in other zebrafish cells was similarly measured.Transcripts detected in zebrafish oligodendrocytes at levels greater than 3 times those found in other cells were selected as candidate myelination genes. Some of the detected genes, including plp1a, Sox10, and mbp, were previously known to be specific to oligodendrocytes, validating the procedure. The cDNA of newly identified candidates was amplified by PCR, and the ends were digested with the restriction enzymes EcoRI and XhoI. Genes were then ligated into the multiple cloning site (MCS) of the pSKII vector, shown in Figures 1 and 2, which had also been digested by EcoRI and XhoI.

Figure 1 Overview of the pSKII vector (plasmid)

Figure 2 The pSKII MCS, showing the restriction sites of several restriction enzymesTable 1 shows the functional parameters of several restriction enzymes.

To confirm that the candidate genes originated in oligodendrocytes, complementary RNA (cRNA) strands were synthesized from the cloned plasmids and hybridized to fixed zebrafish sections. One of the genes that localized to oligodendrocytes, known as cldnk, produces a 915 base pair mRNA transcript from two exons. The cldnk gene is involved in tight junction formation and may be required for myelin sheath development around axons.
-How would expression of cloned cldnk differ from expression of the endogenous cldnk gene?

Quizplus · Accepted Answer

11ecba2d_102f_b7e5_a3bf_db8777394fc2_MD0008_00 Genes within a eukaryotic chromosome typically consist of exons and introns, both of which are transcribed by RNA polymerase to form pre-mRNA. After transcription, pre-mRNA matures through several processes including 5′ capping by 7-methylguanosine, 3′ polyadenylation, and splicing to remove introns. cDNA is derived from mature mRNA through reverse transcription. Because the mature mRNA has already been spliced (ie, introns were removed), the cDNA will not contain any introns. The cDNA can be cloned into expression vectors such as plasmids, which can be introduced into embryonic cells and integrated into the genome. These cloned genes can then be expressed in the organism. However, because the cloned gene does not contain any introns, its expression will not include splicing. (Choice B) Cloned genes may be expressed at lower levels, the same levels, or higher levels than endogenous genes, depending on several factors, including how the cloning was done, how expression was induced, and the type of cell being observed. (Choices C and D) The cDNA was derived from endogenous mature mRNA, so it will encode the same mature mRNA as the endogenous gene. Because mRNA determines the sequence of the protein to be produced, the cloned gene yields the same protein as the endogenous mRNA from which it was derived. Educational objective: RNA polymerase transcribes both exons and introns to form pre-mRNA. During RNA processing, introns are removed by splicing to yield mature mRNA. cDNA is generated from mature mRNA and does not contain introns, so it is not spliced during expression.