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.
-What observation could have led researchers to conclude that cldnk is expressed in oligodendrocytes?

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.
-What observation could have led researchers to conclude that cldnk is expressed in oligodendrocytes?

Quizplus · Accepted Answer

11ecba2d_102d_46e2_a3bf_7da358058e62_MD0008_00 In genetic studies, hybridization refers to the annealing of two complementary nucleic acid strands. Specifically, mRNA is a single-stranded nucleic acid that is present only in the cytosol of cells that express the corresponding gene. To determine whether a cell expresses a specific gene, tissues may be incubated with a labeled RNA strand that is complementary to the mRNA of interest. This labeled RNA hybridizes with the mRNA in cells that express it, but is washed away from other cells, allowing researchers to visualize which cells express a particular gene. For a cell that expresses a gene of interest, one way to determine the cell type is to identify the presence of another mRNA that is known to be specific to the cell type of interest. The passage gives the names of three genes that are specific to oligodendrocytes: plp1a, Sox10, and mbp. If cldnk mRNA is found in the same cells as the mRNA of any of those genes, then cldnk must be expressed in oligodendrocytes. Therefore, if a probe that is complementary to cldnk mRNA hybridizes to cells that alsoexpress plp1A, then cldnk is expressed in oligodendrocytes. (Choice A) The probe must be complementary to the mRNA of interest for hybridization to occur. If the probe is made of cldnk mRNA, it will be identical, not complementary, to cldnk mRNA in the cell and will not hybridize. (Choice C) The affinity of cldnk cDNA for the microarray will be the same regardless of which cells the cDNA came from. Cldnk cDNA derived from oligodendrocytes binds to a greater extent than the same cDNA derived from other cells because there is more of it present, not because it has higher affinity. (Choice D) Gene expression is mediated by mRNA, which is primarily present in the cytosol. If the probe binds to the nucleus, it would indicate the presence of genomic DNA, which may or may not be transcribed to mRNA. Educational objective: In genetic studies, hybridization is the annealing of two complementary nucleic acids. The presence of a particular mRNA within a specific cell type can be assessed by hybridization to a complementary probe.