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.
-Which of the following diagrams shows the structure of the cloned cldnk gene after digestion with EcoRI and XhoI?

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.
-Which of the following diagrams shows the structure of the cloned cldnk gene after digestion with EcoRI and XhoI?

Quizplus · Accepted Answer

11ecba2d_102b_e750_a3bf_6ff69949d945_MD0008_00 Restriction enzymes cleave DNA molecules at specific nucleotide sequences, known as restriction sites. Each restriction enzyme has a unique restriction site that typically consists of a DNA palindrome, in which the sequence of one strand is the reverse of its complement strand. If a restriction enzyme cuts anywhere other than directly in the middle of the palindrome, a few nucleotides will be left on the end of the DNA strand without any complementary bases paired with them. These leftover nucleotides are known as overhangs or sticky ends because they can anneal or "stick to" their complementary bases in another DNA strand. The passage states that the cldnk gene was digested by EcoRI and XhoI. Table 1 shows that EcoRI cuts between the G and the A in GAATTC, leaving an AATT overhang, and XhoI cuts between the C and the T of CTCGAG, leaving a TCGA overhang. Therefore, the digested gene should have an AATT overhang on one end and a TCGA overhang on the other, as shown in Choice B (Note: Because the Xhol overhang is shown as the bottom strand, it is read from right to left [5′ to 3′]). (Choice A) EcoRI breaks the bond between the G and the A in GAATTC, so the G would not be present on the overhang after the digest occurs. Similarly, the terminal C would not be present in the overhang after XhoI digests the CTCGAG sequence. (Choice C) Digestion of one end of the DNA by Xbal (not Xhol or EcoRI) would leave a CTAG overhang (reading from right to left), as shown in this diagram. (Choice D) This diagram shows the wrong overhang for the EcoRI site (GAATT instead of AATT) and the wrong overhang for an Xbal site (TCTAG instead of CTAG). Educational objective: Restriction enzymes cleave DNA molecules at specific sites, known as restriction sites. Each enzyme has a unique restriction site and may cut the DNA such that an overhang, or sticky end, is left on the end of the strand.